Good continuum of HIV care in Belgium despite weaknesses in retention and linkage to care among migrants

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Good continuum of HIV care in Belgium despite weaknesses in retention and linkage to care among migrants

D. Van Beckhoven1*, E. Florence2, J. Ruelle3, J. Deblonde1, C. Verhofstede4, S. Callens5, E. Vancutsem6, P. Lacor7, R. Demeester8, J.-C. Goffard9, A. Sasse1 and For the BREACH (Belgian Research on AIDS and HIV Consortium)

Author Affiliations

1 Epidemiology of Infectious Diseases Unit, Scientific Institute of Public Health, Rue J. Wytsman 14, Brussels, 1050, Belgium

2 Department of Clinical Sciences, Instituut Tropische Geneeskunde, Antwerp, Belgium

3 Institute of Experimental and Clinical Research (IREC), Unit of Medical Microbiology (MBLG), Université Catholique de Louvain, Brussels, Belgium

4 AIDS Reference Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium

5 Department of Internal Medicine, Universitair Ziekenhuis Gent, Ghent, Belgium

6 Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Brussels, Belgium

7 Department of Internal Medicine, Universitair Ziekenhuis Brussel, Brussels, Belgium

8 Department of Internal Medicine and Infectious Diseases, CHU de Charleroi, Charleroi, Belgium

9 Service of Internal Medicine, Hôpital Erasme, Brussels, Belgium

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BMC Infectious Diseases 2015, 15:496  doi:10.1186/s12879-015-1230-3

The electronic version of this article is the complete one and can be found online at:

Received: 26 March 2015
Accepted: 19 October 2015
Published: 3 November 2015

© 2015 Van Beckhoven et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.



The Belgian HIV epidemic is largely concentrated among men who have sex with men and Sub-Saharan Africans. We studied the continuum of HIV care of those diagnosed with HIV living in Belgium and its associated factors.


Data on new HIV diagnoses 2007–2010 and HIV-infected patients in care in 2010–2011 were analysed. Proportions were estimated for each sequential stage of the continuum of HIV care and factors associated with attrition at each stage were studied.


Of all HIV diagnosed patients living in Belgium in 2011, an estimated 98.2 % were linked to HIV care, 90.8 % were retained in care, 83.3 % received antiretroviral therapy and 69.5 % had an undetectable viral load (<50 copies/ml). After adjustment for sex, age at diagnosis, nationality and mode of transmission, we found lower entry into care in non-Belgians and after preoperative HIV diagnoses; lower retention in non-Belgians and injecting drug users; higher retention in men who have sex with men and among those on ART. Younger patients had lower antiretroviral therapy uptake and less viral suppression; those with longer time from diagnosis had higher ART uptake and more viral suppression; Sub-Saharan Africans on ART had slightly less viral suppression.


The continuum of HIV care in Belgium presents low attrition rates over all stages. The undiagnosed HIV-infected population, although not precisely estimated, but probably close to 20 % based on available survey and surveillance results, could be the weakest stage of the continuum of HIV care. Its identification is a priority along with improving the HIV care continuum of migrants.


HIV; Cascade; Continuum of care; Migrants; Belgium


The use of ART (antiretroviral therapy) in HIV-infected patients has shown its efficiency not only in improving the individual outcomes of patients but also in reducing the transmission of HIV [1]–[3]. A persisting challenge of the HIV epidemic is reaching the highest proportion of overall viral suppression among people living with HIV (PLHIV) in order to impact HIV transmission [3], [4].

In Belgium the epidemic is largely concentrated among men who have sex with men (MSM) – mainly of Belgian or European nationality – and Sub-Saharan African (SSA) men and women. The number of annual new HIV diagnoses increased by half between 1997 and 2003 and has since remained stable at 1000 to 1200 new cases per year [5]. The number of PLHIV has steadily increased since introduction of ART. Recent surveys conducted in two large Belgian cities have shown an HIV prevalence among MSM ranging from 6 [6] to 12 % (C. Noestlinger, personnal communication, March 2015). In a survey among SSA migrants in Antwerp, HIV prevalences of 3 % in men and 6 % in women were found [7].

Services for HIV testing in Belgium are available either in primary care, secondary care (specialized outpatient clinics), hospitals and decentralized projects. Three Aids Reference Centers (ARC) are funded to perform low threshold and free anonymous testing. Outreach programs are set up to better target HIV high risk groups. These programs are organized by the ARCs or through a collaborative effort with NGOs working with the specific target population. Compared to other European countries [8], Belgium has a high and relatively stable rate of HIV testing, with a total of 695.433 HIV tests performed (62 per 1000 inhabitants) in 2013.

Access to HIV care in Belgium is, in principle, ensured to all HIV-infected individuals through the compulsory national health insurance. Antiretroviral therapy is reimbursed when a patient’s CD4 count is below or equal to 500 cells/mm 3 or below 25 % and in case of clinical symptoms. There is no national guideline for HIV care, clinicians follow the guidelines of the European AIDS Clinical Society for clinical and laboratory monitoring [9]. Undocumented migrants may access care through a procedure called urgent medical help [10]. Nevertheless, the key populations in the Belgian HIV epidemic, MSM and migrants, differ in socio-demographic characteristics such as nationality, migrant status and social context which may influence their access to HIV testing and care.

In order to reach the best outcomes for HIV-infected individuals and potentially reduce HIV transmission, a continuum of HIV care services needs to be ensured. The HIV care cascade is an illustration of the continuum of HIV care built on estimates of the size of the HIV-infected population at different stages: infection, diagnosis, linkage to care, retention in care, ART uptake and viral load suppression. This approach allows pinpointing possible attrition along the continuum of care and may help to compare and prioritize prevention and care strategies [3]. These results also inform on the potential for domestic transmission of HIV through the estimation of the HIV-infected population without suppressed VL residing in the country. Several countries have already reported their domestic HIV care cascade [11]–[14]. These were built on available national surveillance data and survey results combined with estimates obtained by mathematical data modelling.

In this study, we estimated the proportion in each stage of the continuum of HIV care in Belgium among diagnosed PLHIV and analysed factors associated with attrition at each respective stage.


Data sources and setting

Estimates of the continuum of care from HIV diagnosis to undetectable viral load (VL) were calculated by analysing combined data from two surveillance systems managed by the Scientific Institute of Public Health (WIV-ISP): the registry of new HIV diagnoses and the Belgian HIV Cohort. The national registry of new HIV diagnoses [5] records all newly diagnosed confirmed HIV cases based on exhaustive reporting by the Belgian AIDS Reference Laboratories (ARLs). The Belgian HIV Cohort study [15] collects data on HIV-infected patients in care including data on viral load measurements recorded by the ARLs and CD4 counts and ART data recorded by the ARCs from 2007 onwards. All HIV-infected patients in medical care in Belgium have their VL analysed in the ARLs and around 75–80 % of patients have been followed in the ARCs in recent years. The WIV-ISP is the legal entity in charge of the HIV and AIDS surveillance activities (Royal decree of 8 October 1996). The HIV surveillance system was approved by the ethical committee of Ghent University hospital and authorized by the Privacy Commission. Strict attention to confidentiality is present at every stage of data collection, analysis and storage.


Proportions in each stage of the continuum of HIV care were estimated using the following definitions.

Linkage to HIV care was defined as having at least one VL or CD4 count recorded within 1 year of HIV diagnosis, with a window of 7 days for VL records to prevent incorrectly counting VL measurements performed at the time of diagnosis as initial HIV care visit.

Retention in HIV care was defined as the proportion of patients in care in 2010, those having one CD4 or VL measurement during that year, who had at least one record of CD4 or VL in 2011.

Proportions of patients on ART and with suppressed VL were measured among those in care in the ARCs in 2011. ART was defined as a record of ART prescription at the end of 2011. Suppressed VL was defined as the last measured VL <50 copies/mL.

Continuum of HIV care

Proportions calculated for each stage of the continuum of HIV care were combined to obtain the distribution of the diagnosed HIV individuals living in Belgium in 2011 by stage of the continuum.

The total diagnosed HIV population living in Belgium was obtained by summing the estimated population retained in care in 2011 with the estimated number of HIV-positive persons diagnosed but not linked to care in 2011 and the estimated number of persons previously in HIV care but not retained in care in 2011. The distribution of the diagnosed HIV population through the stages of the continuum of care is schematized in Fig. 1.

thumbnailFig. 1. Diagram representing the distribution of the HIV diagnosed population within the continuum of HIV care

Using 2007 data, we first estimated the proportion of patients that entered in care later than 1 year after diagnosis. We then applied this proportion of late entry into care to patients diagnosed in the years following 2007 to obtain an estimate of the total number of patients entering late in care. From this total we subtracted those who entered in care before the end of 2011 in order to obtain the estimated number of diagnosed patients not linked to care and expected to enter in care after 2011.

Next we estimated for the patients in care in 2007 and not retained in care after 1 year, the proportion who re-entered care later. Using a similar computation, the number of patients entering back in care was calculated for the years following 2007. From the cumulative total, we subtracted patients already re-entered in care by the end of 2011 in order to obtain the estimated number of patients not retained in care and expected to re-enter care after 2011.

Patients estimated to never enter or re-enter care were considered as having left the country. We also hypothesised that persons diagnosed before 2007 were not expected to enter in care after 2011.

The next stages of the continuum of HIV care were estimated by applying the proportions of patients on ART and with VL suppression to the population in HIV care in 2011.

Data management and statistics

Databases were merged using a unique patient identifier. Identification of duplicate patient records was performed by comparing the following set of variables: gender, initials, date of birth, date of HIV diagnosis, presumed mode of transmission, nationality, place of residence and laboratory results.

Socio-demographic factors, time period since diagnosis and CD4 count at first medical consultation (±31 days) were analysed for association with each stage of the continuum of HIV care by multivariate logistic regression. Analyses were performed with Stata 10.1.


A total of 4117 individuals diagnosed with HIV between 2007 and 2010 were analysed for entry in care. Of 11,781 patients in care in 2010, 112 patients died before end of 2011, leaving 11,669 patients analysed for retention in care. ART uptake and VL levels were analysed for 9710 patients in care in the ARCs in 2011. Newly diagnosed individuals and patients in care shared the following similar socio-demographic characteristics (Table 1). Around two thirds were men and median age at diagnosis was approximately 35 years. Probable acquisition of HIV by sexual transmission was reported by more than 90 % of the patients with available information. Nearly half of the patients with reported nationality were Belgians and one third originated from countries in Sub-Saharan Africa.

Table 1. Baseline characteristics of HIV-infected individuals diagnosed between 2007 and 2010 and of patients in medical care in 2010 in Belgium

Among newly diagnosed individuals, 87.0 % entered in HIV care within 1 year of diagnosis. This proportion increased over the years (2007: 84.5 %, 2008: 87.4 %, 2009: 87.0 %, 2010: 89.0 %; p < 0.001). Linkage to care was made within 3 months following HIV diagnosis for 88.1 % of those who entered in HIV care (Fig. 2). Median CD4 value at first medical contact was not significantly different between those who entered in care in the first 3 months following diagnosis (414 CD4 cells/mm 3 (IQR: 248–595)) and those entered in care later (403 CD4 cells/mm 3 (IQR: 221–585)).

thumbnailFig. 2. Distribution of delay between HIV diagnosis and first medical contact in HIV care (recorded CD4 or VL) among patients entered in care

Of those in care in 2010 and not reported to have died, 92.2 % were retained in care in 2011. Among those in care in the ARCs, 84.6 % were receiving ART, of whom 83.4 % were virally undetectable.

Factors associated with entry, time to entry and retention in care

Univariate analyses show that entry and retention in care were lower among women and non-Belgians and higher among MSM (Table 2). Patients infected through injecting drug use (IDU) had lower retention in care. After adjustment for sex, age at diagnosis, nationality and mode of transmission, entry and retention in care remained lower among non-Belgians, retention was still higher among MSM and lower among IDU. Individuals tested for preoperative reasons had lower entry in care, they represented only 2.6 % of those with available data on reasons for testing, and presented significantly longer delay between diagnosis and entry in care than patients with all other reasons for testing (median time: 32 vs 14 days, p < 0.001). Linkage to care within 3 months of diagnosis was slightly lower among non-Belgians than Belgians (respectively 88.2 and 90.5 %; p = 0.049). Longer time period since HIV diagnosis and ART uptake were both independently associated with higher retention in care.

Table 2. Factors associated with entry in care (2007–2010) and retention in care (2010–2011) among HIV-infected patients in Belgium

Factors associated with ART uptake and viral suppression among those on ART

Univariate analyses show that, younger age was associated with lower ART uptake and less viral suppression on ART, MSM had lower ART uptake and more viral suppression, those diagnosed for longer had higher ART uptake and higher VL suppression. Patients diagnosed for clinical reasons and with lower CD4 count at first contact had higher ART uptake. Sub-Saharan African patients on ART had less VL suppression (Table 3).

Table 3. Factors associated with ART uptake and VL suppression among HIV-infected patients in care in the ARCs (2011), Belgium

Multivariate analyses show that younger age remained associated with lower ART uptake and less VL suppression. HIV testing requested for clinical reasons and lower CD4 count at first contact remained associated with higher ART uptake and Sub-Saharan African nationality with less VL suppression. Those with longer time from diagnosis had higher ART uptake and more frequent VL suppression. Multivariate analyses on ART uptake were adjusted for CD4 count at first medical visit. As time since ART initiation was not available, it could not be taken into account in the multivariate analyses on VL suppression.

Proportions of diagnosed patients along the continuum of HIV care in Belgium

We estimated that 11,478 patients were retained in HIV care in 2011 by applying the proportion of retention in HIV care of 92.2 % observed in 2010 to the 12,449 HIV+ patients with at least one laboratory record in 2011.

The proportion of late entry in care of 4.7 % observed in the 2007 data was applied to persons diagnosed in the years following 2007. After subtraction of those already entered in care, we found that 221 patients were expected to enter in care after 2011 (see detailed computation in Additional file 1).

Re-entry in care among patients not retained in care for 1 year after 2007 was estimated at 39.5 %. By applying this proportion of re-entry to the patients not retained in care in the following years and then subtracting those already re-entered in care, we estimated that 940 persons were expected to re-enter in care after 2011 (see detailed computation in Additional file 1).

After combining these results, the population of diagnosed HIV patients living in Belgium at the end of 2011 (n = 12,639) was distributed as follows: 11,478 patients (90.8 %) were estimated to be retained in HIV care, 221 (1.8 %) were not linked to HIV care and 940 (7.4 %) were not retained in HIV care.

The estimated proportions of 84.6 % of patients in care on ART of whom 83.4 % of patients with viral suppression were applied to the population in HIV care in 2011 (n = 12,449). We estimated 83.3 % (n = 10,532) of the diagnosed HIV population living in Belgium to be on ART and 69.5 % (n = 8784) to have suppressed VL.

The continuum of HIV care of the diagnosed HIV patients living in Belgium is illustrated in Fig. 3.

thumbnailFig. 3. Estimated percentage of diagnosed HIV individuals living in Belgium by stage of the continuum of HIV care, 2011


The continuum of HIV care in Belgium presents low attrition rates over all stages of care with 69 % of all HIV diagnosed individuals in Belgium having an undetectable viral load.

HIV care leans on well-organized specialized structures, the ARLs and the ARCs, that offer access to comprehensive care including free psycho-social support, counselling and fully reimbursed antiretroviral therapy. In addition, these specialized structures have developed collaborations with prevention and testing associations that contribute to a more efficient linkage to HIV care.

Vulnerable populations such as non-Belgians and IDU present lower entry and retention in HIV care but, once retention in care is ensured, their access to ART are similar to other HIV-infected individuals. Yet, poorer virological outcome was observed among SSA migrants. Similar findings for lower entry and retention of these populations are reported in other European studies [13], [16], [17], and lower ART uptake or viral suppression among migrants was observed in Spain [18], UK [19] and France [20]. IDU represent only 2.5 % of the HIV-infected patients in care in Belgium with a sustained low HIV transmission reported over the last years [5] due to accessible harm reduction programs. The limited number of IDUs enables more intensive follow-up and counselling to prevent comorbidities and onward HIV transmission by unsafe injecting practices.

Additionally, more frequent delayed diagnoses observed in these two populations (migrants and IDUs) worsen the poorer outcomes observed along the continuum of care [5], [21]. For migrants whose infection was acquired in their country of origin, the diagnosis delay may precede the arrival in Belgium.

Migrants’ lower entry and retention in HIV care may be due to emigration after HIV diagnosis or barriers to access care for those remaining in Belgium. Attrition is similar between Europeans and non-Europeans, but consequences of emigration out of Belgium in terms of access and continuum of care greatly differ. While West-Europeans have reliable access to care in their country of origin, this may not be true for East-European and non-European migrants. In fact, reported ART coverage among those in need of ART in numerous non-European or East-European countries was below 50 % in 2012 [22] and potentially even lower for stigmatized populations like MSM and IDU. This study does not allow differentiation between documented and undocumented migrants although it is mainly undocumented migrants that face disproportionate barriers to medical care [10]. The HIV epidemic in Europe is influenced by its evolution in other countries through travel and migration [23]. The continuum of care for this mobile population could be improved by developing cross-border and national tools to facilitate adequate and individual-tailored care of the migrant population diagnosed with HIV in Belgium.

The results also underline a gap in linkage to care among patients diagnosed for pre-operative reasons due to longer delay between initial diagnosis and entry in care. Timely communication of HIV diagnosis to the patient in specific settings like pre-operative testing would be facilitated by training of caregivers on sexual health counselling and HIV screening. These interventions are part of the recommendations of the Belgian National HIV plan that has been launched in 2013 [24].

Factors impacting ART uptake and VL suppression were logical indicators of the clinico-immunological situation of the patient: duration since HIV diagnosis, older age with associated co-morbidity, HIV diagnosis for clinical reason and low CD4 count. None of the socio-demographic factors studied impacted ART uptake and VL suppression, except the SSA nationality that was associated with lower VL suppression, although the difference with Belgians was small. These results suggest that ARV treatment needs are equally covered for all patients having access to regular HIV care. Suboptimal ART adherence potentially related to socio-economic factors [18] might explain the slightly lower VL suppression among SSA. Other reasons for non VL suppression might be ARV resistance or recent ART initiation at the time of VL measurement but this information was not collected.

Patients on ART have a higher retention in care as also observed in another cohort [17].

The attrition observed along the continuum of HIV care should not be interpreted globally but according to the stage at which it occurs. Indeed, its consequences, both in terms of individual prognosis and potential for HIV transmission are different in early and late stages of the continuum of care. In the early stages of the continuum, HIV-infected individuals who are not diagnosed, not linked or not retained in care have a higher risk of delay in ART initiation, complications and co-morbidities and have no access to regular psycho-social support and counselling. Hence, attrition in the early stages is associated with a higher likelihood of unfavourable clinical evolution and onward transmission [25]. In the late stages of the continuum, patients retained in regular HIV care in Belgium have access to counselling services and timely ART initiation based on their viro-immunological status and their presumed need for ART to prevent transmission. Those untreated or presenting detectable VL among this carefully monitored population are considered as lost in the late stages of the continuum of care according to the cascade analysis definition. Yet, the likelihood of them causing onward transmission or having an unfavourable clinical evolution is lower than among the population lost in the early stages of the continuum [26].

In other European cascades of HIV care, reported proportions of VL suppression among those diagnosed with HIV ranged from 70 % (<500 copies/mL) in Sweden and Denmark in 2010 [13], 72 % (<50 copies/mL) in UK in 2011 [12] and 64 % (<50 copies/mL) in France in 2010 [14]. Given the various definitions used for the study population and for each stage of the continuum of care, international comparison is difficult. Nevertheless Belgian results are close to previously reported European proportions while differing strongly from the recently reported proportion of 35 % of those diagnosed achieving viral suppression (<200 copies/mL) in the United States in 2011 [11].

The UNAIDS has recently published global targets of 90 % of PLHIV knowing their status, 90 % of those receiving ART and 90 % of those having suppressed VL by 2020 [27]. The results presented here show that Belgium is not yet fulfilling these targets. With the recent release of the results of the START study supporting added benefit of offering treatment to everyone with HIV [28] however, we may expect an increase in the proportion of patients on ART in the coming years. This might also increase the retention in HIV care among recently diagnosed patients, who will initiate ART immediately, as ART uptake was associated with higher retention in HIV care. Efforts should then concentrate on the diagnosis of persons unaware of their HIV infection in order to initiate their treatment earlier.

Our understanding of the continuum of HIV care is limited by the lack of precise national estimates of PLHIV unaware of their status. In Europe, 30 % of PLHIV were estimated to be unaware of their infection, with large variations between countries [23]. In Belgium, a local survey limited in sample size and geographical coverage, reported a proportion of undiagnosed HIV-infected MSM of 14 % in 2010 [8]. In the national STI sentinel network, the proportion of patients co-infected with HIV and STI ignoring their HIV status was 14 % in 2011 [29]. Among migrants newly diagnosed in 2013, 93 % of those with known date of arrival (70 %) came to Belgium in the 2 year period preceding the diagnosis in Belgium [30], implying a limited period of residence in Belgium as undiagnosed. Based on these various sources of information, the proportion of undiagnosed PLHIV residing in Belgium is probably not higher than 20 %. This approximate estimate is similar to the proportions of 24 and 19 % found in UK and France respectively [14], [31], [32]. As those infected and remaining undiagnosed for long end up being diagnosed late, the similar proportion of late diagnoses in Belgium as compared to these two countries [8], suggests that the pool of undiagnosed PLHIV in Belgium is not larger than in surrounding countries. When including this estimate in the continuum of care, the proportion of individuals with suppressed VL among all PLHIV residing in Belgium is estimated at 56 %. The proportion of 20 % of PLHIV unaware of their HIV infection could represent the weakest stage of the Belgian HIV care continuum. Given the low attrition observed along the subsequent steps of the continuum of HIV care, identifying the undiagnosed PLHIV could be the intervention with potentially the highest impact on transmission and patient prognosis in Belgium, this coincides with observations made in other countries such as UK, Canada and the US [25], [33]. A project aiming at estimating and characterizing undiagnosed HIV-infected populations by mathematical modelling of routine HIV surveillance data will be conducted in the coming years. These results will be used to target HIV testing to groups that require it the most. Among those undiagnosed, recently HIV-infected individuals appear to be disproportionately involved in onward HIV transmission, likely due to increased viral concentrations, higher viral fitness and more risky behaviour around time of HIV acquisition [2], [34]. In Belgium, the proportion of recently infected individuals among those diagnosed was estimated at 37.5 % in 2012 [35], higher than in UK and France where it ranged from 22 to 30 % [14], [31], [32]. HIV testing strategies, tailored to identify those recently infected as well as those who have remained undiagnosed for long, should be maintained and expanded. Alternative decentralized testing strategies should be further developed along with interventions to reduce missed opportunities for earlier HIV diagnosis in medical settings.

There are some limitations in this study. The results do not inform on the outcomes of the patients after they have left the country. In addition, HIV-infected citizens of surrounding countries are also difficult to capture in the continuum of care as they might be diagnosed and followed in their own country whilst actively contributing to the local epidemic.

We used 2007 data to estimate the proportions of late entry and re-entry into HIV care as time of follow-up for these patients was at least 4 years. We assumed these proportions did not change over the next years and applied them to estimate the number of patients with late entry and with re-entry during the following years. However as linkage to care within 1 year improved slightly after 2007, the proportion of patients expected to enter late in HIV care in the following years might be lower. Hence the population of persons diagnosed and not linked to HIV care living in Belgium might be slightly overestimated based on the proportions estimated in 2007.

This analysis was strongly dependent of a good match between HIV diagnosis data and laboratory data through identifiers built on patients’ date of birth and initials. Errors on these identifiers are not excluded but they are likely to represent a very low proportion of the entire dataset thanks to the thorough annual monitoring.

The analyses of the early stages of the continuum of care included all the individuals diagnosed with HIV and in care in Belgium. Only the analyses on ART coverage and VL suppression were restricted to the patients retained in care in the ARCs. The ARC population differed significantly from those outside of the ARCs for some characteristics such as gender (women: 36 % vs 42 %), nationality (Sub-Saharan Africans: 36 % vs 43 %; Belgians: 50 % vs 45 %), mode of transmission (MSM: 40 % vs 30 %; heterosexuals: 54 % vs 56 %) and median age (34 years vs 32 years). Given the small proportion of patients retained in care outside the ARCs (20 %) and age being the single characteristics associated with ART uptake and VL suppression, the estimates obtained from the ARC population could be used as estimates for the whole study population.

