studentjournalofmedicine

A strange case of ingrown toenail treated with phenol

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Abstract

We experienced a strange case of ingrown toenail, which had developed as a huge mass and enveloped the nail of the left first toe. The patient had self-treated his ingrown toenail for a period of one year with an ointment available over the counter. However, the granulation tissue on both sides of the nail had increased gradually and advanced over the nail plate in the medial direction. Finally, the granulation tissue on both sides had adhered to the nail and epithelial cells advanced over the granulation tissue completely. During surgery, the epithelized granulation tissue was excised at the bilateral terminal base point, and the posterior nail fold and the nail matrix were cauterized completely with phenol. Eighteen months after the operation there was no recurrence of the ingrown toenail.

INTRODUCTION

The ingrown toenail is a common problem which occurs mostly in the first toe and causes a high amount of morbidity in affected patients. In this report we document a strange case of ingrown toenail of the first toe, which had developed as a huge mass and enveloped the nail. We describe here the cauterization of the nail matrix with phenol after surgical resection of the tumor, a treatment that had good results.

CASE REPORT

A 56-year-old man came to our office complaining of pain, offensive smelling discharge and disappearance of the nail of the right great toe. Physical examination showed epithelized mass on the nail plate and fistulation from the proximal nail fold to the tip of the toe, epithelization did not occur on the nail plate side of the mass (Figure 1). The clinical diagnosis of this tumor was epithelized granulation tissue caused by ingrown toenail, however squamous cell carcinoma was not denied completely.

Figure 1

The first toe of a 56-year-old man. Photograph shows epithelized granulation tissue on the nail plate and fistulation from the proximal nail fold to the tip of the toe

The operative treatment involved anesthetizing the toe with a digital nerve block using 1% lignocaine. The tumor mass was excised at the bilateral terminal base point, and was sent to pathology for an immediate diagnosis. The pathological finding was granulation tissue with partial scar formation, and the external surface of the tumor was covered with epithelial cells. Next, the nail plate was incised longitudinally from top to the root at a width of approximately 5mm. The posterior nail fold and the nail matrix were cauterized completely with an 88% phenol-immersed cotton-tipped applicator for five minutes. The excessive skin and soft tissue of the tip of the toe were excised and trimmed (Figure 2).

Figure 2

Bilateral chemical matricectomy of the matrix was done with phenol after resection of the tumor

Eighteen months after the operation, there is no recurrence of the ingrown toenail (Figure 3).

Figure 3

Eighteen months after the operation there was no recurrence of the ingrown toenail

DISCUSSION

Ingrown toenail deformity is a common nail pathology that causes intractable pain and discomfort, hindering normal walking and markedly decreasing the quality of life of patients. Ingrown toenails could be a cause of granulation tissue of the lateral nail fold of the finger or toe (1). However, an ingrown toenail creating such a huge mass on the nail plate, as in this case, is very rare. In the literature, to our knowledge, there have been no reports of cases similar to our patient.

In the case presented here, the patient had treated his ingrown toenail himself for one year with an ointment purchased over the counter before he came to our office. However the granulation tissue of both sides of the nail had increased gradually and advanced on the nail plate in the medial direction. Finally, the granulation tissue from both sides adhered and epithelial cells advanced over the granulation tissue completely. We believe such pathogenesis of this tumor is very uncommon.

Conservative and surgical forms of therapy for ingrown nails have been used, however, there is no common or unique form of treatment (26). In recent years, matrix phenolization of the nail bed has been used increasingly, and has been reported to give less discomfort and lower recurrence rates (7,8). The procedure of phenolization is easy to perform and does not require specialized equipment (9). In our reported case, after resection of the epithelized granuloma, both sides of the posterior nail fold and the nail matrix were cauterized completely with phenol. After this simple treatment, there was no recurrence of the ingrown toenail. The only disadvantage of the treatment was the smallness of the toenail, however, the patient did not complain of this aesthetic disadvantage due to the advantages of freedom from pain and the bad odour.