Information on age and sex was missing in less than 0.5 % of the cases, data on mode of transmission and nationality were missing for approximately 30 % of cases, and up to 60 % for those who never entered in care. The high proportion of missing information among the latter is foreseeable given their medical contact limited to the diagnosis consultation. Such cases included a majority of patients who emigrated after diagnosis as any other patient remaining in Belgium would end up entering in care, thus higher completeness of such data would probably reinforce the strong association observed between no entry and non-Belgian nationality.


The continuum of HIV care in Belgium is well ensured although some barriers to access care for migrants, possibly linked with their legal status, are observed. Migrants who emigrated after diagnosis or those who are in care in neighbouring countries were not captured by this analysis, although these are major actors in the dynamic of the epidemic. Strategies to improve identification of the undiagnosed PLHIV is a priority. Together with the improvement of the HIV care continuum of migrants, these strategies would contribute to move closer to the ambitious “90-90-90” global targets of the UNAIDS [27].


ARC: AIDS Reference Centre

ARL: AIDS Reference Laboratory

ART: Antiretroviral therapy

IDU: Injecting drug use

MSM: Men who have sex with men

PLHIV: People living with HIV

VL: Viral load

WIV-ISP: Scientific Institute of Public Health

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

DVB and AS defined the research theme with the contribution of EF, JR, JD, CV, EV, RD, JCG. DVB developed the study design. DVB and AS performed the data management. DVB performed the statistical analyses. DVB, EF, JR, JD, CV, EV, RD, JCG, AS interpreted the data. DVB drafted the manuscript. EF, JR, JD, CV, SC, EV, PL, RD, JCG, AS read and critically revised the subsequent drafts of the manuscript. All authors approved the final manuscript.

Additional file

Additional file 1:. Computation for estimates of populations in HIV care. (DOCX 14 kb)

Format: DOCX Size: 15KB Download fileOpen Data


The Belgian HIV surveillance including this study is financed by the National Institute for Sickness and Invalidity Insurance (INAMI/RIZIV).

We thank the following members of the Belgian Research on AIDS and HIV Consortium (BREACH) for providing the data and supporting the study: S. De Wit (chair, ARC CHU Saint-Pierre), D. Vogelaers (co-chair, ARC UZ Gent), M.-L. Delforge (ARL Hôpital Erasme), E. Florence (ARC ITG), K. Fransen (ARL ITG), J.-C. Goffard (ARC Hôpital Erasme), M.-P. Hayette (ARL CHU Liège), P. Lacor (ARC UZ Brussel), J.-C. Legrand (ARC CHU Charleroi), M. Moutschen (ARC CHU Liège), D. Piérard (ARL UZ Brussel), J. Ruelle (ARL UCL), D. Vaira (ARL CHU Liège), L. Vandekerckhove (ARC UZ Gent), S. Van den Wijngaert (ARL Hôpital Saint-Pierre), B. Vandercam (ARC Cliniques Universitaires Saint-Luc), M. Van Ranst (ARL KUL), E. Van Wijngaerden (ARC UZ Leuven), C. Verhofstede (ARL UZ Gent). We thank Cristina Valencia for careful reading of the manuscript.


  1. Cohen MS, Chen YQ, McCauley M, Gamble T, Hosseinipour MC, Kumarasamy N et al.. Prevention of HIV-1 infection with early antiretroviral therapy. N Engl J Med. 2011; 365(6):493-505. PubMed Abstract | Publisher Full Text OpenURL
  2. Cambiano V, O’Connor J, Phillips AN, Rodger A, Lodwick R, Pharris A et al.. Antiretroviral therapy for prevention of HIV transmission: implications for Europe. Euro Surveill. 2013; 18(48):20647. PubMed Abstract | Publisher Full Text OpenURL
  3. Nosyk B, Krebs E, Eyawo O, Min JE, Barrios R, Montaner JS. Cost-effectiveness Analysis Along the Continuum of HIV Care: How Can We Optimize the Effect of HIV Treatment as Prevention Programs? Curr HIV /AIDS Rep. 2014; 11:468-78. PubMed Abstract | Publisher Full Text OpenURL
  4. Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-Mangen F et al.. Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med. 2000; 342(13):921-9. PubMed Abstract | Publisher Full Text OpenURL
  5. Sasse A, Deblonde J, Van Beckhoven D. Epidémiologie du SIDA et de l’infection à VIH en Belgique: Situation au 31 décembre 2013. Scientific Institute of Public Health, Brussels; 2014. OpenURL
  6. Vanden Berghe W, Nostlinger C, Buve A, Beelaert G, Fransen K, Laga M. A venue-based HIV prevalence and behavioural study among men who have sex with men in Antwerp and Ghent, Flanders, Belgium, October 2009 to March 2010. Euro Surveill 2011; 16(28).
  7. Loos J, Nostlinger C, Vuylsteke B, Manirankunda L, Namanya F, Muhizi J et al.. HIV-prevalence among sub-Saharan African Migrants (SAM) in Antwerp city. First results of the TOGETHER project. Oral presentation at the BREACH Symposium, Brussels, Belgium; 2014. OpenURL
  8. HIV/AIDS surveillance in Europe 2013. European Center for Disease Prevention and Control, Stockholm; 2014. OpenURL
  9. European AIDS Clinical Society (EACS). Guidelines. Version 8.0. October 2015. Available from:. http://www. OpenURL
  10. Chauvin P, Parizot I, Simmonot N. Access to healthcare for undocumented migrants in 11 European countries. 2008 survey report. Médecins du Monde European Observatory on Access to Healthcare; 2009 Sep.
  11. Bradley H, Hall HI, Wolitski RJ, Van Handel MM, Stone AE, LaFlam M et al.. Vital Signs: HIV diagnosis, care, and treatment among persons living with HIV–United States, 2011. MMWR Morb Mortal Wkly Rep. 2014; 63(47):1113-7. PubMed Abstract | Publisher Full Text OpenURL
  12. Delpech V, Brown A, Conti S, Polavarapu V, Yin Z. Reducing onward transmission: Viral suppression among key population groups living with HIV in the United Kingdom. Oral presentation at the 19th Annual Conference of the British HIV Association (BHIVA), Manchester; 2013. OpenURL
  13. Helleberg M, Haggblom A, Sonnerborg A, Obel N. HIV care in the Swedish-Danish HIV cohort 1995–2010, closing the gaps. PLoS One. 2013; 8(8):e72257. PubMed Abstract | Publisher Full Text OpenURL
  14. Supervie V, Costagliola D. The spectrum of engagement in HIV care in France: strengths and gaps. 20th Conference on Retroviruses and Opportunistic Infections. Atlanta, USA: March 2013. Abstract#1030.
  15. Van Beckhoven D, Buve A, Ruelle J, Seyler L, Sasse A. A national cohort of HIV-infected patients in Belgium: design and main characteristics. Acta Clin Belg. 2012; 67(5):333-7. PubMed Abstract | Publisher Full Text OpenURL
  16. HIV in hiding: methods and data requirements for the estimation of the number of people living with undiagnosed HIV. AIDS. 2011; 25(8):1017-23. OpenURL
  17. Thierfelder C, Weber R, Elzi L, Furrer H, Cavassini M, Calmy A et al.. Participation, characteristics and retention rates of HIV-positive immigrants in the Swiss HIV Cohort Study. HIV Med. 2012; 13(2):118-26. PubMed Abstract | Publisher Full Text OpenURL
  18. Monge S, Alejos B, Dronda F, Del RJ, Iribarren JA, Pulido F et al.. Inequalities in HIV disease management and progression in migrants from Latin America and sub-Saharan Africa living in Spain. HIV Med. 2013; 14(5):273-83. PubMed Abstract | Publisher Full Text OpenURL
  19. Uptake and outcome of combination antiretroviral therapy in men who have sex with men according to ethnic group: the UK CHIC Study. J Acquir Immune Defic Syndr. 2012; 59(5):523-9. OpenURL
  20. Celse M, Geffroy L, Geoffard P, Yeni P, Commission on migrants of the French National AIDS Council (CNS). French policy on access to care for illegal immigrants living with HIV: advantages, limits and improvement levers. 2015. AIDS 2014. Melbourne, Australia: July 2014. Abstract#THPE313.
  21. Pharris A, Spiteri G, Noori T, Amato-Gauci A. Ten years after Dublin: principal trends in HIV surveillance in the EU/EEA, 2004 to 2013. Euro Surveill. 2014; 19(47):20968. PubMed Abstract | Publisher Full Text OpenURL
  22. UNAIDS. Global report: UNAIDS report on the global AIDS epidemic 2013. Joint United Nations Programme on HIV/AIDS (UNAIDS) 2013; 2015 Jul.
  23. Hamers FF, Phillips AN. Diagnosed and undiagnosed HIV-infected populations in Europe. HIV Med. 2008; 9 Suppl 2:6-12. PubMed Abstract | Publisher Full Text OpenURL
  24. HIV Plan 2014–2019. Federal Public Service Health, Food chain safety and environment, Belgium; 2013. OpenURL
  25. Skarbinski J, Rosenberg E, Paz-Bailey G, Hall HI, Rose CE, Viall AH et al.. Human Immunodeficiency Virus Transmission at Each Step of the Care Continuum in the United States. JAMA Intern Med. 2015; 175:588-96. PubMed Abstract | Publisher Full Text OpenURL
  26. Brown AE, Nardone A, Delpech VC. WHO ’Treatment as Prevention’ guidelines are unlikely to decrease HIV transmission in the UK unless undiagnosed HIV infections are reduced. AIDS. 2014; 28(2):281-3. PubMed Abstract | Publisher Full Text OpenURL
  27. 90-90-90 An ambitious treatment target to help end the AIDS epidemic. Joint United Nations Programme on HIV/AIDS (UNAIDS), Geneva; 2014. OpenURL
  28. Initiation of Antiretroviral Therapy in Early Asymptomatic HIV Infection. N Engl J Med. 2015; 373:795-80. Publisher Full Text OpenURL
  29. Verbrugge R, Sasse A. Surveillance des infections sexuellement transmissibles au sein de la population générale en Belgique et dans les régions. Données de 2011. Scientific Institute of Public Health, Brussels; 2012. OpenURL
  30. Sasse A. Epidemiology of HIV infection. Situation in Belgium, Dec. 2013. Oral presentation at the BREACH Symposium, Brussels; 2014. OpenURL
  31. Supervie V, Ndawinz JD, Lodi S, Costagliola D. The undiagnosed HIV epidemic in France and its implications for HIV screening strategies. AIDS. 2014; 28(12):1797-804. PubMed Abstract | Publisher Full Text OpenURL
  32. Yin Z, Brown AE, Hughes G, Nardone A, Gill ON, Delpech VC et al.. HIV in the United Kingdom 2014 Report: data to end 2013. Public Health England, London; 2014. OpenURL
  33. Eyawo O, Hogg RS, Montaner JS. The Holy Grail: The search for undiagnosed cases is paramount in improving the cascade of care among people living with HIV. Can J Public Health. 2013; 104(5):e418-e419. PubMed Abstract | Publisher Full Text OpenURL
  34. Cohen MS, Shaw GM, McMichael AJ, Haynes BF. Acute HIV-1 Infection. N Engl J Med. 2011; 364(20):1943-54. PubMed Abstract | Publisher Full Text OpenURL
  35. Sasse A, Florence E, Pharris A, De Wit S, Lacor P, Van Beckhoven D, et al. Late presentation to HIV testing is overestimated when based on the consensus definition [published online ahead of print July 28, 2015]. HIV Med. doi:. 10. 1111/hiv.12292 OpenURL

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Challenges to the management of curable sexually transmitted infections

Marcus Y Chen12 and Sepehr N Tabrizi345*

Author Affiliations

1 Melbourne Sexual Health Centre, Alfred Health, Carlton 3053, VIC, Australia 2 Central Clinical School, Monash University, Clayton 3800, VIC, Australia 3 Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Australia 4 Department of Microbiology and Infectious Diseases, Royal Women’s Hospital, Melbourne, Australia 5 Murdoch Children’s Research Institute, Melbourne, Australia

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BMC Infectious Diseases 2015, 15:337  doi:10.1186/s12879-015-1061-2 The electronic version of this article is the complete one and can be found online at:

Received: 15 June 2015
Accepted: 23 July 2015
Published: 1 December 2015
© 2015 Chen and Tabrizi. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.


Each year, hundreds of millions of new cases of curable sexually transmitted infections (STIs) occur worldwide resulting in reproductive and other serious sequelae, as well as enhanced transmission of HIV. The clinical management and control of these STIs should include as a minimum access to services that provide timely and accurate diagnostic testing together with effective treatment. The provision of appropriate treatment is challenged by the development of increasing antimicrobial resistance, in particular with gonorrhoea and Mycoplasma genitalium infections, requiring new treatments and management algorithms. In addition, infections such as chlamydia, syphilis and trichomoniasis, which show few signs of resistance, are nevertheless highly prevalent and require better public health control measures. While these may be achievable in high income countries, they are still beyond the reach of many low and middle income countries, making substantial improvements in STI management and reductions in STI prevalence challenging.


In 2008, the World Health Organization (WHO) estimated that globally there were 499 million new cases of curable sexually transmitted infections (STIs), including gonorrhoea, chlamydia, syphilis, and trichomoniasis, occurring every year. Discouragingly, this was even higher than the estimated 448 million cases reported in 2005 [1]. Each of these cases, mostly detected in young men and women, represents a person diagnosed with what is generally a stigmatizing condition with potentially serious reproductive or other sequelae. These include ectopic pregnancy, pelvic inflammatory disease, preterm birth, female infertility, neonatal death, and enhanced acquisition and transmission of HIV, just to name a few [1], [2]. This begs the question, what more can be done to improve the diagnosis, treatment and control these curable infections? This special issue of BMC Infectious Diseases focuses on the challenges confronting the clinical management of several key treatable STI and STI related syndromes with experts in the field offering opinions on where future efforts should be focused. These include infections where antimicrobial resistance is established and increasing as well as infections that show few signs of resistance but are nevertheless highly prevalent with the need for better control. Over several decades, Neisseria gonorrhoeae has successively developed antimicrobial resistance to multiple classes of antibiotics and has been identified by the United States Center for Disease Control as one of the top urgent antibiotic resistance threats [3]. Case reports of high level resistance to ceftriaxone have rung alarm bells and further stimulated efforts towards the discovery of new alternative treatments [4], [5]. In this issue Unemo reviews these and other cases, highlights the need for continued vigilance, and discusses potential new agents in the development pipeline aimed at combatting this mercurial bacterium [6]. Mycoplasma genitalium is a common cause of male urethritis and is found in women with pelvic inflammatory disease [7]. Like gonorrhoea, Mycoplasma genitalium has developed increasing resistance to various antibiotic classes, a problem that is currently under recognized and in pressing need for greater attention. Jensen et al. provide a detailed overview of data pointing to the growing antimicrobial resistance evident with M. genitalium infections [8]. It is difficult to envisage any reversal in these trends without the introduction of fundamental measures such as widespread routine diagnostic testing for M. genitalium, availability of testing for antimicrobial resistance, and ongoing surveillance. In many high income countries these are in place for gonorrhoea but notably absent for M. genitalium. Moi et al. explore the treatment implications of M. genitalium resistance further in their proposed guidance on the clinical management of men presenting with non-gonococcal urethritis, one of the most common STI related syndromes [9]. This currently presents a major clinical challenge as most urethral chlamydia will clear with azithromycin or doxycycline but both of these antibiotics are becoming increasingly ineffective at eliminating M. genitalium urethritis. Moi et al. float the idea of withholding treatment from men presenting with non-gonococcal urethritis until a diagnosis of chlamydia or M. genitalium is confirmed by laboratory testing. This concept is worthy of further exploration and may need to be tailored to different clinical settings. Better drugs and more rational management algorithms for non-gonococcal urethritis are needed. While antimicrobial resistance to chlamydia is fortunately not currently contributing to the treatment failures to any significant extent, there continues to be uncertainty over whether single dose azithromycin, which has been the mainstay of treatment for uncomplicated genital chlamydial infections for many years, or doxycycline, should be the preferred treatment for chlamydia [10]. The question of whether doxycycline should replace single dose azithromycin as first line therapy for non-gonococcal urethritis in men based on efficacy against chlamydia and possible induction of macrolide resistant M. genitalium warrants further investigation [10]. A growing number of observational studies have suggested that azithromycin is inferior to doxycycline for the treatment of rectal chlamydia [11]. Kong et al. delve into these issues in their review of chlamydia treatment and call for randomized controlled trials to definitively determine if doxycycline should replace single dose azithromycin for the treatment of rectal chlamydia [12]. Several studies have pointed to rectal chlamydia being prevalent among not only men who have sex with men, but also women [13]. Globally, syphilis remains a pressing concern with international data indicating a resurgence of syphilis among men who have sex with men and unacceptably high rates of congenital syphilis [14], [15]. To highlight the magnitude of this, the WHO estimated that in 2008 syphilis infections in pregnancy led to 305,000 fetal and neonatal deaths and 215,000 infants at risk of dying from prematurity, low birth weight or congenital syphilis worldwide [1]. Syphilis also drives acquisition of HIV infections, worrying as both syphilis and HIV are overrepresented in many populations of MSM [16]. In their review on the challenges in the management of adult syphilis, Tuddenham et al. revisit whether standard treatments for syphilis are adequate in HIV positive patients [17]. They also discuss uncertainty over the management of patients who remain serofast following syphilis treatment and developments in laboratory diagnostics. Given the extraordinarily high global prevalence of Trichomonas vaginalis and its potential to promote HIV transmission, further investment into the optimal diagnosis, treatment, and control of T. vaginalis is clearly needed. Kissinger provides a comprehensive review on the epidemiology, diagnosis and management of this infection [18]. The fact that T. vaginalis screening of HIV positive women in the US alone would save an estimated $160 million in lifetime costs from new HIV infections prevented underscores the public health importance of the optimal detection and management of this protozoal infection [19]. While not usually regarded as a curable STI, bacterial vaginosis is common among women presenting with vaginal discharge and linked to reproductive sequelae and enhanced HIV transmission [20], [21]. Bradshaw et al. discuss emerging areas for research into bacterial vaginosis aimed at reducing recurrence of this condition which is common following treatment [22]. These include studies to help elucidate the postulated role of reinfection from sexual partners, vaginal microbiota, and vaginal biofilm in the pathogenesis of bacterial vaginosis and possible interventions targeting these. The WHO global strategy for the prevention and control of STI stipulates that comprehensive STI management should include as a minimum, accurate diagnosis by syndrome or laboratory diagnosis, plus effective treatment to prevent complications and further transmission [23]. While laboratory testing is taken for granted in high income countries, they are still beyond the reach of many low and middle income countries. Better performing point of care tests and use of self-collected sampling for several STI bring some hope of more access to diagnostic testing. Similarly, while reasonable adherence by health providers to local treatment guidelines governing antibiotic use for STI should be expected in well-resourced countries, less regulated use of antibiotics in resource limited settings threatens to only compound antimicrobial resistance. Stark differences in levels of health funding, health service infrastructure, together with what is often a low priority for STI on the political agenda, means meaningful improvements in STI management with reductions in STI prevalence will for the foreseeable future remain challenging.

Competing interests

The authors declared that they have no competing interests.

Authors’ contributions

MC and ST both contributed to the drafting and writing of the editorial and approve of the final version.


We wish to thank David Lewis for his comments on the editorial.


  1. Sexually transmitted infections (STIs): the importance of a renewed commitment to STI prevention and control in achieving global sexual and reproductive health. WHO, Geneva; 2013. OpenURL
  2. Wiesenfeld HC, Cates W. Sexually transmitted diseases and infertility. In: Holmes KK, editors. Sexually Transmitted Diseases. 4th ed. McGraw-Hill; 2008. p. 1511–1527.
  3. Goire N, Lahra MM, Chen MY, Donovan B, Fairley CK, Guy R, Kaldor J, Regan D, Ward J, Nissen M, Sloots T, Whiley DM. Molecular approaches to enhance surveillance of gonococcal antimicrobial resistance. Nat Rev Microbiol. 2014; 12:223-9. PubMed Abstract | Publisher Full Text OpenURL
  4. Ohnishi M, Saika T, Hoshina S, Iwasaku K, Nakayama S, Watanabe H, Kitawaki J. Ceftriaxone-resistant Neisseria gonorrhoeae, Japan. Emerg Infect Dis. 2011; 17:148-9. PubMed Abstract | Publisher Full Text OpenURL
  5. Unemo M, Golparian D, Nicholas R, Ohnishi M, Gallay A, Sednaoui P. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure. Antimicrob Agents Chemother. 2011; 56:1273-80. PubMed Abstract | Publisher Full Text OpenURL
  6. Unemo M. Current and future antimicrobial treatment of gonorrhoea: the rapidly evolving Neisseria gonorrhoeae continues to challenge. BMC Infect Dis. 2015. OpenURL
  7. Manhart L, Broad JM, Golden MR. Mycoplasma genitalium: should we treat and how? Clin Infect Dis. 2011; 53(S3):S129-42. PubMed Abstract | Publisher Full Text OpenURL
  8. Jensen JS, Bradshaw CS. Management of Mycoplasma genitalium infections: can we hit a moving target? BMC Infect Dis. 2015. PubMed Abstract | Publisher Full Text OpenURL
  9. Moi H, Blee K, Horner PJ. Management of non-gonococcal urethritis. BMC Infect Dis. 2015. PubMed Abstract | Publisher Full Text OpenURL
  10. Kong FYS, Tabrizi SN, Law M, Vodstrcil L, Fairley CK, Chen M, Guy R, Bradshaw C, Hocking JS. Azithromycin versus doxycycline for the treatment of genital chlamydia infection – a meta-analysis of randomised controlled trials. Clin Infect Dis. 2014; 59(2):193-205. PubMed Abstract | Publisher Full Text OpenURL
  11. Khosropour CM, Dombrowski JC, Barbee LA, Manhart LE, Golden MR. Comparing azithromycin and doxycycline for the treatment of rectal chlamydial infection: a retrospective cohort study. Sex Transm Dis. 2014; 41(2):79-85. PubMed Abstract | Publisher Full Text OpenURL
  12. Kong FYS, Hocking JS. Treatment challenges for urogenital and anorectal Chlamydia trachomatis. BMC Infectious Diseases 2015; doi:10.1186/s12879-015-1030-9
  13. Gratix J, Singh AE, Bergman J, Egan C, Plitt SS, McGinnis J, Bell CA, Drews SJ, Read R. Evidence for increased chlamydia case finding after the introduction of rectal screening among females attending two Canadian STI clinics. Clin Infect Dis. 2015; 60(3):398-404. Publisher Full Text OpenURL
  14. Read P, Fairley CK, Chow E. Increasing trends of syphilis among men who have sex with men in high income countries. Sex Health. 2015; 12:155-63. Publisher Full Text OpenURL
  15. Global guidance on criteria and processes for validation: elimination of mother-to-child transmission of HIV and syphilis. WHO, Geneva; 2014. OpenURL
  16. Solomon MM, Mayer KH, Glidden DV, Liu AY, McMahan VM, Guanira JV, Chariyalertsak S, Fernandez T, Grant RM. Syphilis predicts HIV incidence among men and transgender women who have sex with men in a pre-exposure prophylaxis trial. Clin Infect Dis. 2014; 59:1020-6. PubMed Abstract | Publisher Full Text OpenURL
  17. Tuddenham SA, Ghanem KG. Emerging trends and persistent challenges in the management of adult syphilis. BMC Infectious Diseases 2015; doi:10.1186/s12879-015-1028-3
  18. Kissinger P. Current challenges in the treatment of Trichomonas vaginalis. BMC Infectious Diseases 2015; doi:. 10. 1186/s12879-015-1055-0 webcite OpenURL
  19. Lazenby GB, Unal ER, Andrews AL, Simpson K. Cost-effectiveness analysis of annual Trichomonas vaginalis screening and treatment in HIV-positive women to prevent HIV transmission. Sex Transm Dis. 2014; 41(6):353-8. PubMed Abstract | Publisher Full Text OpenURL
  20. Kenyon C, Colebunders R, Crucitti T. The global epidemiology of bacterial vaginosis: a systematic review. Am J Obstet Gynecol. 2013; 209(6):505-523. PubMed Abstract | Publisher Full Text OpenURL
  21. Cohen CR, Lingappa JR, Baeten JM, Ngayo MO, Spiegel CA, Hong T, Donnell D, Celum C, Kapiga S, Delany S, Bukusi EA. Bacterial vaginosis associated with increased risk of female-to-male HIV-1 transmission: a prospective cohort analysis among African couples. PLoS Med. 2012; 9(6):e1001251. PubMed Abstract | Publisher Full Text OpenURL
  22. Bradshaw CS, Brotman RM. Making inroads into improving bacteria vaginosis treatment – striving for long-term cure. BMC Infectious Diseases 2015; doi:10.1186/s12879-015-1027-4.
  23. Global strategy for the prevention and control of sexually transmitted infections: 2006–2015. WHO, Geneva; 2007. OpenURL

Health Information Technology and Family Physicians – Rebecca Filhart

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Health Information Technology and Family Physicians

Rebecca Filhart

Davenport University

CAPS 799

Professor: Dr. Timothy Delicath

Table of Contents



Abstract 3

Introduction. 4

Review of Literature. 11

Research methodology and design……………………………………………………………….38

Procedures …………………………………………….…………………………………………39

Instrumentation ………………………………………………………………………………….41

Data Collection Process …………………………………………………………………………42

Synthesis of Information………………………………………..……………………………… 45

Data Analysis ………………………………….….……………………………………………..46

Findings …………………………………………………………………………………………46

Conclusion ………………………………………………………………………………………47

Inferences………………………………………………………….……………………………. 49

Recommendations ………………………………………………….……………………………50

References ……………………………………………………………………………………….53

Appendix A………………………………………………………..……………………………. 58

Appendix B………………………………………………………………………………………59



With health information technology making such a huge impact on health care, and with the family physician being on the forefront of health care, the purpose of this project was to examine and analyze the effectiveness, advantages, and disadvantages of combining the two fields in the past, present and future.  The methodology used was to research scholarly articles on health information technology and how this technology pertains to family medicine and the impact it has on physicians and patients.  The researched material and data was then gleaned and synthesized to be presented to the reader in a clear and concise manner.  The results found from the research were that there were indeed many advantages and disadvantages as well as challenges in combining health information technology and family medicine.  In conclusion it was found that health information technology, although still relatively new, can be used as an advantage to the family physician and their patients.  However, it was also concluded that there was much work to do in utilizing health information technology by family physicians in such a way that it could be used to its full potential in family medicine.

Health Information Technology and Family Physicians




As we examine the field of health information technology, and more specifically how it applies to the field of family medicine, we will hopefully be able to identify the challenges, and the benefits that this technology can offer.  Furthermore, if we can identify the challenges that family physician’s and medicine has when it comes to health information technology, perhaps this will also help us to find or propose solutions to help the effectiveness and efficiency of family medicine and thereby increase the quality of patient care.

Health information technology has had a dramatic effect on the field of family medicine for quite some time now.  It has impacted the way that physicians practice medicine, and how they interact with their patients.  It has also influenced how patients receive their care.  Health information technology has helped the patient to become more of an active user in terms of being more proactive and knowledgeable about their health care management.

In the past, family medicine and health information has been mainly on hard copy.  This meaning that the patient records were kept in their file, with handwritten notes by the physician and the health care workers.  Prescriptions were also hand written by the physician.  The prescription would then be handed over to the patient, where the patient would take the piece of paper with the hand written prescription to the pharmacy and the prescription would finally be filled.

With the progress that has been made with health information technology, many family medicine offices are paperless.  Everything is kept in a database.  This database is easily accessible wherever there is an internet connection.  Prescriptions can be sent from the physician directly to the patient’s pharmacy of choice.  Patients can also look up their health records from home to see lab results.  The patient can also be made aware of the other information that may be of interest or benefit to them.

With all of this new technology however, there have also been many challenges.  Some of these challenges include patient confidentiality, as so called hackers can break into health information systems and steal patient information.  This is also amplified by health care workers accidentally leaving their computer screens open to reveal private patient information.  There is also a potentially huge cost to have or participate in a safe and secure health information system that is an operating cost for a lot of family practices today.

An enticing carrot to adapting to all of this technology is that there is a monetary bonus to family practices that use health information technology from the government, especially if the practice deals with Medicare or Medicaid patients.  However, is this bonus enough to offset the costs that come with implementing health information technology, in terms of paying for the software, staff training, and upgrades? Another important question to ask is that if the perceived efficiency really going to make a difference in net gains?

As we discuss and examine these points and many others in this paper, we will hopefully be able to synthesize the best solution to family physicians and the health information technology that will be of most benefit to them, their practice, their staff, and most importantly the highest quality of patient care that they can deliver. The ultimate goal of this paper is to figure out the best way that health information technology can help the family physician to be more effective and efficient in all aspects of their practice to improve the overall delivery of healthcare.



            At the forefront of health care in the United States is the family physician.  This is arguably the most demanding and stressful portion of employment for health care professionals.  This is because of the rising cause of health care, the struggle to get reimbursements from insurance companies, and the desire for more cost effective and higher quality of health care from the patient.  The family physician is pulled in all of these directions, on top of trying to pay their staff and the other overhead costs that come with running a medical practice.

Health information technology, in many ways, can help to lessen the challenges of the practicing family physician.  As the challenges that face family physicians mounts on an ever changing daily basis, health information technology has come at the right time.  With the implementation of new technology however, come the challenges that come from implementing it.  One cannot help but question if the benefits indeed outweigh the costs.

One of the goals of this endeavor is to conclude whether or not health information technology is a benefit that outweighs the costs and challenges to implement it.  If this is successfully accomplished then perhaps this paper may be of benefit to the future of family physicians to help them to more effectively and efficiently manage their patients and practice, while a concomitant effect will be that of a more healthy and satisfied patient.





For the main objectives of this study, it is important to keep in mind that technology in the health care industry is ever changing.  This especially applies to the rapidly growing and evolving segment of health information technology.  When we narrow this study down and focus on how this applies to family medicine, we can see how an important objective would be to try to figure out how health information technology can best help the family physician and all that encompasses, and ultimately to be able to anticipate how or in what direction this technology is going to head towards in the future.

In order to accomplish this objective, we must have the objectives of gathering the pertinent information, gleaning it, and then synthesizing it in such a way that intelligent conclusions can be made in regards to our ultimate objective. If this can be accomplished, it is the hope of the author that another objective will be that of helping family physicians, and or those that manage family physician practices, to be able to find and successfully implement a health information technology system that will best meet their needs.


Problem Statement

The problem this study will address are the growing pains,  perceived advantages, and the disadvantages of health information technology as utilized by family physicians in the realm of family medicine, and how this impacts a family practice and the quality of health care for its patients.   The various problems that will be encountered and discussed in this paper will primarily apply to the challenges that family physicians have, or can potentially have when they strive to improve upon their business, the practice as a whole, while at the same time striving to improve the highest possible quality of patient care.  Specifically, with the advent of the relatively new technology of health information being electronically based, it can be difficult to discern between competitors which health information system would be the most fitting for their specific practice.

This would have to be examined on a more tailored level, as family medicine can have a wide scope as to what their ultimate goals are, and what exactly are the services they provide.  To further amplify this challenge, one would have to also consider the demographic of the patient care, which could be divided into gender, age, and socioeconomic status, just to name a basic few variables (LaTour, 2013).

Because the health care industry is so competitive, and because of the high cost of health care, it is very important for family physicians to address these problems as it could affect their bottom line, their quality of patient care, and their overall business.  There are also many other problems or challenges that are a subset of the aforementioned challenges, such as HIPPA laws, that will also be addressed in this paper (LaTour, 2013).

The family physician is the front line of a patient’s health care, and therefore addressing these problems on this level is probably very important.  At the very least, these problems should be explored and discussed.  This is important to note, as they do apply, and are an integral part to family medicine in the United States today.  Furthermore, health information technology will continue to play a vital role in health care and will be even more so integrated and common in the future of family medicine.


Purpose of the Study

The purpose of this study is to identify health information technology strategies that will be a benefit to the family physician, and or to identify areas in which health information technology needs to be improved upon in order to maximize the quality of patient health care.  This will help to make aware to the family physician, or the family practice manager, of the various aspects of health information technology and its use in family medicine.  This can apply to the family physician or the family practice manager in any stage of their career, be it aspiring, starting out, in their prime, or on their way out and perhaps thinking of selling their practice.

This project will also shed some light on the reader about how integrated health information technology is in health care today.  This is because the information that is gathered for a particular patient, in a particular segment of practice, can potentially be readily available and shared with other portions or practitioners of health care in other specialized fields.  Ultimately, this could equate to less medical errors, and lead to a better quality of patient health care in the United States.



Research Questions

            The way in which the research questions were developed was by keeping in mind the dramatic impact that health information technology has on the family physician.   This lined to the problem and the purpose of this project seamlessly as the main research questions that this project focused on was:  How is health information technology utilized by family physicians today and in the future.  Sub-research questions that stem from the main research question were: what are the advantages and disadvantages of health information technology in family medicine?  How can health information technology be improved upon in family medicine?  And finally, how does health information technology utilized in medicine help to increase the quality of health care for the patient.

When conducting the research on health information technology as it applies to family physicians in medicine, it was fundamental to ask if health information technology was in fact necessary.  If health information technology, as it is widely used today in medicine, why was it necessary?  Or, if it was not necessary, was it beneficial to those that had implemented health information technology into their practice.

It was also important to ask why health information technology was deemed beneficial by those that used health information technology.  What were they measuring, and what were their results?  What were some of the challenges of health information technology? Was it the training of staff and the integration of it? Was it difficult to figure out which system would be best suited for their needs and wants?  What are the problems and challenges that they encounter today?  Is the pace at which the technology evolves to fast?  Is it the government mandates that add to the difficulty?  The most important question for all of this research was however that if health information technology was in fact helping to increase the quality of patient care.


Significance of the Study

The significance in studying health information technology as it applies to family physicians is important because this is basically a new frontier.  With this new frontier comes the challenges of how can we make this beneficial for the patient.  Also, the frontier is vast and ever changing in the form of legislation, novelty, and refinement of what is health information.  The family physician is at the forefront of health care.  If the family physician can utilize health information technology and harness the best parts of it, the quality of patient care could potentially be better than it was, and will continue with the ultimate goal to improve over time (LaTour, 2013).


Literature review


Introduction to Literature Review


The literature review for this worked first focuses on what health information technology is as it is applied to the practice of medicine and health care in general, and more specifically as it applies to the family physician and their role in health care in the United States.  Next the advantages and disadvantages of health information technology are examined.  A closer look is made for certain aspects such as the patient portal as this is an integral part of health information technology in the setting of family medicine.  Also there is a review of which parts of health information technology can be refined or improved upon in order to maximize its effectiveness for family physician.  There is also a good portion dedicated to how and if health information technology in family medicine is utilized effectively to the point that it is in fact raises the level of the quality of patient care.



Health Information Technology and its importance to Family Medicine


Health information technology is an ever growing, and almost all encompassing part of health care today.  Health information technology refers to the electronic health record, and more information than that.  Perhaps one has experienced the new technologies first hand when as a patient; one notices a change in the procedure of seeing their physician.  For instance many patients and family physicians can now communicate via email if there is the health information technology infrastructure on the family physicians end and a personal computer and an internet connection on the patient’s end.

This type of technology was not available at all fifty years ago, and email was not available to the general public until about twenty years ago.  Health information technology is relatively new to the practice of medicine from about five to ten years ago.  This is not only an example of a new technology that can help in family medicine, but it also shows how fast health information technology has really come into the field of medicine.  One of the interesting new concepts in health information technology, if we carefully examine it, is the fact that the patient is a lot more pro-active in their health care as they can access many records from the internet and also learn more about their illnesses and the medications that they are taking.  In other words, the patient today is much more educated about their health care than the patient was fifty years ago.

Patients can also, if they prefer, can make an appoint to see their family physician online if the family physician has the proper health information system. This is another relatively new concept in the world of family medicine as many patients are only accustomed to or grew up having to make an appointment by calling up the medical secretary at the family physician’s office that they wished to go to.  However, as with the email, a patient can now make an appointment if the physician has the health information technology set up and the patient has internet access.

When it comes to health information technology and family medicine, another change that is taking place that may seem novel is the way that a patient’s history is taken.  Even though this may seem somewhat redundant as the physician once again reconfirms the patient’s testimony of their health, there is another way that patient histories are taken.   A patient receiving health care today in the United States may also notice that it is not necessarily the physician that is taking the initial history of their illness or the reason for their visit, but they may find that they are entering their health complaints or concerns on an ipad questionnaire that the physician and the health care team prefer the patient to fill out first.

Obviously this type of technology would not be beneficial if the patient were illiterate, blind, or another disability that would hinder their use of a computer, but this type to health information technology can be of great benefit to a small family medical office as it may cut down on the time needed to see the patient.  It definitely does cut down on the time that is needed to properly check the patient in as the patient has already done a lot of the work on the electronic questionnaire.

Health information technology systems are used so much in health care today because in it one can find the patient record.  The patient record contains a lot of important information on the patient that would pertain to their health care.  For example the patient record would include the proper identification of the patient.  It would also contain the vital records of the patient like their blood pressure, weight, and height.  The patient record would also contain how their breath sounds and heart sounds are on that day and the previous days or appointments that the patient came into the office.  The patient record is also important as it contains a medical history of the patient as given from the patient.

Usually, or in the past, the patient record was kept on a hard copy, or in other words written on paper and kept in a file that was unique to the patient.  With a health information system however all of this patient information and data can be stored on a database and the medical office could be paperless.  Other important information that may be contained in the patient record is the lab results or test results of the patient, the notes the physician has made during the appointment with the patient and the orders from the physician.

Along with the ever important patient record, health information technology encompasses a way to order prescriptions for the patient in a paperless manner.  This can be a great help to the physician as many medications have interactions with each other and or are contraindicated to each other.  The health information technology can have a system implemented where certain medications are flagged or alerts the physician for any adverse effects or contraindications that may occur if the particular medication was prescribed to the patient.

The health information technology usually also has a way in which a medical office can coordinate with a medical laboratory to order labs for the patient.  The health information technology may also help to analyze labs.   One of the most important features or advantages of health information technology is that it could afford a way for one physician to communicate with other physicians or health care teams within their system.  Health information technology also makes it possible for family physicians or their medical team communicates with insurance companies, and as was stated earlier, to communicate with their patients (LaTour, 2013).



Health Information Technology and the Pro-Active Patient


Health information systems are not only for the health care team or professionals either.  An exciting and revolutionary element in health information technology is that it enables the patient to become much more pro-active in their health care by a number of ways.  One of the ways that a patient can become more pro-active in their health care is that patients can often access their own records and health care information.  This means that when a patient for example has a lab drawn, and the physician receives the results of that lab, the patient can look up their patient record online and find out the results of their lab work, thereby becoming more proactive in their health care.  To carry this example a step further, after the patient as looked at their labs, the patient could then learn what the labs are all about, or what the results may mean by doing research online on their own to help them better understand why the labs were ordered in the first place, and why the family physician prescribed the certain or appropriate therapy (Mullner, 2006).



Challenges and Barriers of Health Information Technology


There has also been a downside or challenges that come from the relatively new health information technology.  For example, privacy issues and security are a constant ethical question and patient privacy has to be guarded from white collar criminals and the like.  Patient information security could possibly be the biggest obstacle that health information technology is coming up against as the privacy of the patient is very important to keep confidential in medical health care, especially as it pertains to family medicine.  This is because the patient may not want anyone to know the health problems that they have, as it could be embarrassing, or hurt their reputation socially or professionally.  If there is a breach in patient and physician confidentiality, this could be a huge liability to the health care organization, or even the physician.  It is for this reason that special attention is made to strive to make sure that the highest security standards are met for health information technology (Mullner, 2006).

The access to patient records could be a huge liability for the family physician, or for any health care professional team for that matter.  This is an enormous issue for all of health care technology because a breach in patient confidentiality and security could be a huge liability for not only the family physician, but also all of the auxiliary health care teams that are involved.  If a relative, who was terminally ill for instance, came in to see the physician, this could be a very delicate matter.  The patient could be heading towards the end of life.

The family of the patient could be very stressed out at the thought of losing a loved one and all of the issues and drama that come with such a tragic end of life scenario.  It would be horrifying to find out that after all of the rigorous testing, and therapy, and emotional distress, that somehow the information that was used for the health care of a loved one was exploited in some way for monetary gain, research, or any other reason.  This would be especially disheartening if the patient gave strict orders that this information was not to be shared or leaked to researchers or anyone for whatever reason.  This is the loved ones last and final wish.

The difficulty with this wish is that if a criminal or a careless health care worker made a mistake, the security and the privacy of the patient could in fact be compromised.  Furthermore, what if there was legislation that granted immunity or an exemption to the private records of the patient because of a particular illness that they had, and therefore could be readily accessible to all researchers in the United States of America, or perhaps even worldwide.  This is an ongoing debate that needs to be addressed and the security of the patient needs to be ensured if there is going to be any confidence in the patient, and in the family physician, if there is to be a successful integration and use of a health information system.

Other problems that are arising from health information technology are the fact that there is not a simple standard because the technology is relatively new.  This also means that certain health information systems are not linked together or are very limited in the information that can be shared or exchanged through cross platforms and between physicians or hospitals.

When communication is not hindered however, the health information systems are a great tool in improving the quality of patient care.  This is one of the reasons that health information technology has grown so much in such a short period of time, because it can increase the quality of patient care.  There needs to be a way for the health information systems to be able to exchange information if there is to be any success in a huge health information system that is shared and used by family physicians and other health care workers.

There is a professional organization that was founded to help set up some type of standard for health information technology.  The organization is called the American Health Information Management Association, otherwise known as AHIMA.  AHIMA represents over 63,000 professionals in health information technology management professionals in the United States of America.  Since their inception AHIMA has been trying to set a standard and guidelines to help there to be some kind of consistency in health information technology.

This is done in hopes that this will not only make the standard of health care better, but it will also help various health organizations to be able to communicate one with another, or that their systems will be able to communicate with each other (AHIMA 2012).

AHIMA has even gone so far as to lobby for legislation in Washington because this has been such a challenge.  AHIMA feels that if health information technology was held to some sort of standard that there would be better communication between the physicians, health care teams, the insurance companies, the patients, and all other parts of the health care system in the United States (Two, 2011).

AHIMA also feels that there should be some kind of code of ethics and standards set up for the assurance that the patient’s privacy will be held secure from hackers and other white collar criminals as a lot of very sensitive and person information, as well as financial information is commonly stored in health information technology systems  (AHIMA 2012).  For this reason, this approach may be of benefit as it may help to ensure that at least a minimal standard is held and perhaps preventing certain fly by night companies from ripping off consumers.

For an alternative perspective however, it may be of a hindrance as the author feels that it may be best if government legislation not intervene in this aspect of medicine and let the free market work itself out instead of government mandates that are often ignored or circumvented or become antiquated as soon as they are approved because of the fact that health care information technology is growing and evolving at such a fast pace (Rockefeller, 2012).

In a recent article from the Journal of Medical System, it was proposed that the organizational challenges so rampant in medicine in the United States, that the care is in fact very rarely organized.  Because of this there have been studies conducts as to how a better health information system could be designed and utilized.  This has been found to be a very complicated process however as health care can be very complicated.  Because health care takes the coordination of so many different teams, including the cooperation of the patient, it can be very difficult as time constraints make it difficult to make sure everything is in place when someone’s life is on the line and time is of the essence (Dobalian, 2012).

It can be argued however, that the investment in time and money to organize and execute a properly organized health information system could be of great benefit and worthwhile.  This is because the information, if the different health information systems could communicate with each other, would be available to researchers, physicians, and other members of the health care team.



Precision Medicine, Continuing Education, and Interoperability


There is a movement that is called precision medicine that is gaining steam, and would use in very large par an integrated health information technology system.  The family physician would have to very much be on board with such a system to make strides in its success due to their interaction with patients.

In order to ensure that the health information technology is properly utilized in the United States, it is argued that proper training of physicians and their staff is a very important piece of the puzzle in order to ensure its success.  This is because health information technology has faced some resistance from physicians and their staff when they are not on board to the new way of practicing medicine.

In order to circumvent or augment this challenge, an article in Health Affairs has stated that it may be a great idea to implement training in health information technology as soon as possible, perhaps in the early years of physician training and education.  This would be a way to help shape the future of health care in the United States and to help them know of the importance of evidence based medicine and how health information technology can be utilized for this purpose (Graham-Jones, Friedman, Marcotte, & Blumenthal 2012).

Another interesting way to implement health information technology in a successful way to those that may be well into their medical profession, is to require some knowledge of it on their board certifications or recertification.  This will help to ensure that at least in continuing education that physicians and staff are getting some training and acquiring some knowledge about health information systems (Graham-Jones, et al., 2012).

Graham-Jones suggests that to ensure that physicians are properly trained to use health information technology; this can be acquired in at least two different ways.  This can either occur during the physician’s private time, or it can also be a part of the training of the facility in which they are employed.  Making health information technology a priority in training is very important to ensure the success of this new endeavor (Graham-Jones, et al., 2012).

Senator Alexander out of Tennessee has really made a huge push in wanting to mandate some standards for health information technology.  The senator used the name of Dr. Francis Collins, who is the director of the National Institutes of Health, as his main source of information in regards to the importance of a unified health information system.  The system Senator Alexander argues has to at the very minimum allow for other systems to communicate with each other in the realm of health information systems.

The systems need to do this so that there can be some kind of interoperability between hospitals, offices and smaller physician offices such as can be found in much of family medicine.  If this were to happen then what the Senator refers to as precision medicine could more fully come into fruition.  If the physicians were made accessible to a lot of the genetic information and the information was made available to researchers all across the country then great medical strides would be made in precision medicine.  In other words, for precision medicine to be successful, there needs to be an effective system of communication between health information technology, and the technology has to be accessible and used by as many health professionals as possible, and especially to family physicians (Alexander, 2015).

It is also much more feasible for health information technology to be used especially as it pertains to mapping genomes is concerned.  It was found that just fifteen years ago the cost was actually four-hundred million dollars to map the first genome, and today the cost is approximately one thousand dollars.  This means that more genomes can be mapped and the data entered and shared in a health information system for researchers to use.

For example, this information could be used to target and find a cure, or to reduce the onset of a disease such as Alzheimer’s.  Alzheimer’s is the leading cause of dementia in the United States.  If this were to occur, than the funds that were used for Alzheimer’s and all of the problems that come with it could be used in other ways to help prevent or cure other pathologies.  Dr. Collins has stated that a well mapped out genome can also help physicians to prescribe the medication that will be of most benefit to their patients (Alexander, 2015).



Family Physicians utilizing Health Information Technology


Family medicine is often considered the first line of the health care system.  It is the family medicine physician who is like the quarterback, referring patients to their necessary specialists after they are properly diagnosed by them.  Family medicine physicians also handle a lot of the chronic illnesses and diseases such as hypertension and diabetes.  Family physicians also handle the common clue, influenzae, and other acute problems that may arise.  The family physician may also specialize in weight loss, bariatrics, and even some basic dermatology.  The family physician may also work in the emergency room or even be the town obstetrician and gynecologist.

The realm of family medicine is far and wide, depending largely on the scope of practice that the physician chooses, and the population that they serve.  Because of this, health information technology and its use in family medicine by the family physician is also a very wide window, with many different variables to consider.  This is important to note because this just goes to show how different systems of health information technology may be more important or suited for certain family medical practices (Rogoski, 2006).

It is for this reason that perhaps just one common standard of health information technology will not work for the family physician.  A more viable solution may be to have a more customizable alternative to better personalize and meet the needs of the family physician and their area of practice (Rogoski, 2006).

When health information technology and family medicine are used in the most utopian manner, the family physician can find that his or her practice is also running optimally.  This is because communication is seamless between the disciplines.  Communication is great with the insurance companies (Payer, 2012).