REFERENCES

1. Chapeskie H. Ingrown toenail or overgrown toe skin? Can Fam Physician. 2008; 54:1561–1562. [PMC free article] [PubMed]
2. Moriue T, Yoneda K, Moriue J, et al. A simple therapeutic strategy with super elastic wire for ingrown toenails. Dermatol Surg. 2008; 1729–1732. [PubMed]
3. Arai H, Arai T, nakajima H, et al. Formable acrylic treatment for ingrowing nail with gutter splint and sculptured nail. Int J Dermatol. 2004; 43: 759–765. [PubMed]
4. Matumoto K, Hashimoto I, Nakanishi H, et al. Resin splint as a new conservative treatment for ingrown toenails. J Medical Invest. 2010; 57:321–325. [PubMed]
5. Nazari S. A simple and practical method in treatment of ingrown nails: splinting by flexible tube. J Eur Acad Dermatol Venereol. 2006; 20: 1302–1306. [PubMed]
6. Rounding C, Hulm S. Surgical treatment for ingrown toenails. Cochrane Database Syst Rev. 2005; 2; CD001514. [PubMed]
7. Di Chiaccihio N, Belda W, Jr, Di Chiacchio NG, et al. Nail matrix phenolization for treatment of ingrowing nail: technique report and recurrence rate of 267 surgeries. Dermatol Surg. 2010; 36: 534–537. [PubMed]
8. Tatlican S, Yamangokturk B, Eren C, et al. Comparison of phenol applications of different durations for the cauterization of germinal matrix: an efficicacy and safety study. Acta Orthop Traumatol Turc. 2009; 43: 298–302. [PubMed]
9. Kimata Y, Uetake M, Tsukada S, et al. Follow-up study of patients treated for ingrown nails with the nail matrix phenolization method. Plast Reconstr Surg. 1995; 95: 719–724. [PubMed]

Articles from Journal of Surgical Case Reports are provided here courtesy of Oxford University Press
studentjournalofmedicine

Tinea Incognito in Korea and Its Risk Factors: Nine-Year Multicenter Survey

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Copyright © 2013 The Korean Academy of Medical Sciences.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Tinea incognito (TI) is a dermatophytic infection which has lost its typical clinical appearance because of improper use of steroids or calcineurin inhibitors. The incidence of TI is increasing nowadays. We conducted retrospective review on 283 patients with TI from 25 dermatology training hospitals in Korea from 2002-2010 to investigate the demographical, clinical, and mycological characteristics of TI, and to determine the associated risk factors. More than half (59.3%) patients were previously treated by non-dermatologists or self-treated. The mean duration of TI was 15.0 ± 25.3 months. The most common clinical manifestations were eczema-like lesion, psoriasis-like, and lupus erythematosus-like lesion. The trunk and face were frequently involved, and 91 patients (32.2%) also had coexisting fungal infections. Among 67 isolated strains, Trichophyton rubrum was the most frequently detected (73.1%). This is the largest study of TI reported to date and the first investigational report concerning TI in Korea. We suggest that doctors should consider TI when a patient has intractable eczema-like lesions accompanied by tinea pedis/unguium. Furthermore, there should be a policy change, which would make over-the-counter high-potency topical steroids less accessible in some countries, including Korea.

Keywords: Calcineurin Inhibitor, Dermatophytoses, Korea, Steroid, Tinea, Tinea Incognito

INTRODUCTION

Tinea incognito (TI) is the term given to a dermatophyte infection, which has been modified in appearance by improper use of steroids or calcineurin inhibitors (1, 2). Since it was first described by Ive and Marks in 1968 (3), a few case reports and a number of review articles have been published on TI in English journals (1, 2, 4-6). Though typical dermatophytic infection on most skin surfaces except scalp, volar areas and nails usually present as annular lesions with erythematous scaly border and central clearing. Therefore, dermatophytic infection may be confused with other skin disorders such as granuloma annulare, discoid lupus erythematosus, pityriasis rosea, erythema annulare centrifugum, erythema migrans, or other dermatological lesions (7). Most textbooks or review articles state that mycological confirmation with laboratory testing before the start of antifungal therapy is recommended because the clinical diagnosis of fungal presence could be inaccurate (8). However, this is not easy to do in practice because of time pressure, inadequate access to equipment, lack of experience, low reproducibility, and so on (8, 9). Misdiagnosed dermatophytic disorders could be treated improperly with steroids. As some high-potency topical steroids are easily accessible as over-the-counter (OTC) products and non-dermatologists can also prescribe topical steroids freely without any fungal examination, the incidence of TI seems to be gradually increasing in Korea (10). However, there has been no published large-scale study on TI in Korea, as yet. For this reason, we investigated the demographics and past medical histories of TI patients, and clinical and mycological characteristics of TI in Korea.

MATERIALS AND METHODS

Patients

In the period from 2002 to 2010, cases of TI were collected retrospectively from dermatologic departments of 25 general hospitals in Korea, which had a dermatology training program. We defined 4 criteria for diagnosis of TI: 1) loss of typical clinical appearance of tinea (Fig. 1A); 2) previous history of corticosteroid (topical or systemic) or calcineurin inhibitor application to present lesions; 3) positive for at least 1 mycological evaluation (KOH examination, mycologic culture, or skin biopsy with DPAS stain); and 4) improvement after antifungal treatment. To be enrolled in this study, all patients had to satisfy all the 4 diagnostic criteria listed above. Only the cases confined to dorsum of hands and feet were included to avoid confusion with tinea pedis and tinea manus.