Prescriptions are ordered and ready for the patient at the click of a mouse.  Labs are ordered and analyzed seamlessly.  The staff is therefore more effective and time efficient as the health information system runs seamlessly, and is user friendly.  It would also be great if the health information technology is also easy to learn, and to train the staff to use (Zandieh, 2008).

Another benefit for the family physician is that the time with the patient is also more effective because the patient is more proactive in their health care.  The patient is able to look up their vital information and in some cases meet with health care professionals online.  The patient is therefore a more educated patient and when the patient meets with the physician the patient is able to ask the most important questions, and relay the most important information to the physician.  All of this will ultimately make patient care quality the highest (Electronic 2011).

As far as a business argument is concerned, it seems that the general consensus is that if the implementation is successful, then the health information technology can increase the net value of a family practice.  The important point to be underlined and not underestimated is that it is very important and vital to the success of the health information technology is if it is implemented successfully, and a huge part of a successful implementation is if the physician and the staff are on board and have a good attitude for integrating a new system into their way of practicing medicine (Kumar & Bauer, 2011).

Some of the more specific ways that health information technology makes sense in a business sense is the savings that can be attributed to more effective time management.  For example, a lot of tasks can be streamlined and be less labor intensive, such as writing and delivering prescriptions.  This can be very labor intensive if one is dealing with a class II medication that is highly addictive.

Without the health information technology, one would have to do a lot of phone calls, to law enforcement agencies, other physician, checking various records, just to make sure that the medication is okay for the patient to ensure that the ethical beneficence is being practiced.  However, with health information technology all of the pertinent information as it applies to the patient and the medication can be found with a few simple clicks on the computer screen (Kumar & Bauer, 2011).

The savings from an effective health information system can also be attributed to a more effective and less bloated staff, as theoretically you will have less staffing requirements.  This is because less staff will be needed to do the same job with health information technology (Kumar & Bauer, 2011).  A prime example of this is the aforementioned prescription writing and delivery for a patient that needs highly addictive medication for therapy.

Another way that health information technology can be used effectively to increase the bottom line for the family physician is that when implemented correctly can drastically reduce the costs of charting.  It has been found that paper charts, and the pulling and refiling of them can cost a practice upwards of eight dollars a year per chart to maintain.  A healthy medical practice has approximately two thousand patients per physician.  This would mean that at least sixteen thousand dollars per year can go towards patient charts alone (Kumar & Bauer, 2011).

With health information technology it has been found that because charts do not have to be pulled physically, and with the pertinent patient information found in the records that fewer phone calls have to be made to answer health related questions.  The reduction in cost for maintaining electronic patient charts using health information technology can reduce the costs of patient chart maintenance by fifty to one hundred percent (Kumar & Bauer, 2011).

Medical errors have also been found to be reduced with the use of health information technology.  According to a recent study there was a huge reduction of medial deaths and injuries due to medical errors because of health information systems.  There was a reduction of 45,000 to 98,000 deaths and an astounding 777,000 injuries to adverse drug interactions.  One excellent feature of health information systems is that there can be a flag or a warning when there may be a medical error or an adverse drug interaction (Kumar & Bauer, 2011).

Medical health errors for the most part can be prevented if proper attention to detail is applied.  With the proper knowledge applied to the health care of a patient by the medical health team.   Medical health errors are an important area to look at reducing as it can increase the quality of health care and reduce the cost of health care (Kumar & Bauer, 2011).

In an article published in the Wall Street Journal, it gave great insight as to how health information technology has helped the patient to be much more pro-active in their patient care and how this has helped to reduce medical errors.  The article claims that because patients have been more knowledgeable about their health care with the internet and health information technology, that they are able to make subtle corrections to their health care and this in turn helps the physician to be happier in the care and the responsibility that the patients are showing for their own heatlth care (Landro, 1999).

It was suggested that physicians would not adapt or that they would resist using health information technology in their patient care, and they have to a point.  However, it seems that physicians have been more apt to use health information technology because in a way this helps them to stay on the cutting edge of medicine and also to keep up with whatever information that their patients may have, and to be able to guide their patient as to the validity of the studies or the information that they are reading.  In a way the family physician has used health information technology as a tool of self-defense in some instances.  What is really great however is that the patient and the physician have combined there knowledge and their relationship of respect has strengthened which means that the patient quality of care has ultimately increased and medical errors have decreased (Landro, 1999).

Since the advent of the microprocessor, and its availability to the public, combined with the use of the internet, the medical world  and the knowledge and literature available to the patient and the physician is growing ever so rapidly.  It has been said that even if a physician is fresh out of medical school for only a few years that the physician may be behind in the latest and greatest technology and care if the physician does not keep up with continuing education credits.  Health information technology has been very instrumental in this revolution and arming the patient with more knowledge about their health care (Landro, 1999).



The Importance of an Effective Patient Portal


The use of health information technology by the patient is commonly referred to as the patient portal. Because the patient will be more proactive in their health care, or could potentially be, this also opens up the possibilities of more of a breach in security.  This breach in security would come because the patient would be able to access their records through the patient portal from a computer outside of the health medical office.

For example, the patient could with the proper health information technology, access from the comfort of their own home, the lab results that were just drawn earlier that week.  There are however, even within the realm of the patient portal, so many different levels of functionality between different electronic health record systems (Electronic, 2011).

It must be understood that in order to make an effective patient portal, it is also important to personalize the health information system in such a way that it does indeed meet the needs of the population or demographic that it is serving.   A great example of how personalizing the health information system for specific population was found in a study in the Journal of Community Health, in which a look at Native Americans and Eskimos were found to have a very unique set of needs to their health care (Geana, et al., 2012).

For example it was found that when Native Americans were encountered with health information technology in their patient care, that they were able to receive tailored care and an increased quality of health care for their needs.  However this was not possible unless their health information system was also personalized to meet their needs as acquiring information from this demographic was different than acquiring information from say New York City residents (Geana, et al., 2012).

Another challenge to consider in the patient portal is the computer literacy of the patient.  Some patients may be more willing to deal with their computer, whereas others prefer to deal with a human being.  The family physician may have to ask the important questions, and use professional judgement, as to whether the patient would even benefit the utilization of the patient portal, or would it raise more questions and cause more anxiety to the patient because they have to use a computer.

For this reason it is important to realize that health information technology may not be a solution to those that are computer illiterate or that may have some other underlying conditions that would make it difficult for them to access a computer, or even have the desire to use one.  In some cases health information technology may not be a variable that could increase the quality of patient care, especially as it pertains to the patient portal (Electronic, 2011).

Because of the many challenges that can be presented just in the patient portal alone, it is interesting to note that there are some companies that focus solely on that portion of the health information technology department.  This is because it has been found that patients are much more satisfied with their experience using the health information technology if the experience is user friendly, seamless, and meets their needs.

In a recent editorial by to family physicians from the Virginia Commonwealth University, some very interesting conclusions were made in regards to health information technology.  These family physicians realize the importance and how great health information technology can be especially as it pertained to how the technology could help patients to be more active in their own health care (Information, 2011).

What these physicians found and recommended was that personal health records and the technology that was used for them needed to have one variable at the forefront, and that was to make sure that the technology would keep the patient in mind when designing the software and front end usage.  This is because collecting information from the patients is a great way, and one of the most important ways of helping a family physician to properly diagnose and to treat the patient (Information, 2011).


Keys for Health Information Technology and Family Medicine


The family physicians from Virginia Commonwealth University stated that there were five key functions that were necessary for an effective and efficient health information technology system.  Much of it had to do with the way information was gathered, analyzed, and utilized.  These family physicians stated that the five main functions should be to collect and store information from patients.  The second would be to collect and store the information from the patient’s physician (Information, 2011).

Another key function would be to be able to translate the clinical information into a lay language so that the patient could understand how to improve their health based on this information.  Finally the information had to be actionable for the patients.  By making the information actionable by the patients it is meant that the patients would be able to take the information and make decisions and tailor their health care through education, priorities, and also to be able to integrate and use the information in other areas or specialties of their health care (Information, 2011).

These family physicians felt that although health information technology was great, that the technology was not used to its full potential.  They make some really great points as there is not any health information technology out there that is utilized by the patients in such a way that the technology actually translates the information into language that they will understand, or involves to a deeper level of patient integration where they are making more of the decisions (Information, 2011).



Concerns and Frustrations with Health Information Technology


This is understandable as new technology can be frustrating to begin with, so the more seamless the integration of a new user, the more satisfied that user will be.  This is a very important aspect for the family physician to consider when purchasing or trying to decide which system of health information technology is best suited for their practice.  If the patient is comfortable and satisfied using the patient portal this will be one more area that the patient will be satisfied in their overall health care provided by their family physician (Electronic, 2011).

Another one of the big concerns with health care today is the fact that the delivery of it is often times fragmented.  What is meant by this is for example is that a patient that has testicular cancer may first see a surgeon to remove the cancer.  Then the patient may have to periodically see the oncologist to make sure that the cancer does not return.  In addition to this the patient may be seeing their family physician for other chronic diseases such as diabetes or hypertension (Pawlson, 2007).

The health care delivery fragmentation is exacerbated by the lack of communication between the various specialties of physicians that the patient is seeing.  This can lead to medical errors as one physician may prescribe a medication that could be contraindicated by a physician that is prescribing another medication for a different disease (Pawlson, 2007).

Health information technology, if designed correctly, can help lessen by a great degree the fragmentation of the delivery of health care to a patient.   This would in turn help greatly to reduce medical errors and thus increase the quality of patient care, as well as maximizing resources to more productive endeavors.  This is amplified by an increase in digital communication, in the collection of data, and in the delivery of important data pertaining to the patients’ health.  Another advantage that is coming because of health information technology is more effective follow ups from the patient and the data that can be recorded (Pawlson, 2007).

When there is better communication between physicians, the fragmentation of the patient care is lessened.  This is said to help the physician to increase their learning about the patient.  It is also said that health information technology increases the learning in treating diseases that could be epidemic, and predict how to contain them in such a way that they don’t become pandemic.  Health information technology has really made an impact when it has come to containing certain strains of influenzae.  Health information technology has also helped researchers to predict which strains of influenzae are going to be the most dominant in the flu season (Pawlson, 2007).

As good as the aforementioned utopian health information technology system scenario sounds, it is very rare.  This is because health information technology is so new, and there are so many competitors. Furthermore the standards and ethical questions in regards to health information technology are being defined and figured out as the technology and the needs and wants of the patient, physician, and auxiliary health care is determined.  Not to mention the government mandates and legislation that has made other rules that health information systems must adhere too.  This would not be so difficult if the legislation and mandates did not change so often.



Importance of Cybersecurity in Health Information Technology


Because the health information technology is so new, and still growing and being improved upon, and because of the other aforementioned variables, the family physician is finding them in a situation in which they have to constantly adapt to all of the change.  The other option is to not be as effective with their health information technology, and still another option is to not use it at all.  Some physicians choose not to not use health information technology at all when it comes to patient care because it helps to avoid a lot of problems.  Some of these problems are the privacy of information that their patients desire are not threatened when the information is not stored in the cloud, or a database (Shwayri, 2014).

One of the challenges that really became an issue of health information technology is that of security of the information that is found therein.  There has been a focus that has been highlighted by the Health Information Technology for Economic and Clinical Health Act (HITECH).  This act was developed to help ensure that health information technologies that were being produced and used in the United States were meeting at least a certain amount of security measures to help ensure the privacy of the patient, the health care organization, and academia (Greiman, Zlateva, & Chitkushev, (2012).

Cybersecurity is a very important issue as an integrated health information technology will not, and cannot be in full effect until this issue is solved.  These privacy issues will continue to be an issue not only in the United States, but also if there was a worldwide communication network it would seem that this could be even a bigger problem.

The bigger problem is because a lot of personal information may be utilized and shared without the permission of the patient or whoever has the stewardship over the information.  There is going to be an increased need to use electronic storage databases that are protected and kept safe, and that can be upgraded as cyber systems are continually being compromised (Greiman, et al., 2012).

All of the cybersecurity issues need to be addressed along with maintaining the HIPPA regulations and federal laws that have been put into place to protect the rights of the patients and the citizens of the United States of America.  The HIPPA regulations are even today under scrutiny however as there is currently new legislation that is searching for exemptions for research (Greiman, et al., 2012).



Paper Charts, or the Old Fashioned Way


Sometimes good old fashioned paper charts are the most dependable as they will not be erased or compromised by a breach in electronic security.  The physicians that do not use health information technology also do not have to worry about training their staff and the cost in time and money that takes to learn a new system (Zandieh 2008). Another problem that they would avoid is not having to continually upgrade their software, their system, or their training in order to learn new methods (Spratt, 2008).

For some family physicians avoiding these pitfalls is worth it and they feel that the old fashioned way is indeed more effective and efficient.  This is especially true in areas where Medicaid and Medicare patients are not being seen, and no government funds are being utilized for patient care.  This is because the physician is not held to government standards and mandates in order to get reimbursed for their services, whereas family physicians that do accept Medicare and Medicaid are obligated to be a part of health information technology if they want to get reimbursed, and in fact are given a bonus as of last year if they are properly integrated into the health information system (LaTour, 2013).

Because a lot of patients are on Medicare or Medicaid, a lot of family physicians are finding it necessary to be on the grid so to speak so that they can get properly reimbursed.  Physicians are also seeing that there can be potentially a lot of benefit of having a smooth running health information technology system (Wakefield, 2008).

Proper reimbursement is definitely important in the realm of family practice, because this can increase their revenue by being a more efficient office and team, and also the patient care can be more increased which leads to more satisfied patients and then in turn a good opinion and word of mouth that could also increase the potential patients that a family physician has in the future.




Medical Registries and Family Medicine


Because health information technology is still relatively new, there are a lot of questions as to how in depth the health care teams in the United States, and especially physicians, are willing to delve.  To address this, there was a study conducted in Massachusetts that cost over fifty million dollars.  In this study it was found that physicians that actually used the health information system more effectively were also more apt to participate in medical registries.

Medical registries are important as they help the Center of Disease Control and other organizations including academic organizations to use the data for case series studies or other studies to figure out biostatistics of diseases and other pathologies.  For example the prevalence of diabetes in a population can be tracked, as well as the methods that are effective for treating it.  The yearly incidence rate of chlamydia can be calculated by the information found in the health registries, as well as sensitivities and specificities of diagnostic tests.  For these reasons and others, the strength of the health care registries is very important and vital in health care.  Not to mention, a very drastic reason why health information technology can be utilized to increase the quality of patient care. This is especially true as it pertains to the family physician.

According to the study by Fleurant, it was found that the ability for primary care or family physicians to contribute to the registry could accomplish a great deal to strengthen the sample size thereby increasing the confidence of the data.  As we can see in exhibit 1, the primary care physicians played a vital role in the generation of registries that could be used for health information systems (Fleurant, 2011).

(Please See Exhibit 1 in Appendix A).


To further demonstrate how health care registries are important in the organization of the health information system we can look at a study by Hinman that showed the increase in immunization tracking when this type of system was implemented.  This is not to say that there was necessarily and increase in the immunizations, but it does show that with time and more use of the health care registry that more immunizations were reported.

Immunizations and vaccines are a very important part of the prevention of very dangerous diseases in the United States, and the family physician is the health care professional that is responsible for the delivery of common vaccines.  If this information is properly tracked, than outbreaks and epidemics can be predicted, prevented and anticipated, and hopefully a better quality of life and more lives can be saved.

This is especially true in areas of the United States where there may be a popular trend in not vaccinating children.  When these trends are tracked, the epidemiologist can predict where an outbreak may be for the certain disease.  This is important as epidemics can than possibly be contained and not turned into a pandemic.  The results of the participation of immunization system registry survey can be seen in exhibit 2 where a registry has been able to track immunizations in the United States, which is very important information for the epidemiologist and the Center for Disease Control (Hinman, 2010).

(Please see Exhibit 2 in Appendix B).






Importance of Interoperability in Health Information Technology


In a recent article from Medical Economics, it is predicted that there will in fact be what is called interoperability in the United States between the different health information systems by the year 2024.  What this means is that the health information systems will be able to seamlessly share health data that would allow for the increased quality of patient care (Ritchie, 2014).

This increased interoperability between health information systems will have a drastic impact on the family physician.  According to Ritchie, this will mean that the business model that family physicians have now will of course continue to evolve.  Ritchie predicts that this will be good for the family physician.  It is predicted that the family physician will be even more valuable to the health care system as they will be more integrated and have a deeper collaboration with the hospitals and the communities in which they serve (Ritchie, 2014).



Improved Lifestyle with Health Information Technology


One of the benefits of the predicted business model and role that the family physician will have in the future is that they will not have to deal with many of the administrative headaches that they have to deal with today.  This would mean that there would be less time and resources spent of fighting with insurance companies for reimbursement, and other time consuming tasks that take the family physician away from practicing medicine and seeing patients (Ritchie, 2014).

Perhaps one of the most important reasons that a family physician would want to look into utilizing health information technology is because the technology can help the family physician to have a better and more enjoyable lifestyle.  In a recent article by Montague, it was stated that a lot of physicians are suffering from being overworked and burnt out.  The article also states that many physicians are not satisfied with their work and they feel that they are not making a difference.  Among the physicians that are in the survey, those ranking with the highest amount of dissatisfaction were the physicians that specialized in internal medicine and primary care, both of which contribute to family medicine (Montague, 2014).

The way that Montague reports that health information technology could help the family physician is by solving a lot of the problems that are presented with the shortages of workforce.  The way that health information technology can help to alleviate some of the problems with the shortage of workforce is by allowing the family physician to accomplish more work with less complexity.  This in turn would also allow the family physician to see more patients that may have more difficult problems or diagnoses (Montague, 2014).

Montague claims that in order for health information technology to accomplish the goal of helping the physician and the patient to work together in such a way that health care will improve, that it is vitally important for the health information system to be designed in such a way that the technology is human centered.  The design is imperative to be user friendly for both the family physician and the patient because if it is not, this could lead to frustration and then an ultimate failure of the utilization of health information technology in the realm of family medicine (Montague, 2014).

The reason that it is so important for the family physician to utilize health information technology in this day and age is because it has been found that when there is a good front line of health care, which the front line is the family physician, that this can help to increase the quality of health care.  Serious illnesses can be dealt with at earlier stages.  Emergency rooms with the expenses that come with using them, will be lessened as patients go to their family physicians first, thus health care dollars are not wasted and can be used more efficiently and effectively.  Chronic diseases can be treated in such a way if they are maintained properly in the setting of family medicine, that more drastic treatments and therapies can often be avoided, which ultimately leads to a higher quality of patient care (Montague, 2014).



Research Methodology and Design

Due to the vast amount of information that was available in the area of health information technology, it was vitally important to focus on the scope of the project and to try to stick to the outline, goals, and objectives that the author felt was most important.  Therefore, it was a great asset to have at the forefront, research from scholarly articles that were made available from the Davenport online library databases. This work would have been much more difficult without the access to the scholarly database that was available for student use with internet access.

When searching for information in the databases, it was important to focus the searches as specifically as possible.  A specific search was important because of the incredible amount of articles that would be found with the search of a broad term.  In fact, even with a narrowed search, the amount of articles that was found and made available were really quite numerous and had to be discerning for the benefit of this work.   Then, from the results that would appear, the author would have to peruse the articles to find literature that was most pertinent and that the author felt would be for the benefit of the paper in order to get the most comprehensive information.  Once this was accomplished the author would then try to glean the information and subsequently synthesize the work of the authors to come to some type of conclusion.  Before this was done however, depending on the research, it was also important to compile the literature and research in such a way that it would make sense for the reader of this work to easily understand.

The author felt that in researching literature for this project that it was especially important to consider the source of the information that was used.  For this reason, only scholarly work of the highest level was considered as a source.  The author felt that if the research adhered to the highest standards of quality, that this would then be reflected in the content and validity of the project.  The author maintained this method of research and ensured the highest standards were used steadfastly throughout the entire process of this work.  It is the hopes of the author that the reader will confidently find that the work contained to be credible as the sources from which this work was synthesized were indeed credible.



            The procedures that were used for collecting information and hard data for the project were rather straightforward.  Most of the research was found searching the Proquest Database that is a service of the Davenport University online library source.  The Proquest Database is an invaluable and indispensable resource to use for this project as the Proquest Database has a huge library of scholarly data acquired from various sources scholarly sources and journals from around the world.  Most importantly, there was a huge amount of data on health information technology and family medicine that the author had the pleasure of perusing.   As the author would search through the various articles it was important to find literature that would help the author to demonstrate how health information technology would be beneficial to family physicians, and also look at some of the challenges that this relatively new technology has, and will inevitably continue to have for many more years to come. This is because for one there are many ethical questions and issues that need to be addressed.

Some of these ethical questions are for example if the researcher would be exempt from many of the patient privacy information in order to create a better registry to track the data of certain diseases.  Or would the researcher be able to use data for bio statistical reasons for example to figure out the incidence and prevalence of a new strain of influenzae, and if so, how much personal information are they going to be privileged to acquire.

To go even further in depth, ethical questions can also be spread to a worldwide health information system because health information technology if used on a global scale could really be invaluable to the medical community and for health care in general if it were used correctly.  However the privacy and ethical questions need to be raised and resolved first and foremost.

A great amount of data and literature are submitted and considered scholarly sources which are appropriate for a project of this nature.  This is vitally important when screening sources for scholarly work because if the source has no credibility than the information contained therein is essentially useless, which would in turn would weaken the credibility of this work.  It was for this reason that only the best and highest level of scholarly sources were used when citing work in this project.

A vital part in the procedure of finding the best literature was to search the appropriate key words.  For example, one could not simply search for “information technology” as this would yield far too many results to comb through in the short amount of time that we have for this project.  Therefore the author would have to carefully submit searches that narrowed the results significantly.  Some examples of the searches used were “health information technology family physicians,” and “challenges in health information technology.”

Even when the search was narrowed the results were many which led us to our next challenge.  The author even found it necessary to focus the search of literature to results that were applicable to the United States of America, as the main focus of this project was how health information technology would or could be applied to the family physician in this country.

The biggest challenge when implementing this procedure was using sound judgement as to which sources would be of most value to the work.  This is because there was such a huge array of information of health information technology that it was important to be selective.  Even from the information that was selective it was difficult to glean and choose the information that was the most beneficial to the project, while also trying to be fair and unbiased.

This was undoubtedly a difficult task, as the author definitely has opinions on health information technology and family medicine.  However the author is proud to report that there were points of view and information that help the authors, and hopefully the readers understanding of health information technology as it applies to family medicine really became more in depth and grew to a broader base.



            The instruments that we used were the personal computer to enter and record data. The word processors to compile, write, and edit work.  This was also beneficial in using the graphs and charts creator to illustrate important points in the work.  Also, as mentioned earlier, the author used electronic databases, primarily the Proquest Database, which was an invaluable source of information that was used for this project.  These databases, when searched properly, delivered some of the most respected and scholarly work available on the subjects of health information technology and its various applications to family medicine.

Without the aforementioned instruments, this project would have been much more difficult to come into completion.  It was extremely important that these tools and instruments were in top working order to conduct the research and to put all of the information together and to present the information in a cohesive and in a concise manner.  This was important to the author because one of the main personal objectives of the author was to make a piece of work that would be enjoyable for the reader.  With the combination of the instruments used for this project, it was miraculously possible to accomplish this work to the highest standards as outlined by the professor Dr. Tim Delicath, and Davenport University.


Data Collection Process

Collecting the data was an arduous process, not due to the lack of data, but because of the abundance of it.  In order to collect the appropriate data, one must find it first.  With a wealth of information in the digital age literally available at your fingertips, one must be able to discern the data that is worthwhile or not.  The first step is to look in the proper general area, and this would be a database that contains a library of scholarly articles.  Next would be to effectively search for these items in a focused and concise manner.

As we searched for health information technology and family physicians together, we found most of the best information.  In order to find data for specific areas such as HIPPA regulations and health information technology, the search terms would have to be changed accordingly.  After an effective search was executed, one would have to then peruse through the various scholarly works and articles to find the ones that were most useful and pertinent for the scope of information that would be applicable to the project.  This process was repeated until all of the objectives were covered for the focus of the project.


                        As far as limitations were concerned, there were two obvious ones.  One was the fact that the author was working solo, and already had ideas concerning the project, and personal opinions.  This could very much limit the study as there could have been an unintentional bias when gathering and interpreting information.   In order to ensure or strive to not let this observer bias occur, the author strived to be as open minded in the gathering of information, data, and opinions from a wide range of sources.

This was done with the spirit that a good piece of work could be accomplished from this process due to the aforementioned limitation.  One possible way to resolve this limitation would be to do a group project or if this paper were able to be peer reviewed.  However, due to the logistics and to the time allotted it was found to be a welcomed challenge to the author to strive to make and report a project in which learning was had not only by the author as an open mind and point of view was kept, but to also pass this new knowledge on to the reader of this work.

Another obvious limitation was the fact that because of the rapidly changing field of health information technology, and the constant change in legislation, that it is very possible that the information is outdated as soon as it is published.  For the purposes of this paper however, it is assumed that the highest quality and latest information was used for this project and that the author strived to not be misleading or biased in the gathering and presentation of information.

The author felt that if the latest and most credible sources were used in the gathering of information that this would in fact give more legitimization to the project and greater confidence to the reader that the information presented herein was in fact as accurate as could possibly be, and thus is a means of hopefully minimizing this limitation.  It was for this reason that a considerable amount of time and careful consideration and professional judgement was used to discern which sources of literature would be the best for the scope of this project.

Other limitations were the time constraints that were due to the seven week class time.  It would be very easy to work on this project for another seven weeks to get more in depth first person interviews and other types of research.  The time constraints did however keep the author on task.  Even though a deadline could be perceived as a limitation, it could also be a good way to make sure that the project was completed in a timely manner.  With the time allotted, the author had to be sure to be diligent in the work and strive to maintain a consistent work ethic in order to not have the time constraints to be a hindrance.

It is also important to note that there are limitations to the research of health information systems due to the difficulty of figuring out the actual costs and benefits of implementing health information systems.  Furthermore it is important to note that due to the limitations of this study, that not all of the variables were factored into analyzing every single cost and benefit that a family physician and their practice has.  This is because the variables could be immeasurable intangibles such as bedside manner and being able to personalize health care to individual patient’s personality (Kumar & Bauer, 2008).




Synthesizing Data from the Literature Review

            After the data was collected from credible scholarly sources, it was important for the author to take the time to analytically read and comprehended the material first and foremost.  It was also vitally important to put all of the information together in such a manner that it would be presented to the reader in a way in which they could properly understand the points that the author of this project thought was important and trying to get across to and relay to the reader.

To accomplish this task, was only made possible after the author was able to glean and synthesize the data in a cohesive manner.  The synthesizing of data was also very important as it helped the author to come to specific conclusions as to the effectiveness of health information technology in the field of family medicine.  This is especially true, as it pertained to the family physician, because the family physician is in a unique area of patient health care because of the innumerable amount of variables in the realm of family practice.  One may say that no two family practices are the same, so finding the common threads or areas that may pertain to a great number of family physicians was definitely a lofty but worthy goal of the author.

The author was also able to conclude from synthesized data more specific information such as which health information technology software was popular and what were the reported problems and challenges that family physicians faced in the wake of health information technology.  The proper synthesis of data from literature review was only possible after reading and comprehending the data from the scholarly articles that were researched.

Some of the hard data from graphs and charts that were from other authors were gleaned and used in this work to better illustrate points of health information technology and family medicine, in a more visual and aesthetically pleasing manner.  For example, it was found that many patients appreciated the use of health information technology when they were receiving care from their family physician as they were able to access information about their health care in the privacy of their own home.


Data Analysis

The data analysis was primarily from the secondary research that was found in scholarly articles.  From reading what the authors of the literature concluded about their data analysis, the author of this work tried to understand and comprehend it in such a way that the author was able to then relay the more meaningful points of the data to the readers of this project.  In order to accomplish this goal, the author would also utilize graphs and calculations properly cited from scholarly articles if it would add to the understanding of the data analysis.

The author of this work would then try to amplify and expound on the work of other authors by backing up similar findings by other authors in order to strengthen the data analysis. The author felt that it was important to analyze the data in this way because it would help strengthen the conclusion of the author if other scholarly works were found to have the same conclusion.  This in turn would give the reader a higher confidence level that the work concluded in this work was in fact legitimate.




It was found that there were many positive benefits to health information technology that would be applicable to family physicians.  There was also many challenges to overcome and to work out before health information technology and family medicine could be combined and work in the best possible manner. Before the challenges and ethical questions can be worked out however, there needs to be a time for growth and a time to see in the real world how these challenges can be overcome.  This will no doubt take many years to finally come up with a near perfect system.

Although paper charts can still have a place in medicine, it seems that the more beneficial way to practice medicine and to increase the quality of health care would be to integrate health information technology into the family practice, while at the same time being able to customize the system as no one system will be able to fit every family physician and their practice. This is vitally important as there are many different ages, demographics, and cultures in the United States that the family physician serves.

Being able to be trusted and being able to communicate with the patient is one of the most important variables in successfully diagnosing and treating a patient.  The family physician is usually the first medical care provider that the patient comes across, and it is for this reason that if a family physician is to use health information technology it has to be in a way that the patient can trust the physician in such a way that they will be confident that the physician will be ethical in confidentiality, privacy, beneficence and in informed consent to the patient.  In other words, it would be a very great thing for the patient to feel that the physician has their best interests at heart and will do the patient not harm in any way, shape or form.



It is important to note that in order to have an integrated and successful health information system that the physicians and their staff need to be enthusiastic and onboard.  It would definitely be beneficial for proper training of health information technology to occur as soon as possible.  This could occur even during the early years of medical training.  To ensure that additional training is happening requirements in board certification may in the future also be able to require the family physician to have a working knowledge of health information technology (Graham-Jones, et al., 2012).

We need to also remember the importance of personalizing the health information system in such a way that it will be most beneficial to the patient population that is being served.      A great example of how personalizing the health information system for specific population was found in a study in the Journal of Community Health, in which a look at Native Americans and Eskimos were found to have a very unique set of needs to their health care.  For example it was found that when Native Americans were encountered with health information technology in their patient care, that they were able to receive tailored care and an increased quality of health care for their needs.  In other words, creating a health information system can be a very complex and arduous process as there are so many different cultures and demographics with variables to consider.  However this was not possible unless their health information system was also personalized to meet their needs as acquiring information from this demographic was different than acquiring information from say New York City residents (Geana, et al., 2012).

Cybersecurity is an area of health information technology that also needs to be addressed if there is going to be a successful and fully integrated system that will be used by all family physicians.  This is very important because it is imperative that the rights of the patients are protected first and foremost.  This standard however may not be made for some time as the debate as to what is ethical and what should be available for researchers as far as patient data is concerned, has not been legislated as of yet (Greiman, et al., 2012).

It would also be important to remember that there has to be some kind of way to customize health information technology to fit the needs of different family physicians, as well as the culture or demographics that they serve.  If this were to happen, the implementations and integration of a health information system would be able to successfully acquire the pertinent information to add to medical registries and also be available to researchers.  If this were to somehow happen, then great strides in healthcare could really take place (Geana, et al., 2012).



            It is very important to note that family physicians and those that are managing a family practice need to be made aware of the great tool that health information technology can be to a family practice in modern medicine today. If they were to implement health information technology into their family practice they would see an overall increase in production by the physician and their office staff, and more importantly in the quality of health care delivered to their patients.  With this increase in productivity would also mean that the family practice would make more money and have and increase in net revenue.

It is also important for these parties to understand that there will be many costs and expenses associated with implementing such a complex system.  One major cost will be that of training the staff, as well as the physician.  This is difficult to gauge how much this will cost as some people have a higher aptitude for computers and some people are faster learners.  Another major cost will be that of the actual software program.  Depending on what is needed and wanted in a health information system, and how many work stations it will be connected to, they system could cost as little as a thousand dollars to upwards of tens of thousands of dollars.

The family physician also has to realize as well and be braced for the future upgrades that will inevitably be required to stay current in the future.  As far as health information technology is concerned, especially in the growing stage that it is in now, upgrades are a very common occurrence in this field.  For this reason it is very important to have planned in the budget a portion for upgrades in order to ensure that this would not be an unforeseen expense in the future.

These upgrades can range from increased cyber security to ensure patient and organizational safety, and to minimize liability, to new and improved software features.  Other reasons to upgrade could be a change in government mandates and legislation requirements to the health information system that needs to be upgraded or else there may be a fine or a penalty applied which could in the worst case scenario mean that the family physician would not be able to effectively care for their patients (Kumar & Bauer, 2008).



            It is strongly recommended by the author that family physicians in the United States get on board with health information technology.  The sooner the integration of a health information technology system into their practice, the sooner the customizations and growth can begin in order to better suit their needs, and the needs of their patients and staff.  It would be much more feasible to grow with the technology than to be left behind and not reap the benefits that health information technology can offer family physicians today.

It is also recommended by the author that the training of family physicians or any physicians for that matter would include training in health information technology as this is surely a way to move the practice of medicine forward in a more rapid and positive direction.  This will help to ensure that the new physician will then better be able to integrate health information technology into their practice more seamlessly.

Another recommendation would be to make sure first and foremost that patient privacy will be protected, because without this the health information technology will never be all that it could be.  If the patients do not trust their privacy will be protected, they will feel vulnerable and look for alternatives.  If this were the case, then medicine would not move forward as swiftly as it could with the help of health information technology.

This would be because the old paper-chart system, although very secure, would not be readily accessible to those that may research, or to pharmacists, or to the wealth of knowledge that is available to help the physician and the patient to use evidence based medicine to properly treat the patient.

A paper chart system may be recommended for very specific and private health care such as a family physician run weight loss clinic.  This may be of benefit to use a paper chart, as the weight loss medication is rarely covered by insurance in the first place.  This would mean that reimbursements using the health information technology would be non-existent making this type of family medicine cash only.  Also, due to the high confidentiality requirements of patients seeking weight loss, a paper chart is really secure.

However, some other portions of health information technology could be used in this realm of family medicine such as emails and appointment settings. This would in large part depend on the physician and the patients in regards to how private the practice would want to be, because it could be argued that an appointment book being hacked could just as well be considered compromised privacy.

Perhaps the most important and final recommendation would be is to have an open mind and a positive attitude toward health information technology.  If the family physicians and their staff can buy into the bigger picture of health information technology, and how it can improve the many facets of their medical practice, they may be more apt to integrate, change and to learn new things.  The same recommendation also goes to the patients of these family physicians.

Though it may be difficult to actually realize how perhaps being generous with a little information may be, that the patient population as a whole could greatly help to progress the practice of evidence based medicine.  It is important to see that this benefit could in fact have a huge impact on their posterity and also help to solve some of the problems that have been plaguing health care today and that have been a hindrance to the practice of evidence based medicine for a long time.

In order for health information technology to become an amazing system of health care benefit, it is important for and recommended that globally there be some kind of communication between health information systems and data. Although this is a very lofty goal that will undoubtedly take years to achieve, it is definitely a worthwhile goal to work towards for the greater good of human kind.  It all starts today with using the health information technology that we now have and improve try to improve upon it.  It also starts at the front lines of medicine, and that is with the family physician.





Alexander: Failing electronic health records program may stand in way of precision medicine. (2015). (). Lanham: Federal Information & News Dispatch, Inc. Retrieved from

AHIMA calls for improved health information governance to unify standards for EHR use. (2012, Oct 01). PR Newswire Retrieved from

Dobalian, A., Claver, M. L., Pevnick, J. M., Stutman, H. R., Tomines, A., & Fu, P. (2012). Organizational challenges in developing one of the nationwide health information network trial implementation awardees. Journal of Medical Systems, 36(2), 933-40. doi:

Electronic medical records; origin healthcare solutions provide advanced patient portal functionality within electronic health record system. (2011). Computers, Networks & Communications, 490. Retrieved from

Fleurant, M., Kell, R., Love, J., Jenter, C., Volk, L. A., Zhang, F., . . . Simon, S. R. (2011). Massachusetts E-health project increased physicians’ ability to use registries, and signals progress toward better care. Health Affairs, 30(7), 1256-64. Retrieved from

Geana, M. V., Greiner, K. A., Cully, A., Talawyma, M., & Daley, C. M. (2012). Improving health promotion to American Indians in the Midwest United States: Preferred sources of health information and its use for the medical encounter. Journal of Community Health, 37(6), 1253-63. doi:

Graham-Jones, P., Jain, S. H., Friedman, C. P., Marcotte, L., & Blumenthal, D. (2012). The need to incorporate health information technology into physicians’ education and professional development. Health Affairs, 31(3), 481-7. Retrieved from

Greiman, V., Zlateva, T., & Chitkushev, L. (2012). Protecting health information privacy and safety on the internet: United States eHealth systems and legal perspectives. Paper presented at the 122-X. Retrieved from

Hinman, A. R., & Ross, D. A. (2010). Immunization registries can be building blocks for national health information systems.Health Affairs, 29(4), 676-82. Retrieved from

Information technology; making health information technology more patient-centered. (2011). NewsRx Health & Science, , 520. Retrieved from

Kumar, S., & Bauer, K. (2011). The business case for implementing electronic health records in primary care settings in the united states. Journal of Revenue and Pricing Management, 10(2), 119-131. doi:

Landro, L. (1999, Oct 18). Health & medicine (A special report): Living with change — the decision is yours: Doctors are starting to embrace information technology — and it’s changing their relationship with patients. Wall Street Journal Retrieved from

LaTour, K. (2013). Health information management: Concepts, principles, and practice (4th ed.). Chicago, Ill.: AHIMA.

Lee, K., Chul-young Roh, M. P., & , H. (2007). The impact of payer-specific hospital case mix on hospital costs and revenues for third-party patients. Journal of Medical Systems, 31(1), 1-7. doi:

Lifland, S. A. (2012). The corporate soap-opera “as the cash turns”: Management of working capital and potential external financing needs. Review of Business, 32(1), 35-46. Retrieved from

Lorenzen, M. (2011). Gulf coast health center serves its community with a new EMR system. Health Management Technology,32(8), 22. Retrieved from

Marco, D. (2006). Understanding data governance and stewardship, part 1. DM Review, 16(9), 28. Retrieved from

Montague, E. (2014). The promises and challenges of health information technology in primary health care. Primary Health Care Research & Development, 15(3), 227-230. doi:

Mullner, R. M., & Chung, K. (2006). Current issues in health care informatics. Journal of Medical Systems, 30(1), 1-2. doi:

Norberto (bert) robles – SVP, information technology and corporate chief information officer, new york city health and hospital corporation (2013). . San Francisco: Boardroom Insiders, Inc. Retrieved from

Pawlson, L. G. (2007). Health information technology: Does it facilitate or hinder rapid learning? Health Affairs, , W178-W180. Retrieved from

Payer mix soon will be more complex. (2012). Hospital Access Management, Retrieved from

Ritchie, A. (2014). The future of family medicine. Medical Economics, 91(18), 35-38. Retrieved from

ROCKEFELLER CALLS ON FORTUNE 500 COMPANIES TO PRIORITIZE CYBERSECURITY. (2012). (). Lanham: Federal Information & News Dispatch, Inc. Retrieved from

Rogoski, R. R. (2006). As easy as… EMR. Health Management Technology, 27(5), 10-5. Retrieved from

Shin, D. Y., Menachemi, N., PhD., Diana, M., PhD., Kazley, A. S., PhD., Ford, E. W., PhD., & Allen, Ryan L,C.P.H.I.M.S., C.H.P.S. (2012). Payer mix and EHR adoption in Hospitals/PRACTITIONER

Shwayri, R. N., J.D. (2014). Balancing the risks and rewards of cloud-based healthcare information. Information Management,48(3), 42-45,47. Retrieved from

Spratt, A. D., & Dickson, K. E. (2008). CHANGE FACTORS AFFECTING THE TRANSITION TO AN EMR SYSTEM IN A PRIVATE PHYSICIANS’ PRACTICE: AN EXPLORATORY STUDY. Academy of Health Care Management Journal, 4(2), 41-88. Retrieved from

Two AHIMA certification programs granted NCCA accreditation. (2011, Nov 30). Targeted News Service Retrieved from

Wakefield, W. (. W. (2008). Medicare physician reimbursement policy and type of service volume sensitivity (Order No. 3301326). Available from ProQuest Central. (304552969). Retrieved from

Zandieh, S. O., Yoon-flannery, K., Kuperman, G. J., Langsam, D. J., Hyman, D., & Kaushal, R. (2008). Challenges to EHR implementation in electronic- versus paper-based office practices. Journal of General Internal Medicine, 23(6), 755-61. doi:









Appendix A


Figure Captions

Exhibit 1



(Fleurant, 2011).







Appendix B


Exibit 2


(Hinman, 2010).

Survival of Bacillus spp. SUBB01 at high temperatures and a preliminary assessment of its ability to protect heat-stressed Escherichia coli cells

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Survival of Bacillus spp. SUBB01 at high temperatures and a preliminary assessment of its ability to protect heat-stressed Escherichia coli cells

Md. Sakil Munna, Jannatun Tahera, Md. Mohibul Hassan Afrad, Ifra Tun Nur and Rashed Noor*

Author Affiliations

Department of Microbiology, Stamford University, 51 Siddeswari Road, Dhaka 1217, Bangladesh

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BMC Research Notes 2015, 8:637  doi:10.1186/s13104-015-1631-9

The electronic version of this article is the complete one and can be found online at:

Received: 20 November 2014
Accepted: 26 October 2015
Published: 3 November 2015

© 2015 Munna et al.

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The bacterial stressed state upon temperature raise has widely been observed especially in Escherichia coli cells. The current study extended such physiological investigation on Bacillus spp. SUBB01 under aeration at 100 rpm on different culture media along with the high temperature exposure at 48, 50, 52, 53 and 54 °C. Bacterial growth was determined through the enumeration of the viable and culturable cells; i.e., cells capable of producing the colony forming units on Luria–Bertani and nutrient agar plates up to 24 h. Microscopic experiments were conducted to scrutinize the successive physiological changes. Suppression of bacterial growth due to the elevated heat was further confirmed by the observation of non-viability through spot tests.


As expected, a quick drop in both cell turbidity and colony forming units (~10 4 ) along with spores were observed after 12–24 h of incubation period, when cells were grown at 54 °C in both Luria–Bertani and nutrient broth and agar. The critical temperature (the temperature above which it is no longer possible to survive) of Bacillus spp. SUBB01 was estimated to be 53 °C. Furthermore, a positive impact was observed on the inhibited E. coli SUBE01 growth at 45 and 47 °C, upon the supplementation of the extracellular fractions of Bacillus species into the growing culture.


Overall the present analysis revealed the conversion of the culturable cells into the viable and nonculturable (VBNC) state as a result of heat shock response in Bacillus spp. SUBB01 and the cellular adaptation at extremely high temperature.


High temperature stress; Bacillus spp.; Colony forming units (CFUs); Viable but non culturable (VBNC) cells


Bacillus species are well known spore-forming pathogenic bacteria which are frequently found in the environment. Like Escherchia coli, these bacterial species may encounter a number of growth retarding stress factors, including nutrient depletion, temperature fluctuation, variation in pH and redox potential, limited water activity (a w ), elevated level of reactive oxygen species (ROS), osmotic imbalance along with unusual solute concentrations, etc. [1]–[15]. In response to such stress causing stimuli, different bacterial species have been observed to employ various defensive strategies to cope with the stress signals [11], [16], [17]. To deal with the heat stress, a number of reports suggested the expression of the global molecular chaperones and other components (CspB and CspE in Bacillus spp. cells, GroEL and DnaK proteins in Salmonella spp., rpoE, rpoS and rpoH genes in E. coli and Pseudomonas spp.) to combat against the stress as well as to maintain the cellular homeostasis [5], [6], [13], [18]–[34].

Our earlier studies unraveled the influence of the temperature up-shift with the generation of oxidative stress retarding the amount of viable and culturable bacterial cells [5], [12], [13], [35]; spontaneous accumulation of the ROS not only at the beginning of the early stationary phase but also by the supplementation of hydrogen peroxide (H 2 O 2 ) in the growing culture [2], [5], [32], [34] and the effect of different aeration speed on the cellular capability to produce the colony forming units on agar plates [33]. In all instances, the physiological response of E. coli SUBE01, Pseudomonas spp. SUBP01, Salmonella spp. SUBS01 and Bacillus spp. SUBB01 against oxidative stress was observed through their sustainability in retaining the culturable cells [5], [12], [13], [32]–[34], [36]. Besides, the information on the defense strategy of these bacteria especially those belonging to Bacillus spp. SUBB01 under the static condition was evidently noticed through their phenotypic behavior [34]. Along these lines of information, current study was conducted to further scrutinize the heat-shock response in Bacillus spp. SUBB01 under the shaking condition at 100 rpm in different culture media.


Demonstration of culturable Bacillus spp. SUBB01 under heat stress

Laboratory stock culture of Bacillus spp. SUBB01 and E. coli SUBE01 were used in this study. Experiments demonstrating the bacterial growth in terms of cell turbidity (optical density at 600 nm) and colony forming units (CFUs) were conducted as described earlier by Nur et al. [34]. Nutrient agar (NA), Luria–Bertani (LB) agar, nutrient broth (NB) and Luria–Bertani broth were used for the assay of culturability [13]. After 24 h of incubation on nutrient agar plates at 37 °C, one loopful of the bacterial culture was introduced into 5 ml nutrient broth followed by incubation at 37 °C for 4–6 h at 100 rpm (pre-culture). After adjusting the optical density of the pre-culture at 600 nm (OD 600 ) to 0.1, 30 µL each was introduced into 2 different sets of 30 ml of nutrient broth and Luria–Bertani broth and incubated at 48, 50, 52, 53 and 54 °C at shaking condition (100 rpm). At the time points of 12 and 24 h, the cell growth was monitored by measuring OD 600 and by counting the colony forming units (CFUs) [34]. All the experiments were conducted three times. Statistical analysis regarding bacterial growth was performed by determining P value through t test. Standard deviations for all data have been indicated by error bars.

Assessing cell viability was further confirmed by the spot tests [13], [32]–[34]. As described previously, each the culture suspension was serially diluted in 9 ml nutrient broth to obtain up to 10 −4 fold dilution. From each dilution, an aliquot of 5 µl was dropped on to the nutrient agar and Luria–Bertani agar, dried off for 15 min, and finally the plates were incubated at 37 °C for 24 h. Spotting on the agar was accomplished at 24 h of growth.

Demonstration of morphological changes

Simple staining (Crystal Violet, Hucker’s Solution) was conducted to assess the viability and the cellular morphology as previously done [32]–[34]. Spore staining (malachite green oxalate, safranin O) was conducted to differentiate the bacterial spores from vegetative cells following standard procedures [37]. An aliquot of 10 µl from the bacterial culture suspension was removed at 24 h of growth, and the cellular morphology, shape and organization were observed under the light microscope (Optima Biological Microscope G206, manufactured in Taiwan) at 1000× magnification [32].

Preparation of organic and inorganic supplements

To prepare the extracellular fractions of bacteria (E. coli and Bacillus species), cells were grown separately in 6 different sets of Durham’s bottle containing 5 ml minimal broth, which were kept in a shaking water bath at 100 rpm for 24 h at 37 °C (optimum growth temperature) [13]. Subsequently, actively growing bacterial cells were centrifuged at 4000 rpm for 15 min, and the resulting pellets were collected. Afterward pellets were centrifuged at 4000 rpm for 15 min for 3 times with 10 % glycerol and 50 mM CaCl 2 , respectively. The resulting supernatants were collected and used as organic supplements to observe the possible retrieval of E. coli cell viability both at high temperature stress (45 °C) and around the critical temperature (47 °C). Subsequently, a mixture of 20 mM MgSO 4 and 5 mM ethylene diamine tetraacetic acid (EDTA) were used as inorganic supplement to conduct a similar experiment [12].