Fig. 1

Various features of tinea incognito (A-G). Vitiligo-like (A; pre-treatment, B; after 4 weeks of application of topical pimecrolimus, C; 6 weeks after topical antifungal treatment), contact dermatitis-like (D), nonspecific eczema-like (E), seborrheic dermatitis-like

Ethics statement

The study protocol was approved by Institutional Review Boards or Ethics Committees of Pusan National University Hospital and informed consent was obtained.

Demographics, past histories, and clinical characteristics

Clinical data including charts and clinical photos from 25 hospitals were systematically and retrospectively reviewed. Demographic information included age, gender, coexisting diseases, and other dermatologic diseases. Past medical histories included the duration of TI, how the patients obtained the topical steroid or calcineurin inhibitor, and treatment modality. After dividing the patients into 3 groups (dermatologist-treated, non-dermatologist-treated, and self-treated TI groups), the duration of TI and treatment modality were compared among the 3 groups. Regarding the clinical characteristics of TI, the distribution, the most likely clinical feature, and coexisting fungal infections were investigated.

Mycological data

The KOH examination (20% potassium hydroxide) was performed to check for the presence of fungi. Mycological culture was performed on Sabouraud dextrose agar with chloramphenicol and cycloheximide. After incubation at 25℃ for at least 3 weeks, dermatophytes were identified by means of gross morphology, light microscopy, and/or biopsy with PAS stain (11).

Statistical analysis

Pearson’s chi-square test was used to compare the frequency of treatment modalities and one-way ANOVA was used to compare the duration of TI among dermatologist-treated, non-dermatologist-treated, and self-treated TI patients group. A P value of less than 0.05 was considered statistically significant.

RESULTS

Demographics

After thorough review, 283 patients fulfilled the diagnostic criteria of TI in this study. The mean age was 44.0 ± 22.5 yr (range 3-94) and 125 patients (44.3%) were female. Table 1 shows the age distribution of TI patients with a slightly lower frequency of patients with TI under 10 and over 80 yr old. Sixty-five patients (23.0%) had coexisting diseases at first clinic visit such as hypertension in 37 (13.1%), diabetes in 23 (8.1%), and hepatitis in 7 (2.5%). Five patients had underlying malignancy (1.8%), 2 patients suffered from angina, and 2 patients had asthma. In addition, 1 patient had adrenal insufficiency, 1 patient had myasthenia gravis, 1 had depression, and 1 had epilepsy. Sixteen patients (5.7%) had coexisting dermatologic diseases including 5 patients with atopic dermatitis (1.8%), 4 patients with psoriasis (1.4%), 3 with systemic lupus erythematosus (1.1%), and 2 with seborrheic dermatitis (0.7%). There was 1 patient with rosacea, and 1 patient with bullous pemphigoid.

Table 1

Demographics and past histories of 283 cases of tinea incognito in Korea during 2002-2010

Past medical histories

The mean duration of TI in the study patients was 15.0±25.3 months. While mean duration of self-treated TI patients was 9.0±11.1 months, that of TI patients treated by dermatologists and non-dermatologists was 16.4±25.8 and 15.7±28.1 months, respectively. There was no statistical significance among the 3 groups (P = 0.234) (Table 2).

Table 2

Mean duration of the disease and previous treatment modalities according to past physician’s specialty

Before coming to the teaching hospital, 40.6% of TI patients received treatment from a dermatologist, 43.8% from non-dermatologists, and another 15.5% were self-treated. While all of self-treated patients used topical steroids only, people treated by dermatologists or non-dermatologists used various treatment modalities such as topical/systemic steroids, topical/systemic antibiotics, topical calcineurin inhibitor, steroid intralesional injection, or a combination of aforementioned agents. Overall, most of TI patients were treated with topical steroids only (86.9%), and other treatment modalities included topical and systemic steroids (6.4%), topical steroid and topical calcineurin inhibitor (1.4%), and topical calcineurin inhibitor (0.7%), etc. There were no significant differences in treatment modalities according to past physician’s specialty (p > 0.05).