Results and discussions

Growth retardation of Bacillus spp. SUBB01upon heat shock

As stated earlier, our former studies demonstrated the effective defense strategies of Bacillus spp. SUBB01 in response to the oxidative stress artificially created by the supplementation of 6 mM H 2 O 2 into the growing culture at an aeration speed of 100 rpm, while the E. coli SUBE01 and Pseudomonas spp. SUBP01 were found to loose culurability under the same condition. Interestingly, as has been noticed in our earlier study, a decrease in the amount in the culturable cells of Bacillus spp. SUBB01 was observed when challenged with an increased concentration (21 mM) of H 2 O 2[34]. Apart from this stressed condition, the present study also employed a state of heat stress in the Bacillus cells since the increase in temperature is known to induce the accumulation of ROS inside the cells, which in turn largely accounts for losing cellular viability and culturability [35]. In this investigation, when the Bacillus spp. SUBB01 cells were grown at 48–53 °C, around 4-log reduction in cell turbidity (Fig. 1a, b) as well as in the generation of the colony forming units (CFUs) were observed (Fig. 1c, d) up to 24 h of incubation periods in both nutrient and Luria–Bertani (LB) agar and broth. Notably, even as high as at 53 °C, cells were noted to produce the CFUs up to 10 2 CFU/ml. Interestingly, a complete elimination in both cell turbidity and colony forming units (CFUs) were observed when the cells were challenged at 54 °C, and hence indicating the critical temperature for Bacillus spp. SUBB01 to be at 53 °C (Fig. 1).

thumbnailFig. 1. Growth retardation of Bacillus spp. SUBB01 at extended temperature. Assessment of growth of Bacillus spp. SUBB01 at 48, 50, 52, 53 and 54 °C in terms of cell turbidity (a, b) and colony forming unites (c, d) at 100 rpm. Cells were grown aerobically as stated in “Methods”. Aliquots were removed at 12 and 24 h for the assay of culturable cells. Retardation of growth was slightly observed in case of Bacillus spp. SUBB01 after 12–24 h in both LB broth and nutrient broth

Consistent with the results acquired in the growth related experiments, no morphological changes were observed under light microscope when cells were subjected to growth temperatures of 48, 50, 52, 53 °C in both LB and nutrient broth for up to 24 h of incubation (Fig. 2). However, sporulation was observed at 54 °C. Earlier studies showed that the stressosome signaling complex of Bacillus spp. is activated in response to several environmental stresses including the housekeeping σ factor (σ A ) and alternative sigma factor (σ B ) during the early or mid stationary phase, providing the protective adaption from environmental changes [16], [38]–[44]. Several other studies showed that in response to heat shock, σ F has been found to be activated, which in turn may protect cells from heat shock through sporulation means [45]. Although such genetic analyses were not conducted in the present investigation; the results presented in our study clearly indicate that the cells lose their culturability at 54 °C, which indicates the dormant but viable state through sporulation.

thumbnailFig. 2. Cells were grown and aliquots were removed from the respective bacterial cultures for light microscopy as stated in the “Methods” section. However, no significant morphological or arrangement changes were observed regardless of heat shock at 48, 50, 52, 53 and 54 °C at 100 rpm (aj)

Confirmative demonstration of loss of culturability of Bacillus spp. SUBB01cells

The sharp decline in culturable cell population fraction (Fig. 1), as well as the presence of spores under light microscope due to extreme high temperature (imposed by 54 °C) in Bacillus spp. SUBB01 cells (Fig. 2), led us to further cross-check of the stressed physiology of the cells through the spot tests as corroborated earlier [13], [32]–[34]. Consistent to the growth experiments as shown in Fig. 1, a steady growth was observed at 48, 50 and 52 °C with minimal variation or growth reduction in both nutrient and LB agar (Fig. 3) and a relatively slower growth was observed when bacterial cells were grown at 53 °C on both nutrient and LB agar, whereas a complete growth cessation was observed at 54 °C (Fig. 3).

thumbnailFig. 3. Confirmative demonstration of culturability and survival potential of Bacillus spp. SUBB01 cells upon heat shock. Cells were grown aerobically as stated earlier, and aliquots were removed at 24 h for spot tests. A progressing decline in bacterial growth was observed at 53 °C and the complete abolishment of cells was found at 54 °C after 24 h of incubation period in both nutrient agar media and Luria–Bertani agar media

As stated earlier, our previous investigations on stress responses in E. coli SUBE01 showed the influence of the temperature up-shift resulting in oxidative stress [13] which further led us to investigate the stress response against external and internal oxidative stress stimuli within Bacillus spp. SUBB01, E. coli SUBE01, Pseudomonas spp. SUBP01 and Salmonella spp. SUBS01 [5], [12], [13], [32]–[34], [36]. Those studies clearly revealed that E. coli SUBE01 lost viability upon heat shock [13]. Moreover, the external and internal oxidative stresses in the early stationary phase of E. coli SUBE01 and Pseudomonas spp. SUBP01 culture were found to influence the formation of culturable cells; i.e., capable of forming colonies [2], [32]–[34]. With a succession of those work, the results presented in this study showed that Bacillus spp. SUBB01 is likely to exhibit the alteration in cellular homeostasis and culturability due to heat shock at 48–53 °C, under aeration (100 rpm) condition on different culture media. Notably, cells were found to lose culturablility completely at 54 °C, wherein spores were observed under light microscope (Fig. 2). Previously several studies reported that Bacillus cells exhibits six classes of heat shock genes upon environmental stress and the activation of the heat shock genes of Bacillus species especially depends on the commencement of specific temperature [45], [46].

Demonstration of growth retrieval

Another remarkable aspect was to evaluate the possible positive effect on the high temperature stressed E. coli SUBE01 growth at critical (45 °C) and above critical (47 °C) temperature [13] upon the supplementation of the extracellular fractions of Bacillus species as organic supplement which was further compared to the inorganic supplement into the cultures in the conditions as stated above (Figs. 4, 5). Previously it has been reported that Mg 2+ and EDTA were capable of protecting the outer membrane from cell burst upon heat shock [12]. Concerning that factual report in the current investigation, when the stressed culture of E. coli SUBE01 cells were treated with the extracellular fractions of Bacillus spp. around their critical temperature in both minimal agar and broth media, cells were surprisingly found to retrieve their growth after certain incubation periods (Figs. 4, 5). Moreover, no significant changes were observed, when cells were grown with the extracellular fractions of E. coli SUBE01 compared to those treated with inorganic supplement as illustrated in Fig. 6.

thumbnailFig. 4. Growth revival assay of E. coli SUBE01 at critical growth temperature (45 °C). Effect of high temperature on growth of E. coli: a impact on cell turbidity and influence on the formation of CFUs, (b) and the demonstration of loss of cell culturability by means of spot test (c) upon supplementation with organic and inorganic supplement. Cells were grown in minimal at 45 °C up to 72 h and after 10 h of growth organic supplement (the extracellular fractions of Bacillus species) and inorganic supplement (20 mM MgSO 4 with 5 mM EDTA) were added and untreated cells were referred as control. At every 12 h interval, cells were diluted and grown in minimal agar at 37 °C for 24 h. A growth revival was observed to be increased by approximately four logs for E. coli cells at different period of incubation. All experiments were carried out three times and the standard deviations for all data have been indicated by error bars

thumbnailFig. 5. Growth revival assay of E. coli SUBE01 at above critical growth temperature (47 °C). Effect of high temperature on growth of E. coli: a impact on cell turbidity and influence on the formation of CFUs, b and the demonstration of loss of cell culturability by means of spot test c upon supplementation with organic and inorganic supplement. Cells were grown in minimal agar at 47 °C up to 72 h and after 10 h of growth organic supplement (the extracellular fractions of Bacillus species) and inorganic supplement (20 mM MgSO 4 with 5 mM EDTA) were added and untreated cells were referred as control. At every 12 h interval, cells were diluted and grown in minimal agar at 37 °C for 24 h. A growth revival was observed at 47 °C (c, d), when cells were grown with inorganic supplement. All experiments were carried out three times and the standard deviations for all data have been indicated by error bars

thumbnailFig. 6. Impact of extracellular fractions of E. coli on cell culturability of E. coli at 45 °C. Effect of high temperature on growth of E. coli SUBE01: a impact on cell turbidity b and influence on the formation of CFUs, upon supplementation with the extracellular fractions of E. coli SUBE01 as organic supplement and 20 mM MgSO 4 with 5 mM EDTA as inorganic supplement and untreated cells were referred as control. Cells were grown in minimal agar at 45 °C up to 72 h and after 10 h of growth organic supplement and inorganic supplement were added. At every 12 h interval, cells were diluted and grown in minimal agar at 37 °C for 24 h. No significant changes were observed at 45 °C (a, b), when cells were grown with the extracellular fractions of E. coli SUBE01. All experiments were carried out three times and the standard deviations for all data have been indicated by error bars

As stated earlier, our previous studies unraveled the defensive strategies of various bacterial species against heat shock and oxidative stress [5], [12], [13], [32]–[36]. Recently the response within yeast cells against heat stress and osmotic shock has also been observed [47]. While the mechanisms of survival of E. coli cells have clearly been chalked out very recently [48], the retrieval of a heterogeneous E. coli population consisting of viable cells and defective cells (incapable of forming colonies on agar plates) by the Bacillus extracts as found in the current study is being reported for the first time so far to our knowledge. Such an experimental demonstration could be of significance to understand the cellular survival strategies mediated by different bacterial species.


Despite the lack of molecular investigation as well as an apparent impression of descriptive nature of research stipulation, the data in the current study is quite consistent to the previous findings with the novel projection on the critical growth temperature of Bacillus strain. Moreover, the work clearly illustrated the phenotypic changes in the bacterial cell caused by the heat shock at the optimum speed of aeration which, unlike to that of E. coli, is relatively new in the field of heat shock response in Bacillus cells. Such preliminary findings could be worth incrementing the existing knowledge on bacterial cell biology and signal transduction. Finally, the observation of E. coli growth retrieval upon supplementation of the extracellular fractions from Bacillus spp. have been indeed interesting to ponder on the heat stress resistance mechanisms of Bacillus spp. However, further molecular studies on the genetic makeup of such stress responses as well as the growth retrieval mechanisms by means of exogenous organic factors (Bacillus extracts) would be of greater effectiveness.

Authors’ contributions

This work was carried out in collaboration between all authors. Author MSM managed the analyses of the study and wrote the first draft of the manuscript. Authors JT and MMHA performed the experiments. Author ITN managed the literature searches. Author RN designed the study and critically revised the manuscript. All authors read and approved the final manuscript.


We thank Stamford University Bangladesh for providing us the facilities to carry out the experiments. However, the authors received no specific funding for this work.

Authors’ information

All the authors are from Department of Microbiology, Stamford University Bangladesh. Authors MSM, JT and ITN are thesis students of MS program and MMHA is thesis students of BSc. (Hons) program of the department. Author RN is corresponding author of the manuscript, has been working as Associate Professor and Chairman of the Department of Microbiology, Stamford University Bangladesh.

Competing interest

The authors declare that they have no competing interest.


  1. Givskov M, Eberl L, Moller S, Poulsen LK, Molin S: Responses to nutrient starvation in Pseudomonas Putida KT2442: analysis of general cross-protection, cell shape, and macromolecular content.

    J Bacteriol 1994, 176:7-14. PubMed Abstract | Publisher Full Text OpenURL

  2. Kabir MS, Yamashita D, Noor R, Yamada M: Effect of σ S on σ E -directed cell lysis in Escherichia coli early stationary phase.

    J Mol Microbiol Biotechnol 2004, 8:189-194. PubMed Abstract | Publisher Full Text OpenURL

  3. Nystrom T: Role of oxidative carbonylation in protein quality control and senescence.

    EMBO J 2005, 24:1311-1317. PubMed Abstract | Publisher Full Text OpenURL

  4. Den Besten HMW, Mols M, Moezelaar R, Zwietering MH, Abee T: Phenotypic and transcriptomic analyses of mildly and severely salt-stressed Bacillus cereus ATCC 14579 cells.

    Appl Environ Microbiol 2009, 75:4111-4119. Publisher Full Text OpenURL

  5. Noor R, Murata M, Yamada M: Oxidative stress as a trigger for growth phase-specific sigma-E dependent cell lysis in Escherichia coli.

    J Mol Microb Biotech 2009, 17:177-187. Publisher Full Text OpenURL

  6. Noor R, Murata M, Nagamitsu H, Klein G, Rain S, Yamada M: Dissection of sigma-E dependent cell lysis in Escherichia coli: roles of RpoE regulators RseA, RseB and periplasmic folding catalyst Ppid.

    Genes Cells 2009, 14:885-899. PubMed Abstract | Publisher Full Text OpenURL

  7. Ju KS, Parales RE: Nitro-aromatic compounds, from synthesis to biodegradation.

    Microbiol Mol Biol R 2010, 74:250-272. Publisher Full Text OpenURL

  8. Fuchs G, Boll M, Heider J: Microbial degradation of aromatic compounds—from one strategy to four.

    Nat Rev Microbiol 2011, 9:803-816. PubMed Abstract | Publisher Full Text OpenURL

  9. Kivisaar M: Evolution of catabolic pathways and their regulatory systems in synthetic nitroaromatic compounds degrading bacteria.

    Mol Microbiol 2011, 82:265-268. PubMed Abstract | Publisher Full Text OpenURL

  10. Deepika G, Karunakaran E, Hurley CR, Biggs CA, Charalampopoulos D: Influence of fermentation conditions on the surface properties and adhesion of Lactobacillus rhamnosus GG.

    Microb Cell Fact 2012, 11:116. PubMed Abstract | BioMed Central Full Text OpenURL

  11. Huillet E, Tempelars MH, Andre-Leroux G, Wanapaisan P, Bridoux L, Makhzamis S, Panbangred W, Martin-Verstraete I, Abee T, Lereclus D: PIcRa, a new quorum-sensing regulator from Bacillus cereus, play a role in oxidative stress response and cystein metabolism in stationary phase.

    PLoS One 2012, 7:e51047. PubMed Abstract | Publisher Full Text OpenURL

  12. Murata M, Noor R, Nagamitsu H, Tanaka S, Yamada M: Novel pathway directed by sigma-E to cause cell lysis in Escherichia coli.

    Genes Cells 2012, 17:234-247. PubMed Abstract | Publisher Full Text OpenURL

  13. Noor R, Islam Z, Munshi SH, Rahman F: Influence of temperature on Escherichia coli growth in different culture media.

    J Pure Appl Microbiol 2013, 7:899-904. OpenURL

  14. Den Besten HMW, Effraimidou S, Abee T: Catalase activity as a biomarker for mild stress-induced robustness in Bacillus weihenstephanensis.

    Appl Environ Microbiol 2013, 79:57-62. Publisher Full Text OpenURL

  15. Shimizu K: Regulation systems of bacteria such as Escherichia coli in response to nutrient limitation and environmental stresses.

    Metabolites 2013, 4:1-35. PubMed Abstract | Publisher Full Text OpenURL

  16. Price CW, Fawcett P, Ceremonie H, Su N, Murphy CK, Youngman P: Genomewide analysis of the general stress response in Bacillus subtilis.

    Mol Microbiol 2001, 41:757-774. PubMed Abstract | Publisher Full Text OpenURL

  17. Phillips ZE, Strauch MA: Bacillus subtilis sporulation and stationary phase gene expression.

    Cell Mol Life Sci 2002, 59:392-402. PubMed Abstract | Publisher Full Text OpenURL

  18. Ananthan J, Goldberg AL, Voellmy R: Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes.

    Science 1986, 232:522-524. PubMed Abstract | Publisher Full Text OpenURL

  19. Sarniguet A, Kraus J, Henkels MD, Muehlchen AD, Loper JE: The sigma factor σ S affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5.

    Proc Natl Acad Sci USA 1995, 92:12255-12259. PubMed Abstract | Publisher Full Text OpenURL

  20. Mayr B, Kaplan T, Lechner S, Scherer S: Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201.

    J Bacteriol 1996, 178:2916-2925. PubMed Abstract | Publisher Full Text OpenURL

  21. Ramos-Gonzalez MI, Molin S: Cloning, sequencing, and phenotypic characterization of the rpoS gene from Pseudomonas putida KT2440.

    J Bacteriol 1998, 180:3421-3431. PubMed Abstract | Publisher Full Text OpenURL

  22. Jorgensen F, Bally M, Chapon-Herve V, Michel G, Lazdunski A, et al.: RpoS-dependent stress tolerance in Pseudomonas aeruginosa.

    Microbiology 1999, 145:835-844. PubMed Abstract | Publisher Full Text OpenURL

  23. Suh SJ, Silo-Suh L, Woods DE, Hassett DJ, West SHE, et al.: Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa.

    J Bacteriol 1999, 181:3890-3897. PubMed Abstract | Publisher Full Text OpenURL

  24. Whistler CA, Stockwell VO, Loper JE: Lon protease influences antibiotic production and UV tolerance of Pseudomonas fluorescens Pf-5.

    Appl Environ Microbiol 2000, 66:2718-2725. PubMed Abstract | Publisher Full Text OpenURL

  25. Miller CD, Kim YC, Anderson AJ: Competitiveness in root colonization by Pseudomonas putida requires the rpoS gene.

    Can J Microbiol 2001, 47:41-48. PubMed Abstract | Publisher Full Text OpenURL

  26. Miller CD, Mortensen WS, Braga GUL, Anderson AJ: The rpoS gene in Pseudomonas syringae is important in surviving exposure to the near-UV in sunlight.

    Curr Microbiol 2001, 43:374-377. PubMed Abstract | Publisher Full Text OpenURL

  27. Periago PM, Schaik WV, Abee T, Wouters JA: Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579.

    Appl Environ Microbiol 2002, 68:3486-3495. PubMed Abstract | Publisher Full Text OpenURL

  28. Stockwell VO, Loper JE: The sigma factor RpoS is required for stress tolerance and environmental fitness of Pseudomonas fluorescens Pf-5.

    Microbiology 2005, 151:3001-3009. PubMed Abstract | Publisher Full Text OpenURL

  29. Heeb S, Valverde C, Gigot-Bonnefoy C, Haas D: Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0.

    FEMS Microbiol Lett 2005, 243:251-258. PubMed Abstract | Publisher Full Text OpenURL

  30. Akerfelt M, Morimoto RI, Sistonen L: Heat shock factors: integrators of cell stress, development, and lifespan.

    Nat Rev Mol Cell Biol 2010, 11:545-555. PubMed Abstract | Publisher Full Text OpenURL

  31. Morimoto RI: The heat shock response: systems biology of proteotoxic stress in aging and disease.

    Cold Spring Harb Symp Quant Biol 2012, 76:91-99. Publisher Full Text OpenURL

  32. Munna MS, Nur IT, Rahman T, Noor R: Influence of exogenous oxidative stress on Escherichia coli cell growth, viability and morphology.

    Am J BioSci 2013, 1:59-62. Publisher Full Text OpenURL

  33. Munna MS, Tamanna S, Afrin MR, Sharif GA, Mazumder C, et al.: Influence of aeration speed on bacterial colony forming unit (CFU) formation capacity.

    Am J Microbiol Res 2014, 2:47-51. Publisher Full Text OpenURL

  34. Nur IT, Munna MS, Noor R: Study of exogenous oxidative stress response in Escherichia coli, Pseudomonas spp., Bacillus spp. and Salmonella spp.

    Turk J Biol 2014, 38:502-509. Publisher Full Text OpenURL

  35. Yamada M, Noor R, Nagamitsu H, Murta M. The higher temperature, the more oxidative stress and lysis in Escherichia coli. In: The 3rd International Conference on Fermentation Technology for Value Added Agricultural Products; Khon Kaen; 2009.
  36. Nitta T, Nagamitsu H, Murata M, Izu H, Yamada M: Function of the sigma-E regulon in dead-cell lysis in stationary phase Escherichia coli.

    J Bacteriol 2000, 182:5231-5237. PubMed Abstract | Publisher Full Text OpenURL

  37. Cappuccino JG, Shermen N: Microbiology; laboratory manuals. Benjamin/Cummings Publishing Company Incorporated, San Francisco; 1996. OpenURL
  38. Hecker M, Volker U: General stress response of Bacillus subtilis and other bacteria.

    Adv Microb Physiol 2001, 44:35-91. PubMed Abstract | Publisher Full Text OpenURL

  39. Price CW: General stress response in Bacillus Subtilis and its closest relatives: From Genes to Cells. Washington, DC. Am Soc Microbiol 2002: 369–84.
  40. Helmann JD, Wu MFW, Gaballa A, Kobel PA, Morshedi MM, Fawcett P, Paddon C: The global transcriptional response of Bacillus subtilis to peroxide stress is coordinated by three transcription factors.

    J Bacteriol 2003, 185:243-253. PubMed Abstract | Publisher Full Text OpenURL

  41. Petersohn A, Brigulla M, Haas S, Hoheisel JD, Völker U, Hecker M: Global analysis of the general stress response of Bacillus subtilis.

    J Bacteriol 2001, 183:5617-5631. PubMed Abstract | Publisher Full Text OpenURL

  42. Hecker M, Pane-Farre J, Volker U: SigB-dependent general stress response in Bacillus subtilis and related Gram-positive bacteria.

    Annu Rev Microbiol 2007, 61:215-236. PubMed Abstract | Publisher Full Text OpenURL

  43. Hardwick SW, Pané-Farré J, Delumeau O, Marles-Wright J, Hecker M, Lewis RJ: Structural and functional characterization of partner switching regulating the environmental stress response in Bacillus subtilis.

    Am Soc Biochem Mol Biol 2007, 283:11562-11572. OpenURL

  44. Nannapaneni P, Hertwig F, Depke M, Hecker M, Mäder U, Volker U, Steil L, van Hijum SA: Defining the structure of the general stress regulon of Bacillus subtilis using targeted microarray analysis and random forest classification.

    Microbiology 2012, 158:696-707. PubMed Abstract | Publisher Full Text OpenURL

  45. Schumann W: The Bacillus subtilis heat shock stimulon.

    Cell Stress Chaperone 2003, 8:207-217. Publisher Full Text OpenURL

  46. Versteeg S, Escher A, Wende A, Wiegert T, Schumann W: Regulation of the Bacillus subtilis heat shock gene htpG is under positive control.

    J Bacteriol 2003, 185:466-474. PubMed Abstract | Publisher Full Text OpenURL

  47. Munna MS, Humayun S, Noor R: Influence of heat shock and osmotic stresses on the growth and viability of Saccharomyces cerevisiae SUBSC01.

    BMC Res Notes 2015, 8:369. PubMed Abstract | BioMed Central Full Text OpenURL

  48. Noor R: Mechanism to control the cell lysis and the cell survival strategy in stationary phase under heat stress.

    SpringerPlus 2015, 4:599. BioMed Central Full Text OpenURL

Targeting glycolysis by 3-bromopyruvate improves tamoxifen cytotoxicity of breast cancer cell lines

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Targeting glycolysis by 3-bromopyruvate improves tamoxifen cytotoxicity of breast cancer cell lines

Yasmin M. Attia1, Hanan S. EL-Abhar2, Mahmoud M. Al Marzabani1 and Samia A. Shouman1*

Author Affiliations

1 Pharmacology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Kasr Al Eini Street, Fom El Khalig, Cairo 11796Egypt

2 Pharmacology and Toxicology Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St, Cairo 11562Egypt

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BMC Cancer 2015, 15:838  doi:10.1186/s12885-015-1850-4
Mahmoud M. Al Marzabani Deceased

The electronic version of this article is the complete one and can be found online at:

Received: 22 June 2015
Accepted: 26 October 2015
Published: 3 November 2015

© 2015 Attia et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.



Tamoxifen is the standard endocrine therapy for ER+ breast cancer; however, many women still relapse after long-term therapy. 3-Bromopyruvate, a glycolytic inhibitor, has shown high selective anti-tumor activity in vitro, and in vivo. The aim of this study was to evaluate the possible augmentation of the effect of tamoxifen via reprograming cancer cell metabolism using 3-bromopyruvate.


An in vitro screening of antitumor activity as well as the apoptotic, anti-metastatic, and anti-angiogenic potentials of the combination therapy were carried out using different techniques on breast cancer cell lines MCF7and T47D. In addition the antitumor effect of the combined therapy was done on mice bearing tumor.


Our results showed modulation in apoptosis, angiogenesis and metastatic potential by either drug alone; however, their combination has surpassed that of the individual one. Combination regimen enhanced activated caspases-3, 7 and 9, as well as oxidative stress, signified by increased malondialdehyde and decreased glutathione level. Additionally, the angiogenesis and metastasis markers, including hypoxia inducing factor-1α, vascular endothelia growth factor, and metaloproteinases-2 and 9 were decreased after using the combination regimen. These results were further confirmed by the in vivo study, which depicted a decrease in the tumor volume and angiogenesis and an increase in oxidative stress as well.


3-bromopyruvate could be a valuable compound when added with tamoxifen in breast cancer treatment.


Breast cancer; Tamoxifen; 3-bromopyruvate; Apoptosis; Angiogenesis; MMPs


Breast cancer was estimated one of the most commonly diagnosed cancers worldwide among women (11.9 %). It is the most common cause of cancer death and the most frequently diagnosed cancer in 140 out of 184 countries worldwide [1] including Egypt, where there were an estimated 49.5 cases of breast cancer per 100,000 adults in 2012 [2]. Among the different molecular subtypes of breast cancer, estrogen (ER) positive comprises ~70 % of all breast cancers cases [3].

Tamoxifen (TAM), a synthetic nonsteroidal anti-estrogen, has been used widely as the gold standard endocrine therapy for most women with ERα + breast cancer. Five years of TAM treatment reduced the risk of relapse of 10 years by 37 % in females aged 50-59 years, and by 54 % in females aged 60–69 years [4]. The antiproliferative effects of TAM may relate to its antiestrogenic effect via binding competitively to estrogen receptor, thereby blocking the mitogenic effect of estrogens [5]. TAM also induces apoptosis of cancer cell through several distinct mechanisms including its inhibition of protein kinase C and its binding to calmodulin, a protein that plays a role in DNA synthesis [6]. Although TAM is an extremely effective treatment for millions of patients with breast cancer, a significant proportion, as much as 30 % of women still relapse during or after long-term therapy [7]. Besides, some patients display de novo or acquired resistance [5].

The competency to increase response and reduce chemoresistance of cancer therapeutics via the use of the combination therapy as well uncovering underlying mechanisms of chemoresistance would be a significant advantage for cancer patients. The development of a combination therapy that increases the efficacy of TAM has been investigated in several studies, using vitamin E [8] and green tea [9]. Moreover, mounting evidence supports, that reprogramming of cellular metabolism in cancer cells is linked to failure of treatment, and drug resistance in cancer therapy [10].

The glycolysis pathway is one of the main characteristics of tumor cells, which increases dramatically with malignancy [11]. Such increased aerobic glycolysis has been observed in a variety of cancer types; hence, targeting this pathway in cancer cells provides a biochemical basis for developing new chemotherapeutic strategies. 3-Bromopyruvate (3-BP) is an inhibitor of the glycolysis process that has shown remarkable anti-tumor efficacy, documented by both in vitro[12] and in vivo[13] studies. 3-BP mediates its effect by causing cell cycle arrest, inducing apoptosis and inhibiting angiogenesis activity, which closely related to glycolysis inhibition [14]. Therefore, we hypothesized that the use of glycolytic inhibitor (3 BP) could increase TAM efficacy on MCF-7 and T47D cell lines, as well as on mice -bearing Ehrlich solid tumor as a model established in studying the effect of chemotherapy in vivo.



Tamoxifen (TAM) and 3-BP were obtained from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Each vial of TAM contains one gm white powder. It was dissolved in DMSO to yield 50 μM then serially diluted in RPMI-1640 medium immediately before use to yield a concentration range of 10–50 μM. 3-Bromopyruvate (3-BP) was obtained in a vial containing 10 g white powder. It was dissolved in saline to yield 50 μM then serially diluted in RPMI-1640 supplemented medium immediately before use to yield a concentration range of 10–50 μM.


RPMI-1640 Medium, fetal bovine serum, dimethylsulfoxide (DMSO), Ellman’s reagent [5,5-Dithio-bis-(2-nitro benzoic acid)], β-mercaptoethanol, reduced glutathione, sodium dodecyl sulfate (SDS), sodium bicarbonate, 1,1.3,3-tetramethoxypropane, trichloroacetic acid (TCA) and thiobarbituric acid were all purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Triton X-100 was procured from MP Biochemical (Santa Ana, California, USA). All other chemicals and reagents were from standard analytical grade.

Cell lines and animals

Cell lines

Breast carcinoma estrogen receptor positive (ER+) cell lines MCF-7 and T47D were used in this study. They were obtained frozen in liquid nitrogen (−180 °C) from the American Type Culture Collection Organization (USA). The tumor cell lines were maintained by serial sub-culturing at the National Cancer Institute, Cairo, Egypt. They were cultured in a humidified incubator at 37 °C and 5 % CO 2 in RPMI-1640 medium supplemented with 10 % fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 3 mM/l glutamine. The cells were trypsinized every 3 days.


24 Female Swiss albino mice, weighing 22–25 g, were obtained from the National Cancer Institute, Cairo, Egypt. All of the animal handling and study procedures were approved by the research ethics committee of Faculty of Pharmacy, Cairo University, Cairo, Egypt (Permit Number: PT 661), and was conducted with the “Guide for the Care and Use of Laboratory Animals”. Animals were kept under suitable laboratory conditions of temperature and humidity. They were provided with standard chow and water and housed in plastic cages.

In-vitro parameters

Cytotoxicity assay

To study the antitumor activity of TAM, 3BP, and their combination on breast cancer cells, sulphorhodamine-B (SRB) method as described by Skehan et al. [15] was used. In brief; cells were seeded at a density of 3 × 10 3 cells/well in 96-well microtiter plates. They were left to attach for 24 h before incubation with drugs. Next, cells were treated with different concentrations of TAM, 3BP (10, 20, 30, 40 and 50 μM). The combination regimens was designed using IC 50 of TAM with different concentrations (10, 20, 30, 40, 50 μM) of 3BP. For each concentration, three wells were used and incubation was continued for 48 h. A control wells containing, vehicles DMSO (1 % v/v) for TAM, and media for 3-BP were used. At the end of incubation, cells were fixed with 20 % trichloroacetic acid (TCA), stained with 0.4 % SRB dye. The optical density (O.D.) of each well was measured spectrophotometrically at 570 nm using ELISA microplate reader (TECAN sunrise™, Germany).

The mean survival fraction at each drug concentration was calculated as follows: O.D. of the treated cells/O.D. of the control cells. The IC 50 (concentration that produce 50 % of cell growth inhibition) value of each drug was calculated using sigmoidal dose response curve-fitting models (Graph Pad Prizm software, version 5).

In all the following mechanistic experiments, we used the first concentration of 3BP that produced significant decrease of survival with IC 50 of TAM in both cell lines. Therefore, we used in MCF-7, 20 μM of TAM, 3BP and their combination, while in T47D it was 30 μM of 3-BP and 20 μM TAM and their combination.

Real time polymerase chain reaction (qPCR)

In order to study the effect of different treatments on angiogenesis, metastasis and apoptosis, the gene expression levels of mRNA of hexokinase (HK2), hypoxia inducing factor (HIF1-α), and metalloproteinase (MMP 2 and 9) as well as caspase 9 were assessed using q PCR. The total cellular RNA was extracted following the protocol of the RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse transcription was completed using High capacity cDNA archive kit (Applied Biosystem, California, USA). Real time PCR of GAPDH, caspase 9, HK2, HIF1-α, and (MMP 2 and 9) were performed in triplicate on an ABI 7500 Fast Real-Time PCR System using the GoTaq PCR master mix (Promega, Madison, U.S.A). Fast amplification parameters were as follows: one cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. All primers used in this study were purchased from Invitrogen (California, USA) (Table 1). Quantitative analysis of data was performed by using the∆∆ Ct method [16]. Values were normalized to GAPDH and were expressed as relative expression levels.

Table 1. The primer sequences of GAPDH, Caspase-9, HK-2, HIF-1 α, MMP-2 and 9 genes

Assay of caspase-3 activity

To confirm our data different techniques as ELISA, gelatin zymography and western method were used

Caspase 3, the executioner caspase, was assessed spectrophotometrically at 450 nm in cell lysate using ELISA kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions [17]. Cells were cultured in 75 cm 3 flasks, left till 70–80 % confluent, cells were treated with the different drug for 48 h. The treated and control cells were lysed in a RIPA lysis buffer containing protease inhibitors. Each concentration repeated two times and the experiment was carried out three independent times. The activity was calculated relative to the corresponding protein content.

Protein concentration assay

Protein concentrations were measured in the medium and cell lysate by the method described previously by Bradford [18] using kit (Pierce, Rockford, IL, USA). The method depends on the binding of Comassie Brilliant Blue G-250 dye with protein and forming a complex which can be measured spectrophotometrically at 595 nm then the concentration was determined using a standard calibration curve.

Assay of VEGF-A level

VEGF was determined in cell culture medium using eBioscience (San Diego, CA, USA) ELISA kit. MCF-7 and T47D cells were plated in 6 well plate with 5*10 4 / well. After treatment with drugs, the medium was aspirated, centrifuged at 10,000 rpm for 10 min at 4 °C to remove any dead cells. The clear supernatant was used for assay following the manufacturer’s instructions [19].

Determination of matrix metalloproteinases (MMP)-2 and 9 activities by gelatin zymography

Cells were seeded in 75 cm 3 flasks, left for 24 h, and then treated with TAM, 3 BP, or their combination for 48 h. Cells were harvested and protein concentration of each sample was determined by Bradford method [18]. Briefly, 20 μg protein /lane was prepared in a non-reducing loading buffer consisting of 63 mM Tris–HCl pH 6.8, 10 % glycerol (v/v), 2 % sodium dodecyl sulphate (SDS) (w/v), 0.0025 % bromophenol blue (w/v), and electrophoresed on 10 % SDS-polyacrylamide gels containing 0.1 % gelatin. After electrophoresis, SDS was removed from gels by incubation with renaturation buffer (2.7 % TritonX-100) for 1 h, then incubated for 24 h at 37 °C in developing buffer (50 mM Tris–HCl, pH 7.5, 0.2 M NaCl, Triton-X 5 ml and 5 mM CaCl 2 ), stained with coomassie brilliant blue and destained using destaining solution (10 % methanol, 5 % acetic acid). Enzyme-digested regions were observed as clear bands against a dark blue background. Gels were scanned using image Scanner III LabScan6.0 and the subsequent. In order to determine mean intensity of each band (mean pixel), the band densities were measured with Scion Image Beta 4.0.2 (Scion Co., Frederick, MD, U.S.A.) software. For the quantitative analysis, each of the bands was compared with β-Actin taken as a control.

Western blot

Cells were washed twice with PBS and lysed in cell lysis buffer (150 mM NaCl,10 mM Tris, 0.2 %TritonX-100, 0.3 %nonylphenoxy-polyethoxyethanol-40, 0.2 %mM Na 3 VO 4 , protease inhibitor cocktail). The cell lysates were centrifuged and the protein concentration was measured as previously mentioned. Each sample was separated by electrophoresis using 8 % SDS-PAGE gel and analyzed by Western blotting using the following antibodies: primary rabbit anti-human MMP-9 (Novusbio, Colorado, USA), and β-HK2 (Cell signaling, Beverly, Massachusetts, USA), as well as primary mouse anti-human HIF-1α (eBioscience, CA, USA), MMP-2 (Invitrogen, CA, USA), caspase-7 (Novusbio, Colorado, USA), and β-Actin (Sigma-Aldrich Chemical Co., USA). Horseradish peroxidase linked to the corresponding secondary antibody was used at 1:5000 dilution. The membrane was visualized by exposure to Kodak XAR film. For the quantitative analysis, the mean intensity of each band (mean pixel), was compared with β-Actin band using with Scion Image Beta 4.0.2 (Scion Co., MD, U.S.A.) software.

Oxidative stress markers (reduced glutathione and lipid peroxide)

In order to explore the role of oxidative stress in drug – induced cytotoxicity, levels of lipid peroxide and reduced glutathione (rGSH were determined. Glutathione content was determined according to the method of Ellman [20]. The treated and control cells were collected in phosphate buffer, protein was precipitated with trichloroacetic acid (TCA) and centrifuged. The supernatant was treated with Ellman’s reagent, the developed color was measured spectrophotometrically at 405 nm using a spectrophotometer (Spectronic, Milton Ray Co., USA). Lipid peroxidation products were quantified by measuring malonaldialdehyde (MDA) level to the method described by Draper and Hadley [21]. Treated and control cells were mixed well with of 20 % (w/v) trichloroacetic acid (TCA) containing 0.8 % (w/v) thiobarbituric acid (TBA), incubated in a boiling water bath for 1 h. The absorbance of the supernatant was determined at 535 nm using a spectrophotometer (Spectronic, Milton Ray Co., USA). The concentrations were calculated using MDA standard calibration curve by preparing a serial dilutions of 1,1,3,3- tetraethoxypropane.

In-vivo parameters

Assessment of the antitumor activity in mice-bearing solid Ehrlich carcinoma (EAC)

Ehrlich carcinoma (EAC)-cells (2 × 10 6 ) were transplanted subcutaneously in the right thigh of the lower limb mice. 24 Mice with a palpable tumor mass (approximate 100 mm 3 ), which developed within 7 days after implementation, were divided randomly and blindly into 4 groups each 6 animals. Group one injected i.p with 5 mg/kg TAM, group two injected with 3-BP (10 mg/kg), group three treated with their combination and control group received saline. Treatment continued twice/weekly for 3 weeks. The change in tumor volume was measured using venire caliber and calculated by the following formula according to Osman et al. [22].

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Where A and B denote the minor and major tumor axis, respectively.

Reduced glutathione (rGSH) and MDA contents in solid tumor tissue

Twenty four hours after the last treatment, animals were anesthetized with sodium pentobarbital 100 mg/kg i.p, then cervical dislocation was done with high degree of proficiency to anesthetized animals according to Euthanasia guidelines. Tumors were quickly excised, washed with saline, blotted with a piece of filter paper, and homogenized using a Branson sonifier (250, VWR Scientific, Danbury, Connecticut, USA). The homogenates were centrifuged at 800 g for 5 min at 4 C° to separate the nuclear debris, then supernatant was again centrifuged at 10,500 g for 20 min at 4 C°. Levels of glutathione and MDA were determined as previously described.

Immunohistochemical staining (IHC) of VEGF

Representative tissue samples were fixed in 10 % neutral phosphate-buffered formalin, embedded in paraffin, and sectioned at 5 μm thickness. Sections were incubated with monoclonal mouse anti-VEGF antibody (Sigma Aldrich Chemical Co., USA) as a primary antibody at a dilution of 1:150 overnight at 4 °C then rinsed three times. Sections were incubated with polymer horseradish peroxidase HRP secondary antibody (Sigma Aldrich Chemical Co., USA) for 1 h. Immuno-reactivity was detected by the standard avidin–biotin immunoperoxidase method. Counterstaining with Meyer’s hematoxylin was then performed for 5 min. Thereafter, they were evaluated under light microscope (Olympus, Japan) and analyzed with Scion Image Beta 4.0.2 (Scion Co., Frederick, MD, U.S.A.) software.

Statistical analysis

All data were expressed as mean ± S.D. The difference between the treated samples and the untreated controls was analyzed by one way ANOVA followed by Tukey multiple comparison test in which p < 0.05 was considered as significant. To test for interaction between individual treatments when given in combination, a factorial design test is used. All statistical analysis was performed using GraphPad In Stat, version 5.0 (GraphPad, San Diego, California, USA). Compusyn software was used to determine the interaction between the two drugs in the combination. Statistical significance was set at p < 0.05.


In vitro

3-BP enhances cytotoxicity of TAM on MCF7 and T47D cells

Figure 1 showed that treatment of MCF7 [A] and T47D [B] cells with various concentrations (10–50 μM) of TAM or 3-BP for 48 h caused a concentration dependent decrease in cell survival. The IC 50 of TAM was 20 and 23 μM, while that of 3-BP was 36 and 33 μM in MCF7 and T47D, respectively. Addition of 20 μM of 3BP increased significantly cytotoxicity of 20 μM TAM in MCF-7 cells, while, T47D cells required 30 μM of 3BP to produce significant increase in cell death compared to TAM alone (Fig. 2a and b).

thumbnailFig. 1. Cytotoxicity of TAM and 3-BP on MCF7 and T47D breast cancer cell lines after 48 h. Surviving fraction and I.C 50 of MCF-7 (a) and T47D (b), cells treated with TAM and 3-BP after 48 h. Results are expressed as the mean ± SD of 5 independent experiments performed in triplicate. * Significantly different from control at P < 0.05

thumbnailFig. 2. Effect of addition of 3-BP on the cytotoxicity of 20 μM TAM on MCF-7 and T47D cell lines. Cells were treated with different concentrations of 3-BP and 20 μM TAM (a, b, respectively). Results are expressed as the mean ± SD of 5 independent experiments performed in triplicate. The statistical significance of the results was analyzed by one way ANOVA using Tukey multiple comparison test using one way analysis of variance (ANOVA). “ a ” Significantly different from its control and “ b ” from 20 μM TAM at P < 0.05

3-BP synergizes oxidative stress and activates apoptotic machinery of TAM on MCF7 and T47D cells

Both TAM and 3-BP increased significantly the MDA level (Fig. 3a, b), but leveled off the rGSH content (Fig. 3c, d) significantly in the two breast cancer cell lines. The addition of 3-BP to TAM caused synergistic effect on the oxidative stress (lipid peroxidation) in both cell lines and a synergistic effect on glutathione content in MCF-7 but in T47D cells, the interaction was potentiation. Treatment of breast cell lines with TAM, 3-BP and their combination has switched on the apoptotic activity assessed as caspases 3, 7 and 9. The effect of the different treatment regimens had activated caspase-3 (Fig. 4a, b), with the 3-BP showing the least effect and the combined treatment showing the highest action with synergistic interaction. The same pattern was mirrored in the 2 cell lines. The same effect was observed on the expression of caspase-9 (Fig. 4c, d) but the interaction was synergistic on MCF-7 and potentiation on T47D cells. Additionally, the three treatments succeeded to cleave caspase-7 as shown in (Fig. 4e, f) using western blot.

thumbnailFig. 3. Oxidative stress markers following treatment with TAM, 3-BP and their combination. Effect of different regimen on lipid peroxidation in MCF-7 (a) and T47D (b). Figure (c) and (d) show the content of reduced glutathione (rGSH) in MCF-7 and T47D, respectively after 48 h treatment with 3-BP, TAM and their combination. Results are expressed as means ± SD of 2 independent experiments performed in duplicates. Statistical significance of results was analyzed by one way ANOVA using Tukey’s multiple comparison test. “ a ” Significantly different from control, “ b ” from 3-BP and “ c ” from TAM at P ≤ 0.05. means synergistic and * means potentiating interaction when TAM and 3-BP where combined using factorial design

thumbnailFig. 4. Effect of 48 h treatment with 3-BP, TAM and their combination on apoptosis markers. Caspase-3 activity in MCF-7 cells (a) and T47D cells (b). Expression of Caspase −9 gene using qPCR in MCF-7 (c) and T47D (d). Caspase 7 protein level was done by western in MCF-7 (e) and T47D (f). Results are expressed as means ± SD of 2 independent experiments performed in duplicates. Significance was determined with one way ANOVA using Tukey’s multiple comparison test. “ a ” Significantly different from control, “ b ” rom 3-BP and “ c ” from TAM at P ≤ 0.05. means synergistic and * potentiation interaction when TAM and 3-BP where combined using factorial design

Combined treatment of TAM and 3BP inhibits VEGF-A, HIF-1α, HK-2 and metalloproteinases 2, 9

As depicted in Fig. 5a, b, VEGF-A activity was inhibited by the combined regimen showing the best effect with synergistic interaction on MCF-7 and potentiating interaction on T47D. Regarding the effect on the HIF-1α expression (Fig. 6a, b), TAM and/or 3-BP showed the same previous pattern with a more pronounced effect on the MCF-7 cell line. Nevertheless, these results were not reflected exactly on the HIF-1α protein content assessed by the western blot technique (Fig. 6c, d) as the interaction was synergistic in the expression level but it was potentiation one in protein level. The expression and the protein level of HK2 were presented in Fig. 7a-d. As expected the inhibitory effect of 3-BP on the HK2 surpassed that of TAM alone in the 2 breast cell lines studied herein. Despite the combination effect added a further inhibition in the HK2 expression as compared to the 3-BP alone with synergistic interaction, however, this effect was lost in the protein verification (Fig. 7c, d). TAM increased MMP 2 and 9. Surprisingly, 3-BP caused a sharp decline in the MMPs in the two breast cell lines to reach even a lower level below the untreated control group. The combination regimen succeeded to lower the TAM effect on the secreted MMP 2 and 9 (Fig. 8a, b); the same pattern was observed by the q-PCR technique (Fig. 8c, d) and the Western blot assay (Fig. 9a-d) with antagonistic interaction.

thumbnailFig. 5. Levels of VEGF in breast cancer cell lines following treatment with 3-BP, TAM and their combination. Effect of TAM, 3-BP and their combination on level of VEGF-A in the MCF-7 (a) and T47D (b) cells media. Results are expressed as means ± SD of 2 independent experiments performed in duplicates. Significance was determined with one way ANOVA using Tukey’s multiple comparison test. “ a ” Significantly different from control, “ b ” rom 3-BP and“ c ” from TAM at P ≤ 0.05. means synergistic and * potentiation interaction when TAM and 3-BP where combined using factorial design

thumbnailFig. 6. Effect of TAM, 3-BP and their combination on the level HIF-1α. The expression level of HIF-1α in MCF-7 and T47D cells (a, b). The effect of different treatments on the protein level (c, d). Results are expressed as means ± SD of 2 independent experiments performed in duplicates for qPCR experiment. The results for western blot are expressed as means ± SD of 3 independent experiments. Significance was done by one way ANOVA using Tukey’s multiple comparison test. “ a ” Significantly different from control, “ b ” from 3-BP and “ c ” from TAM at P ≤ 0.05. means synergistic and * potentiation interaction when TAM and 3-BP where combined using factorial design

thumbnailFig. 7. Effect of TAM, 3-BP and their combination on the level Hexokinaes-2 (HK-2). HK-2 gene expression of 3-BP, TAM and the combination regimen (a, b). The effect of different treatments on the HK-2 protein level. (c, d). Results are expressed as means ± SD of 2 independent experiments performed in duplicates for qPCR experiment and for western blot the results are expressed as means ± SD of 3 independent experiments. Significance was done by one way ANOVA using Tukey’s multiple comparison test. “ a ” Significantly different from control, “ b ” from 3-BP and “ c ” from TAM at P ≤ 0.05. means synergistic and * potentiation interaction when TAM and 3-BP where combined using factorial design

thumbnailFig. 8. Effect of TAM, 3-BP and their combination on the extracellular level and the expression of Metastasis markers. After adding 3-BP to TAM succeeded to decrease the extracellular level of MMP-2 and 9 using gelatin zymography in MCF-7 cells (a) and T47D (b) cells. The analysis was done by image software. The effect of this combination on the secreted MMPs was reflected on their genes expression using qPCR in MCF-7 (c) and T47D (d). Results are expressed as means ± SD of 2 independent experiments for zymography but for qPCR results are expressed as means ± SD of 2 independent experiments performed in duplicates. Significance was determined with one way ANOVA using Tukey’s multiple comparison test.“ a ” Significantly different from control, “ b ” from 3-BP and “ c ” from TAM at P ≤ 0.05. Significant interaction (antagonism) when TAM and 3-BP where combined using factorial design

thumbnailFig. 9. Effect of TAM, 3-BP and their combination on the protein level of the Metastasis markers. The results of zymography and qPCR were confirmed also by western technique for MMP-2 and 9 in the cells of MCF-7 (a, c) and T47D (b, d). Results are expressed as means ± SD of 2 independent experiments western. The analysis was done by image software. Significance was determined with one way ANOVA using Tukey’s multiple comparison test.“ a ” Significantly different from control, “ b ” from 3-BP and “ c ” from TAM at P ≤ 0.05. Significant interaction (antagonism) when TAM and 3-BP where combined using factorial design

In vivo

3-BP enhances the antitumor effect, increases oxidative stress and inhibits VEGF of TAM in vivo

The results of in vitro are also documented in vivo, the volume of Ehrlich tumor was decreased by 52 and 37 % in individually treated TAM or 3-BP respectively; however, the combination regimen caused a further decrease reaching 80 % as compared to the control untreated group (Fig. 10). An increase in MDA and decrease rGSH with synergistic interaction in the combination using factorial design was also observed (Fig. 11a, b). Moreover, as presented in Fig. 12a-d, all the treatment regimens lowered the level of VEGF expression to different extent when compared to the control group. Moreover, in the combination treated group the expression was even less than either treatment alone.

thumbnailFig. 10. Tumor volume of solid Erlich carcinoma-bearing mice after treatment with 3-BP, TAM or their combination. The tumor volume was markedly reduced in mice treated 3-BP (10 mg/kg), TAM (5 mg/kg); however the best result was observed in group treated with combination of both drugs. Results are expressed as means ± SD of tumor volume from 6 mice. Results are analyzed by one way ANOVA using Tukey’s multiple comparison test. “ a ” Significantly different from control,“ b ” from 3-BP and “ c ” from TAM at P < 0.05. Significant interaction when TAM and 3-BP where combined using factorial design

thumbnailFig. 11. Effect of TAM, 3-BP and their combination on oxidative stress in vivo. a shows the level of the Glutathione level and b shows the lipid peroxidation following treatment with 3-BP and TAM and their combination. Results are expressed as means ± SD of tumor volume from 6 mice. Results are analyzed by one way ANOVA using Tukey’s multiple comparison test. “ a ” Significantly different from control,“ b ” significantly different from 3-BP and “ c ”significantly different from TAM at P < 0.05. Significant interaction when TAM and 3-BP where combined using factorial design

thumbnailFig. 12. VEGF expression using immunohistochemsitry. a: Paraffin section photograph of mouse solid tumor (control group) showing the increased VEGF expression in most tumor cells, the large capillaries and hyperchromatic cells were present (4/5 of the field). b: Paraffin section photograph of mouse solid tumor (group treated with 10 mg/kg3-BP) showing the VEGF expression in most tumor cells absent the apoptotic cells (3/5 of the field). c: Paraffin section photograph of mouse solid tumor (group treated with 5 mg/kg) showing the VEGF expression in some tumor cells, absent in mitotic and apoptotic cell, large conjugated blood vessels were observed. (1/5 of the field). d: Paraffin section photograph of mouse solid tumor (group treated with 3-BP and TAM) showing the VEGF expression in some tumor cells (1/5 of the field)


Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer-related deaths among females worldwide [1]. ER status is the most important and primary determinant of treatment options through targeting ER functions by TAM or synthesis by aromatase inhibitors [23]. TAM is the first endocrine therapy; it acts as an antagonist for estrogen receptors in pre and postmenopausal breast cancer by controlling the binding of estradiol to the ER and forms a TAM-ER complex which then binds to DNA. This leads to the failure of transcriptional activation and growth inhibition in estrogen-dependent cells [5].