Clinical characteristics

Overall, the trunk (30.4%) is the most commonly affected area of TI followed by the face (24.4%), foot (13.8%), multiple involvements (13.8%), the groin (9.9%), and hand (7.8%) (Table 3). The clinical features were variable, but regardless of distribution, over more than three-quarters of all study patients showed eczema-like (82.0%) lesions which included nonspecific eczema, contact dermatitis, seborrheic dermatitis, and atopic dermatitis. Less often, TI mimicked psoriasis (6.0%), lupus erythematosus (2.5%), impetigo (1.4%), urticaria (1.2%), folliculitis (0.7%), and other dermatological lesions (Table 3). According to the anatomical distribution, facial TI presented as eczema-like (76.8%), lupus erythematosus-like (8.7%), impetigo-like (2.9%), and vitiligo-like (2.9%) lesions. Trunk TI presented as eczema-like (79.1%) and psoriasis-like (10.5%) lesions, and almost all of groin, hand, and foot TI resembled eczema. When TI involved multiple sites, it appeared similar to eczema (69.2%), psoriasis (15.4%), folliculitis (2.6%), and other dermatological lesions (Table 3). In children, TI was most likely to be found in the facial area (11.6%), and the trunk (11.6%), and least likely to be found in the groin (3.6%).

Table 3

Clinical and mycological characteristics of 283 cases of tinea incognito in Korea during 2002-2010

In 91 cases (32.2%), other fungal diseases such as tinea pedis (42.9%), tinea unguium (31.9%), tinea pedis et unguium, or tinea unguium/tinea corporis (25.3%) were diagnosed apart from TI sites (Table 3). According to anatomical distribution, TI of the trunk, groin, or hand was commonly seen with tinea pedis (>50.0%), TI involving foot or multiple areas usually accompanied tinea unguium, and facial TI was strongly associated with tinea pedis et unguium.

Mycological data

Direct microscopic examination was performed in all cases and 260 cases (91.9%) were positive. Of 49 biopsied specimens, 42 (85.7%) showed fungal hyphae and/or spores by D-PAS stain. Sixty-seven cases (23.7%) were cultured in our study and Trichophyton rubrum was the most frequently detected dermatophyte (49/67, 73.%), regardless of TI distribution. Trichophyton mentagrophytes (6/67, 9.0%) and Microsporum canis (6/67, 9.0%) were the second-most frequently detected causative agents, and T. tonsurans, T. verrucosum and M. gypseum were also isolated in a few cases. While only 1 or 2 species of dermatophytes were found in groin, hand, and foot TI, various kinds of fungi were identified in face or trunk TI (Table 3).

DISCUSSION

Tinea incognito had been defined as tinea modified by the improper use of systemic or topical corticosteroids. However, as the use of topical calcineurin inhibitors has been increasing gradually in many dermatologic diseases such as atopic dermatitis, seborrheic dermatitis, intertriginous psoriasis, contact dermatitis and other dermatological lesions (12), the number of cases of modified tinea has also increased (7, 13, 14). Thus, we propose that TI be defined as certain dermatophytoses which have lost their usual clinical manifestation because of erroneous use of systemic/topical corticosteroids or topical calcineurin inhibitor, as in 1 recent article (2). In addition, we think that TI, which involves the hand or the foot, should be confined to the dorsal surface, because tinea pedis and tinea manus cannot be definitively differentiated from TI involving the palm or the sole. It has been suggested that the use of immunosuppressants decreases the fungus-induced local inflammation, and this may allow the fungus to grow slowly with less erythema or scaling causing a “modification” of the typical manifestation of tinea (7).

While TI seems to be common in dermatology practices currently, only a few numbers of large scale studies have been reported (1, 2, 4). These studies were done in Italy, Spain, and Iran. Our study in Korea was designed to be the largest scale study on TI. While previous case reports of TI in Korean literature (10, 15-21) (Table 4) showed female predominance, this study showed relatively equal gender distribution and relatively uniform age distribution (mean: 44.0±22.5 yr) were found except for patients over 80 yr. A recent article regarding TI in Italy (1) also reported equal gender distribution and similar mean age (42 yr), and another article in Iran (4) also revealed equal gender distribution with slightly younger mean age (32.6 yr). Based on these data, we can postulate that TI is common in middle-aged persons with little difference in gender. Moreover, 65 (23.0%) patients in our study had coexisting non-dermatologic diseases such as hypertension, diabetes, hepatitis, malignancy, and so on, and 16 (5.7%) patients had coexisting dermatologic disorders requiring systemic steroids or other immunosuppressants. This was lower than the previous Italian report in that 40% of patients with TI had non-dermatological pathologies which required treatment with systemic steroids (1). Though the percentage of the patients in this study who received immunosuppressive therapy was lower than in the Italian report, the possibility of TI should be kept in mind whenever the patient with skin lesions is on immunosuppressant medications.

Table 4

Previous reports of tinea incognito in Korean literatures

There have been no published data regarding TI according to past treating physician’s specialty or treatment modalities, as yet. Based on our study, over half of the patients were either treated by non-dermatologists (124/283, 43.8%) or self-treated (44/283, 15.5%). TI was thought to be associated with easy access to high-potency OTC topical steroids such as betamethasone valerate by patients and with lack of understanding of tinea by non-dermatologists. Therefore, there should be swift policy changes to limit OTC access of high-potency steroids to patients in Korea. This would limit inappropriate tinea treatment by patients. Furthermore, to reduce the number of cases of TI caused by non-dermatologists, education regarding skin diseases including fungal infections could be provided by Korean Dermatologic Associations.