Our data showed either TAM or 3BP alone or in combination inhibited the survival of breast cancer cell lines as well as in mice bearing EAC tumor. The combination regimen enhanced significantly the growth inhibition both in vitro and in vivo. TAM was reported as effective anticancer against many types of cancer other than breast including hepatocellular carcinoma, lung cancer [24], [25] and colon cancer [26] cell lines. The in vitro findings were further elucidated in an in vivo model of EAC bearing mice. The present study showed that TAM and 3-BP can reduce the volume of solid tumor in mice bearing tumor. Several studies have also documented the antitumor effect of TAM and 3BP in vivo[13], [50]. Moreover, the combination of both drugs reduced the tumor significantly from TAM or 3-BP treated groups. It increases the level of p53 which is responsible for activation of many genes to induce apoptosis [27]. In addition, TAM causes induction of c-Myc, activation of members of mitogen-activated protein kinase (MAPK) family as well as increased accumulation of ceramide which serves as a second messenger in cell survival [28]. Moreover, 3-Bromopyruvate (3-BP) is a promising glycolytic inhibitor, in this study; it increases significantly the cytotoxicity of TAM. 3BP was found to have anticancer effects on many types of cancer including; leukemia [29], breast cancer cell line and hepatocellular carcinoma [30]. This may be related to the ability of 3BP to act as multi-targeted inhibitor of glycolytic pathway and mitochondria. It covalently binds to the glycolytic enzymes; hexokinase-2 [31], Glyceraldehyde-3-phosphate dehydrogenase [32] and mitochondrial; succinate dehydrogenase [33], in addition, to the endoplasmic reticulum [27] and the lysosomes [32] resulting in severe depletion in ATP and cancer death [34].

The antitumor effects of TAM observed in this study, was accompanied by significant increase in ROS and activation of different caspases at both m RNA and protein levels resulting in induction of apoptosis. Additionally, both the individual drug and combination treated mice showed increase in the oxidative stress markers in vivo. TAM increases mitochondria oxidative stress markers in vitro and in vivo[35] and induces collapse of mitochondrial transmembrane potential [36] that triggers release of cytochrome c from mitochondria which activates pro-caspase-9,7 and 3 leading to apoptosis [37]. In addition, oxidative stress increases intracellular Ca 2+ concentrations leading to leak in the plasma membrane [38] and activation of endonucleases which degrade DNA and, ultimately, contribute to cell death [39].

In our study the apoptotic effect of TAM is enhanced upon its combination with 3BP in both breast cancer cell lines compared to each drug individually. 3-Bromopyruvate, as a member of the mitocans, it exerts its pro-apoptotic mechanism on cells via disruption the mitochondria membrane potential causing the generation of mitochondrial ROS [40]. One of the major consequences of the disruption of the mitochondrial membrane potential by reactive oxygen species (ROS) is the release of the cytochrome c [41] in the cytosol and initiation of the caspase cascade by activating pro-caspase-9. Mature caspase-9 then activates the executioner caspases including caspase-3which is a point of no return in apoptosis. Caspase-3 then cleaves a variety of vital biological macromolecules [42].

As the tumor cells proliferate, they are subjected to hypoxia and undergo biological changes to adapt themselves to the hypoxic conditions. HIF-1α mediates many of the changes, [43] which regulate many genes in glycolytic pathway as hexokinase-2 (HK2) which plays a key step in glycolysis and angiogenesis as (VEGF) [44]. HIF-1α has been reported to be over expressed in various malignant tumors and cancer cell lines [45]. In our study TAM, 3BP as well as their combination inhibited the expression and the protein content of HIF-1 α with concomitant inhibition of HK and VEGF. However, the expression levels of both HIF-1 α and HK of the combined treatment in both cell lines showed synergistic effect which did not appear in the protein level carried out by Western blotting. Such difference between mRNA expression and protein level may be due to several biological and methodological constraints that play a role when comparing mRNA to protein levels [46]. The most prominently influences the correlation between mRNA and protein are the translation efficiency or protein half-life. Individual protein half-lives range from several seconds to tens of hours [47], a more than 1000-fold range. Hence protein turnover is probably influencing the correlation between mRNA and protein abundances to a greater degree. Minor effects are attributed translation initiation, start codon, stop codon and stop codon context [48] and [49]. HIF-1α plays an important role in tumor angiogenesis and high levels of HIF-1 α can predict an early relapse and metastatic disease [50]. Additionally, HIF-1α overexpression is associated with increased VEGF expression in many different types of cancer such as; breast cancer, colon cancer and hepatocellular carcinoma [51]. However, the role of estrogens and tamoxifen and in the clinic and HIF-1 α modulation in breast cancer is unclear [52]. 3-BP was reported to decrease the level of HIF-1 α [53] and covalently binds to HK-2, causing its dissociation from VDAC [31]. 3BP causes cancer cell death by rapid depletion of ATP and suppresses tumor growth in animal model [54]. The combined treatment produced significant decrease in VEGF compared to either drug alone, the effect was synergistic in MCF7, while it was additive in T47D. This difference in drug interaction between the two types could be attributed to the aggressive nature of T47D compared to MCF7. According to their biological functions, the proteins involved in cell growth stimulation, anti-apoptosis mechanisms and carcinogenesis are more strongly expressed in T47D than in MCF7 [55]. Moreover, in vivo the expression level of VEGF was decreased in mice treated combination regimen significantly compared to TAM or 3-BP treated groups.

Metalloproteinase degrade extracellular matrix components enabling tumor cell invasion and metastasis. It was found that estradiol and TAM regulate MMP-2, MMP-9 and extracellular endostatin in ER + PR + human breast cancer cells and in vivo[56], [57]. A significant increase of intracellular and secreted protein levels upon TAM exposure whereas, estradiol induced a significant decrease [56]. The authors suggested a possible role of MMP in regulation the bioavailability of a variety of biologically active molecules such as anti-angiogenic fragments, which may be beneficial for the host [56], [57]. Surprisingly in this study, 3-BP caused a sharp decline in the MMPs in the two breast cancer cell lines, contrary to TAM which caused increased levels of both enzymes. However, the combination regimen succeeded to lower the increased effect of TAM by all tested techniques (Fig. 7a-h) with antagonistic interaction. Therefore TAM and 3BP the ability to modulate MMP-2/MMP-9 activity and VEGF levels in human breast cancer in vitro.


3-BP is a promising antitumor, it improved antitumor effect of TAM on breast cancer cell lines and in mice bearing-Ehrlish carcinoma. The combination regimen increases the antitumor effect via activation of apoptotic machinery, decreases angiogenesis markers HIF, HK2 and VEGF. Moreover 3BP modulates MMPs 2 and 9 which makes its combination with TAM promising treatment to be applied clinically.


3-BP: 3-Bromopyruvate

BC: Breast cancer

EAC: Ehrlish ascites carcinoma

ER+: Estrogen receptor positive

HIF: Hypoxia inducing factor

HK2: Hexokinase2

MDA: Malondialdehyde

MMPs: metalloproteinases

rGSH: Reduced glutathione

ROS: Reactive Oxygen Species

TAM: Tamoxifen

VEGF: Vascular endothelial growth factor

Competing interests

The authors declare no conflict of interest.

Authors’ contribution

YA performed the experimental work and drafted the paper. HE participated in analysis of data and revision of the manuscript. MM provided some of reagents and chemicals to conduct the experiments and provided the data analysis tools. SS designed the experimental work, analyzed the data and revised the manuscript. All authors contributed to revise the manuscript and approved it.


This research received no specific grant from any funding agency in the public, commercial, or non-profit sectors.


  1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al.: GLOBOCAN 2012 v1.0, cancer incidence and mortality worldwide: IARC cancer base no. 11. International Agency for Research on Cancer, Lyon, France; 2013.

    Available from


  2. United Nations, Department of Economic and Social Affairs, Population Division. 2013; UN World Population Prospects: The 2012 Revision..


  3. Jordan VC: Tamoxifen: the herald of a new era of preventive therapeutics.

    J Natl Cancer Inst 1997, 89:747-9. PubMed Abstract | Publisher Full Text OpenURL

  4. Davies C, Pan H, Godwin J, Gray R, Arriagada R, Rania V, et al.: Adjuvant Tamoxifen: Longer Against Shorter (ATLAS) Collaborative Group: Long-term effects of continuing adjuvant tamoxifen to 10 years versus stopping at 5 years after diagnosis of oestrogen receptor-positive breast cancer: ATLAS, a randomised trial.

    Lancet. 2013, 381:805-16. PubMed Abstract | Publisher Full Text OpenURL

  5. Aesoy R, Sanchez BC, Norum JH, Lewensohn R, Viktorsson K, Linderholm B: An autocrine VEGF/VEGFR2 and p38 signaling loop confers resistance to 4-hydroxytamoxifen in MCF-7 breast cancer cells.

    Mol Cancer Res 2008, 6:1630-8. PubMed Abstract | Publisher Full Text OpenURL

  6. Lagadec C, Adriaenssens E, Toillon RA, Chopin V, Romon R, Van Coppenolle F, et al.: Tamoxifen and TRAIL synergistically induce apoptosis in breast cancer cells.

    Oncogene 2008, 27:1472-7. PubMed Abstract | Publisher Full Text OpenURL

  7. Gonzalez-Angulo AM, Morales-Vasquez F, Hortobagyi GN: Overview of resistance to systemic therapy in patients with breast cancer.

    Adv Exp Med Biol 2007, 608:1-22. PubMed Abstract | Publisher Full Text OpenURL

  8. Peralta EA, Viejas ML, Louis S, Engle DL, Dunnington GL: Effect of vitamin E on tamoxifen-treated breast cancer cells.

    Surgery 2006, 140:607-14. PubMed Abstract | Publisher Full Text OpenURL

  9. Sartippour M, Pietras R, Marquez-Garban D, Chen H, Heber D, Henning S, et al.: The combination of green tea and tamoxifen is effective against breast cancer.

    Carcinogenesis 2006, 27:2424-33. PubMed Abstract | Publisher Full Text OpenURL

  10. Zhao Y, Liu H, Liu Z, Ding Y, Ledoux SP, Wilson GL, et al.: Overcoming trastuzumab resistance in Breast cancer by targeting dysregulated glucose metabolism.

    Cancer Res 2011, 71:4585-97. PubMed Abstract | Publisher Full Text OpenURL

  11. Seyfried TN, Shelton LM: Cancer as a metabolic disease.

    Nutr Metab 2012, 7:7-29. BioMed Central Full Text OpenURL

  12. Pelicano H, Martin DS, Xu RH, Huang P: Glycolysis inhibition for anticancer treatment.

    Oncogene 2006, 25:4633-46. PubMed Abstract | Publisher Full Text OpenURL

  13. Mathupala S, Ko Y, Pedersen P: Hexokinase-2 bound to mitochondria: cancer’s stygian link to the ‘Warburg effect’ and a pivotal target for effective therapy.

    Semin Cancer Biol 2009, 19:17-24. PubMed Abstract | Publisher Full Text OpenURL

  14. Ko Y, Smith B, Wang Y, Pomper M, Rini D, Torbenson M, et al.: Advanced cancers: eradication in all cases using 3-bromopyruvate therapy to deplete ATP.

    Biochem Biophys Res Commun 2004, 324:269-75. PubMed Abstract | Publisher Full Text OpenURL

  15. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al.: New colorimetric cytotoxicity assay for anticancer drug screening.

    J Natl Cancer Inst 1990, 82:1107-12. PubMed Abstract | Publisher Full Text OpenURL

  16. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2 − ΔΔCT.

    Methods 2001, 25:402-8. PubMed Abstract | Publisher Full Text OpenURL

  17. Casciola-Rosen L, Nicholson DW, Chong T, Rowan KR, Thornberry NA, Miller DK, et al.: Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death.

    J Exp Med 1996, 183:1957-64. PubMed Abstract | Publisher Full Text OpenURL

  18. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, j.

    Anal Biochem 1976, 72:248-54. PubMed Abstract | Publisher Full Text OpenURL

  19. Kim KJ, Li B, Houck K, Winer J, Ferrara N: The vascular endothelial growth factor proteins: Identification of biologically relevant regions by neutralizing monoclonal antibodies.

    Growth Factors 1992, 7:53-64. PubMed Abstract | Publisher Full Text OpenURL

  20. Ellman GL: Tissue sulfhydryl groups.

    Arch Biochem Biophys 1959, 82:70-7. PubMed Abstract | Publisher Full Text OpenURL

  21. Draper HH, Hadley M: Malondialdehyde determination as index of lipid peroxidation.

    Methods Enzymol 1990, 186:421-31. PubMed Abstract | Publisher Full Text OpenURL

  22. Osman AM, Sayed-Ahmed MM, Khayal MT, El-Merzabani MM: Hyperthermic potentiation of cisplatin on solid Ehrlich carcinoma.

    Tumori 1993, 79:268-72. OpenURL

  23. Zhang MH, Man HT, Zhao XD, Dong N, Ma S: Estrogen receptor-positive breast cancer molecular signatures and therapeutic potentials.

    Biomed Rep 2013, 2(1):41-52. PubMed Abstract | Publisher Full Text OpenURL

  24. Nowak AK, Stockler MR, Chow PK, Findlay M: Use of tamoxifen in advanced-stage hepatocellular carcinoma. A systematic review.

    Cancer 2005, 103:1408-14. PubMed Abstract | Publisher Full Text OpenURL

  25. Shen H, Yuan Y, Sun J, Gao W, Shu YQ: Combined tamoxifen and gefitinib in non-small cell lung cancer shows antiproliferative effects.

    Biomed Pharmacother 2010, 64:88-96. PubMed Abstract | Publisher Full Text OpenURL

  26. Lee YH, Kang BS, Bae YS: Premature senescence in human breast cancer and colon cancer cells by tamoxifen-mediated reactive oxygen species generation.

    Life Sci 2014, 97:116-22. PubMed Abstract | Publisher Full Text OpenURL

  27. Han P, Kang JH, Li HL, Hu SX, Lian HH, Qiu PP, et al.: Antiproliferation and apoptosis induced by tamoxifen in human bile duct carcinoma QBC939 cells via upregulated p53 expression.

    Biochem Biophys Res Commun 2009, 385:251-6. PubMed Abstract | Publisher Full Text OpenURL

  28. Mandlekar S, Kong AN: Mechanisms of tamoxifen-induced apoptosis.

    Apoptosis 2001, 6:469-77. PubMed Abstract | Publisher Full Text OpenURL

  29. Akers LJ, Fang W, Levy AG, Franklin AR, Huang P, Zweidler-McKay PA: Targeting glycolysis in leukemia: a novel inhibitor 3-BrOP in combination with rapamycin.

    Leuk Res 2001, 35:814-20. Publisher Full Text OpenURL

  30. Ganapathy-Kanniappan S, Geschwind JF, Kunjithapatham R, Buijs M, Syed LH, Rao PP, et al.: Bromopyruvate induces endoplasmic reticulum stress, overcomes autophagy and causes apoptosis in human HCC cell lines.

    Anticancer Res 2010, 30:923-35. PubMed Abstract | Publisher Full Text OpenURL

  31. Chen Z, Zhang H, Lu W, Huang P: Role of mitochondria-associated hexokinase-II in cancer cell death induced by 3-bromopyruvate.

    Biochim Biophys Acta 1787, 2009:553-60. OpenURL

  32. Dell’Antone P: Targets of 3-bromopyruvate, a new, energy depleting, anticancer agent.

    Med Chem 2009, 5:491-6. PubMed Abstract | Publisher Full Text OpenURL

  33. Da-Silva WS, Gomez-Puyou A, de Gomez-Puyou MT, Moreno-Sanchez R, De Felice FG, de Meis L, et al.: Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria.

    Biol Chem 2004, 279:39846-55. Publisher Full Text OpenURL

  34. Pedersen PL: Voltage dependent anion channels (VDACs): a brief introduction with a focus on the outer mitochondrial compartment’s roles together with hexokinase-2 in the “Warburg effect” in cancer.

    J Bioenerg Biomembr 2008, 40:123-6. PubMed Abstract | Publisher Full Text OpenURL

  35. Nazarewicz RR, Zenebe WJ, Parihar A, Larson SK, Alidema E, Choi J, et al.: Tamoxifen induces oxidative stress and mitochondrial apoptosis via stimulating mitochondrial nitric oxide synthase.

    Cancer Res 2007, 67:1282-90. PubMed Abstract | Publisher Full Text OpenURL

  36. Hirsch T, Decaudin D, Susin SA, Marchetti P, Larochette N, Resche-Rigon M, et al.: PK11195, a ligand of the mitochondrial benzodiazepine receptor, facilitates the induction of apoptosis and reverses Bcl-2-mediated cytoprotection.

    Exp Cell Res 1998, 241:426-34. PubMed Abstract | Publisher Full Text OpenURL

  37. Tuquet C, Dupont J, Mesneau A, Roussaux J: Effects of tamoxifen on the electron transport chain of isolated rat liver mitochondria.

    Cell Biol Toxicol 2001, 16:207-19. Publisher Full Text OpenURL

  38. Huang CC, Cheng HH, Lin KL, Cheng JS, Tsai JY, Liao WC, et al.: Tamoxifen-induced [Ca2+]i rise and apoptosis in corneal epithelial cells.

    Toxicology 2009, 255:58-64. PubMed Abstract | Publisher Full Text OpenURL

  39. Annunziato L, Amoroso S, Pannaccione A, Cataldi M, Pignataro G, D’Alessio A, et al.: Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions.

    Toxicol Lett 2003, 139:125-33. PubMed Abstract | Publisher Full Text OpenURL

  40. Neuzil J, Dong LF, Rohlena J, Truksa J, Ralph SJ: Classification of mitocans, anti-cancer drugs acting on mitochondria.

    Mitochondrion 2013, 13:199-208. PubMed Abstract | Publisher Full Text OpenURL

  41. Gogvadze V, Orrenius S, Zhivotovsky B: Multiple pathways of cytochrome c release from mitochondria in apoptosis.

    Biochim Biophys Acta 1757, 2006:639-47. OpenURL

  42. Chandra J, Orrenius S: Mitochondria, oxygen metabolism and the regulation of cell death.

    Intern Congress Series 2002, 1233:259-72. Publisher Full Text OpenURL

  43. Greijer AE, van der Groep P, Kemming D, Shvarts A, Semenza GL, Meijer GA, et al.: Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1).

    J Pathol 2005, 206:291-304. PubMed Abstract | Publisher Full Text OpenURL

  44. Kim W, Yoon JH, Jeong JM, Cheon GJ, Lee TS, Yang JI, et al.: Apoptosis-inducing antitumor efficacy of hexokinase II inhibitor in hepatocellular carcinoma.

    Mol Cancer Ther 2007, 6:2554-62. PubMed Abstract | Publisher Full Text OpenURL

  45. Keith B, Johnson RS, Simon MC: HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression.

    Nat Rev Cancer 2012, 12:9-22. OpenURL

  46. Semenza G: HIF-1: upstream and downstream of cancer metabolism.

    Curr Opin Genet Dev 2010, 20:51-6. PubMed Abstract | Publisher Full Text OpenURL

  47. Maier T, Güell M, Luis SL: Correlation of mRNA and protein in complex biological samples.

    FEBS Lett 2009, 583:3966-73. PubMed Abstract | Publisher Full Text OpenURL

  48. Doherty MK, Hammond DE, Clague MJ, Gaskell SJ, Beynon RJ: Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC.

    J Proteome Res 2009, 8:104-12. PubMed Abstract | Publisher Full Text OpenURL

  49. Lithwick G, Margalit H: Hierarchy of sequence-dependent features associated with prokaryotic translation.

    Genome Res 2003, 13:2665-73. PubMed Abstract | Publisher Full Text OpenURL

  50. Nie L, Wu G, Zhang W: Correlation of mRNA expression and protein abundance affected by multiple sequence features related to translational efficiency in Desulfovibrio vulgaris: a quantitative analysis.

    Genetics 2006, 174:222-43. Publisher Full Text OpenURL

  51. Mahon PC, Hirota K, Semenza GL: FIH-1. a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity.

    Genes Dev 2001, 15:2675-86. PubMed Abstract | Publisher Full Text OpenURL

  52. Kazi AA, Jones JM, Koos RD. Chromatin immunoprecipitation analysis of gene expression in the rat uterus in vivo: estrogen-induced recruitment of both estrogen receptor alpha and hypoxia inducible factor 1 to the vascular endothelial growth factor promoter. Mol Endocrinol. 2005;19:1–19.
  53. Xiao H, Li S, Zhang D, Liu T, Yu M, Wang F: Separate and concurrent use of 2-deoxy-D-glucose and 3-bromopyruvate in pancreatic cancer cells.

    Oncol Rep 2013, 29:329-34. PubMed Abstract | Publisher Full Text OpenURL

  54. Buijs M, Wijlemans JW, Kwak BK, Ota S, Geschwind JF: Antiglycolytic therapy combined with an image-guided minimally invasive delivery strategy for the treatment of breast cancer.

    J Vasc Interv Radiol 2013, 24:737-43. PubMed Abstract | Publisher Full Text OpenURL

  55. Aka JA, Lin SX: Comparison of functional proteomic analyses of human breast cancer cell lines T47D and MCF7.

    PLoS One 2012, 24:1-9. OpenURL

  56. Nilsson UW, Garvin S, Dabrosin C: MMP-2 and MMP-9 activity is regulated by estradiol and tamoxifen in cultured human breast cancer cells.

    Breast Cancer Res Treat 2007, 102(3):253-61. PubMed Abstract | Publisher Full Text OpenURL

  57. Nilsson UW, Dabrosin C. Estradiol and tamoxifen regulate endostatin generation via matrix metalloproteinase activity in breast cancer in vivo. Cancer Res. 2006;66(9):4789–94.