Surprisingly, about 40% of the patients were treated by dermatologists in this study. Even though tinea can mimic many other skin disorders and there could be selection bias, this ratio seems to be too high. This may mean a lack of mycological evaluation and carelessness of dermatologists when diagnosing tinea infection. It is important for dermatologists to consider fungal infection in the differential diagnosis of skin disorders, and increase the use of laboratory tests for mycological evaluation. In practice, the medical cost for mycological examinations is very low in Korea. However, the patient load is high and doctors are pressed for time. This could be the prime reason for misdiagnosis of fungal infections (22). Therefore, we think that if physicians were better paid for mycological evaluations there might be more active mycological examinations and fewer misdiagnoses of fungal infections.

On the aspect of distribution, the trunk was the most commonly involved site of TI and the face was another commonly involved area, as reported in other original articles (1, 4) and previous Korean literatures. Another recent study regarding 54 childhood TI cases also reported similar results with highest incidence in the trunk and face (2), and our study backed it up with the same results. From these findings, we can postulate that the most common sites of TI are trunk and face regardless of age.

The clinical features of TI were reported to be variable, and the most prevalent features seen are eczema-like disorders such as nonspecific eczema, contact dermatitis, and atopic dermatitis (1, 4, 7), and previous Korean reports about TI also in accordance with it. Similarly, 232 TI patients in this study showed quite various clinical features such as eczema-like, psoriasis-like, lupus erythematosus-like, and etc. Specifically, nearly all cases of hand and foot TI showed eczema-like features. Therefore, when dealing with recalcitrant eczematous lesions on the hand or foot, mycological examination should always be considered. Compared to TI of groin, and hand and foot, where the eczema-like features were quite high, TI of the face, trunk, or multiple areas showed more variable features. Therefore, not only eczema-like lesions but also other recalcitrant skin manifestations resembling psoriasis, lupus erythematosus, impetigo, urticaria, etc., should also be carefully evaluated to rule out TI especially when skin diseases involve the face, the trunk, or multiple areas (Table 5).

Table 5

Suggested risk factors of tinea incognito

Moreover, one-third of our study population (32.2%) had combined fungal diseases, which involved distant areas from present TI, and most of them had dermatophytic infection on their feet regardless of affected areas, including tinea pedis and tinea unguium. Therefore, in patients with refractory skin disease especially resembling eczema and also with concomitant tinea pedis or tinea unguium, TI should be ruled out because these coexisting fungal infections could be an autoinoculation source of superficial dermatophytic infection in another body part at any time.

As many previous studies confirmed (1, 23-25), Trichophyton rubrum (T. rubrum) was also the most frequently identified dermatophyte. T. rubrum is one of the anthropophilic dermatophytes and the most common pathogen in tinea corporis, tinea cruris, tinea manus, tinea pedis, and tinea unguium (26). Since TI affecting the trunk, groin, hands, and feet accounted for almost 60% of T1 in our study, it would not be surprising that T. rubrum was the most commonly isolated dermatophyte. Moreover, the high prevalence of combined tinea pedis and tinea unguium might also have contributed to the high isolation rate of T. rubrum. Anthropophilic dermatophytes have adapted to humans and elicit a mild to non-inflammatory host response unlike zoophilic and geophilic infections (26). This mild inflammatory response might be the cause of the long duration of TI because topical corticosteroids or topical calcineurin inhibitor could alleviate the inflammation, which could be the main mechanism of disguising the typical manifestation of tinea.

In summary, our research was the largest study of TI in Korea to date. We investigated the characteristics of TI according to the primary physician’s specialty though the clinical and mycological results were similar to previous studies. From this study, we can suggest that long-lasting erythematous scaly skin lesions unresponsive to steroids or calcineurin inhibitor as the most important risk factors of TI. Not only truncal or facial involvement, but also combined tinea pedis/unguium or the history of immunosuppressant treatment could also be a good clue in diagnostic approach of TI. Moreover, we can suggest several things to reduce the incidence of TI based on our results. First, reform of OTC sales system of high-potency topical steroids is needed so that they are not as easily available to the public in some countries including Korea. Second, non-dermatologists need to be informed and educated that superficial dermatophytic infection could appear in a variety of forms. Care by experienced dermatologists could also be needed especially when dealing with long-lasting erythematous scaly skin lesions, which have proven to be unresponsive to steroids or calcineurin inhibitor treatment. Finally, we recommend dermatologists not to neglect TI as a possibility in cases of recalcitrant variable skin lesions, not hesitating to do active mycological examinations, which would give them some critical clues in diagnosis of TI, and doing careful clinical examinations when finding combined tinea pedis or tinea unguium.

ACKNOWLEDGMENTS

The authors have no conflicts of interest to disclose.

Footnotes

This work was subsidized by a research grant from the Janssen Award in 2010.

References

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Articles from Journal of Korean Medical Science are provided here courtesy of Korean Academy of Medical Sciences
studentjournalofmedicine

Characteristics of Androgenetic Alopecia in Asian

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Won-Soo Lee, M.D., Ph.D.corresponding author and Hae-Jin Lee, M.D.
Copyright © 2012 The Korean Dermatological Association and The Korean Society for Investigative Dermatology
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Androgenetic alopecia (AGA), or pattern hair loss, is a common disorder in Asian men and women, with a reported incidence of up to 73% among general population. There are several descriptions regarding the characteristics of AGA in patients of European descent. Asian patients with AGA have different types of hair loss and family histories from Europeans, which may affect treatment response. Therefore, in this review, prevalence, hair loss patterns, familial factors, androgen receptor gene polymorphisms of Asian AGA patients, and management based on algorithmic guidelines for AGA are discussed. This review may be useful for dermatologists in clinical practice for diagnosing and designing management approaches for Asian patients with AGA.

Keywords: Androgenetic alopecia, Asians

INTRODUCTION

The term “androgenetic alopecia (AGA)” was introduced by Orentreich1 in 1960, but the same condition in men has also been termed male pattern alopecia, common baldness, male pattern baldness, and male pattern hair loss (MPHL). Androgen dependence and hereditary factors are less obvious in affected women than in affected men, thus the term pattern hair loss, which is a much broader concept, is preferred for women. AGA is the most common type of alopecia that occurs after puberty in both sexes. Patients typically present with progressive thinning and shortening of hair in affected areas. AGA is clearly a stressful experience for both sexes, but it may be substantially more distressing for women. In this review, Asian characteristics of prevalence, hair loss patterns, familial factors, androgen receptor (AR) gene polymorphisms, and management of AGA will be discussed.

PREVALENCE OF AGA IN ASIAN

There are population differences in the prevalence and types of AGA. In individuals of European descent, the prevalence of AGA has been well documented by Hamilton2 and Norwood3. A study of European-American men in the USA revealed a predominance for frontal baldness (Type A variant in Norwood-Hamilton classification) in 12% and a Type III or worse pattern in 16% of males aged 18~29 years old, which increased progressively to 53% in those aged 40~49 years old4. A study of 20~50 year old Norwegian men most commonly reported Type I (31%), followed by Type II (26%) and Type V or worse (20%)5.

AGA is also a common disorder in Asian people. Takashima et al.6 and Kakizo7 studied AGA in Japanese and found that it was minimal before the age of 40 and that, although the incidence increased with age, it remained lower than in Europeans. Japanese men develop AGA approximately one decade later than Europeans, and the prevalence is 1.4-fold lower in each decade of life6. In Korean men, the prevalence of AGA (Norwood III or above) at all ages was 14.1% and increased steadily with advancing age, but remained lower than that of Europeans: 2.3% in the third decade, 4.0% in the fourth decade, 10.8% in the fifth decade, 24.5% in the sixth decade, 34.3% in the seventh decade and 46.9% over 70 years. Type III vertex involvement was the most common type in the third decade to the seventh decade in Korean men; over 70 years, type VI was most common. A ‘female pattern’ was observed in 11.1% of cases8. In Korean women, the prevalence of AGA (Ludwig I or above) at all ages was 5.6%, and also increased steadily with advancing age: 0.2% in the third decade, 2.3% in the fourth decade, 3.8% in the fifth decade, 7.4% in the sixth decade, 11.7% in the seventh decade and 24.7% over age 70 years. Type I was the most common type up to the sixth decade; over 60 years, Type I and II were similar in prevalence. Type III was not observed8.

The age specific prevalence of AGA in Taiwanese men was compatible to that among Korean men but was lower than that among individuals of European descent9. Smoking status, current amount of cigarette smoking, and smoking intensity were statistically significant factors for AGA after controlling for age and family history9. A population-based cross sectional study was carried out in 7,056 subjects (3,519 men and 3,537 women) in Shanghai, China10. The prevalence of AGA in Chinese men was 19.9%, and the prevalence of female pattern hair loss (FPHL) in men was 0.1%. The most common type of hair loss in men was type III vertex (3.5%). The prevalence of AGA in Chinese men was lower than in European men but was similar to that in Korean men. However, over the age of 60 it approached the prevalence in European men and was higher than in Korean men. The prevalence of AGA in Chinese women was 3.1%, while MPHL in Chinese women was found in those aged over 50 years (0.4%), and the most common type was Ludwig type I (1.4%). Interestingly, the prevalence of AGA in Chinese women was lower than that in Korean women and European women, and type I was the most common type10.

In Singapore, Tang et al.11 reported a prevalence of 63% for Norwood type I to VII. The prevalence increased with age from 32% among young adults aged 17 to 26 years to almost 100% for those in their eighties. In Thailand, Pathomvanich et al.12 conducted a randomized study including 1,124 Asian men (local Thai and Chinese) between the ages of 18 and 90. The prevalence of baldness was reported as 38.52%; this figure approached that of Europeans, rather than the one fourth to one third reported in previous studies of Asians6. The prevalence increased with age, affecting 11% of young adults aged over 20 years and reaching 61.78% at 70 years of age. There are, however, two limitations of this survey. First, the small number of men included over 80 years of age (31 men) might have affected the results when compared to the Norwood study of the same age group. In addition, there were two Asian subgroups involved in this study, Thai and Chinese.

According to these studies mentioned, the prevalence of AGA in Chinese and Korean men was similar to, but significantly lower than, the prevalence in Thailand. The highest prevalence among the Asian groups studied was the 63% observed in Singapore; this discrepancy may be attributed to the diverse populations residing in the country or the inclusion of the almost normal Norwood type I in the Singapore study. In contrast, type II was the most common pattern among Indian males until the sixth decade, followed by type III Vertex after the sixth decade. The type A variant was only seen in 1% of Indian males, and FPHL was only observed in 0.2% of Indian males. These results suggest a less extensive balding pattern in Indian male population than in other Asian populations13.

In summary, Asian men with AGA have different characteristics from those of men of European descent. There are similar increases in prevalence with age among all the Asian groups studied. This high prevalence in older men suggests that this form of hair loss may be a normal consequence of aging. However, particularly in younger men, hair loss can have significant psychosocial manifestations, and can in turn have a significant economic impacts on household health expenditures14. The wide variation in prevalence rates in the current Asian studies would require a more standardized protocol.

AR GENE POLYMORPHISM IN AGA

Balding scalps are characterized by high levels of the potent androgen dihydrotestosterone (DHT) and increased expression of the androgen receptor gene. Most AGA patients have an androgen dependent trait, although it is thought to be under the control of multiple genes, such as genes for the AR, insulin-like growth factor-1, and DHT regulations15,16. The human AR gene is on the X chromosome at Xq11-12. The AR is a structurally conserved member of the nuclear receptor superfamily. The amino terminal domain is required for transcriptional activation and contains a region of polyglutamine that is encoded by CAG trinucleotide repeats. In humans, the number of CAG repeats is polymorphic. Expansion of CAG repeats in the AR has clinical implications for human disease17. A low number of CAG repeats in the AR gene implies increased risk factors for coronary heart disease18 and prostate cancer19. In recent studies, neurotrophic factors, especially brain-derived nerve factor, were found to have potential importance mediating the effects of androgens on hair follicles, serving as negative regulatory control signals20. These findings suggest that other regulatory signals may affect the pathogenesis of AGA, as well as AR gene polymorphisms.

The ubiquity of the AR gene Stu I restriction site, and the higher incidence of shorter triplet repeat haplotypes in bald men, suggests that these markers are very close to a functional variant that is a necessary component of the polygenic determination of male pattern baldness. A meta-analysis study by Zhuo et al.21 suggests that the G allele of the AR Stu I polymorphism might be a potential risk factor for AGA, especially in subjects of European descent. Functional mutations in or near the AR gene may explain the high reported levels of expression of this gene in the balding scalp22. Shorter CAG repeat lengths may be associated with the development of androgen mediated skin disorders such as AGA, hirsutism, and acne in men and women23. These findings suggest that the CAG repeat length in AR may affect androgen mediated gene expression in hair follicles and sebaceous glands in men and women with androgenic skin disorders14,23. Interestingly, when the number of triplet repeats (CAG+GGC) was plotted against degree of symptom improvement after treatment with finasteride, a broad correlation between these variables was observed24. The smaller the repeat number, the greater the improvement with finasteride. The group of patients with shorter repeat regions in the AR gene responded better to finasteride than did those with longer repeat regions, although patients with shorter repeats tended to have severe initial symptoms. The determination of such polymorphisms is thought to be useful in drug choice for AGA patients24.

Jung et al.25 compared CAG repeat numbers within the AR genes of 64 male Korean AGA patients with those of 40 normal male controls in a preliminary study. There was no significant difference in the number of CAG repeats between the Korean AGA patients and controls. There were no correlations between CAG repeat numbers and age of onset or severity of AGA in Korean AGA patients. These results suggests that AR receptor CAG polymorphisms in the Korean male population might not play a major role in AGA susceptibility. Nevertheless, a more extensive study to clarify whether there are real population-based differences in AR gene polymorphisms is needed.

Recently, advanced genetic studies of AGA have been published. Hayes et al.26 reported that the gene locus of E211A is significantly lower in proportion in the vertex and vertex balding group and frontal balding group compared with the no balding group. Therefore, the AR-E211 A allele, in linkage with the functional repeat sequences, is associated with a lower risk of metastatic prostate cancer and a lower risk of alopecia. Moreover, a study using genome-wide linkage study revealed a locus associated with AGA on chromosome 3q2627.

Hillmer et al.28 investigated the signatures of genetic variants of AR and their relationships to the AGA risk haplotype. Haplotype homozygosity suggested that the AGA risk haplotype was driven to high frequency by positive selection in Europeans, although a low meiotic recombination rate contributed to high haplotype homozygosity. Further, they detected high levels of population differentiation and a series of fixed derived alleles along an extended region centromeric to AR in the Asian HapMap sample.

RACIAL DIFFERENCES IN PATTERNS OF HAIR LOSS IN AGA AND THE BASP CLASSIFICATION FOR AGA

Asian men with AGA display different characteristics compared with men of other ethnicity. However, there is an increase in prevalence with age among all the Asian groups studied similar to that observed in other ethnic population8-12,29. The reason for this increase rate in the prevalence of AGA compared to Europeans remains unknown, but a transition toward a more Western diet and lifestyle may play a role.

There are also populational differences in the patterns of hair loss in AGA. In a previous study, FPHL was observed in 11.1% of Korean males with AGA8. In a Chinese study, MPHL was found in 13 of 108 (12%) women with AGA, all of whom were over 50 years of age10. In an Indian study, although it was possible to classify 80% of cases of AGA and II (28%) and III (15%) were the most common types of AGA, 27 patients of 150 male subjects (18%) did not fit into specific patterns according to the Norwood Hamilton classification30. In addition, the type ‘a’ variant was noted in 20% of patients, clearly indicating the limitations of the existing classifications. There is considerable overlap in types IV, V and VI in the Norwood classification, with the ‘a’ variants further confusing the picture30.

Various classification methods have been proposed for describing AGA. In 1950, Beek31 published a classification system, based on 1,000 males of European descent, which used two evolutionary aspects: frontal and frontovertical baldness. In the following year, the first systematic classification of AGA was established by Hamilton2, who sub-classified patterns of baldness based on frontoparietal, frontal recession, and vertex thinning, then evaluated a large group of men and women for the presence of specific patterns of hair loss from the prenatal period through the tenth decade of life. In 1975, Norwood3 refined Hamilton’s classification by emphasizing temporofrontal or vertex only subcategories of hair loss into seven types with a type A variant and reported the incidence of male pattern baldness at various ages in 1,000 adult male subjects of European descent. An additional pattern was introduced as the Norwood-Hamilton classification in a clinical trial of finasteride in MPHL32. Olsen33,34 proposed assigning separate designations (temporal, frontal, mid and vertex) to areas of the scalp that bald at different rates in different individuals with MPHL. Ludwig35 presented quite a different picture of hair loss in women from that described by Hamilton2. He emphasized preservation of the frontal fringe despite progressive centrifugal loss over the top of the scalp and arbitrarily designated three gradations of hair loss. Olsen36 proposed that frontal accentuation (or the ”Christmas tree” pattern) should be considered an additional pattern of hair loss in women, which helps to distinguish AGA from potential hair loss mimicries in women. Presently, the Norwood-Hamilton classification32 for MPHL and the Ludwig classification35 for female AGA are the most commonly used classification methods for assessing AGA worldwide.

Each of these existing classifications has substantial limitations in clinical setting. The Norwood-Hamilton classification (Fig. 1)3 is very detailed and is less stepwise classifications in its descriptions, making it difficult to memorize for common use. It does not list some non-typical types of baldness, such as FPHL in men. Additionally, many women with MPHL cannot be classified using the Ludwig classification system (Fig. 2)35. For most of these classification systems, clinicians must use different classification systems for each gender in order to correctly classify patterns34.

Fig. 1

The Norwood-Hamilton classification of male balding defines two major patterns and several less common types. Thinning starts in both temples as well as the crown/vertex and slowly progresses to encompass the entire top of the scalp (cited from Ref. 3
Fig. 2

The Ludwig pattern of hair loss (3-point). There are three main classes, each with increasing hair loss (cited from Ref. 35).

Thus, a more widely accepted, accurate, and stepwise method for classifying AGA would be of great benefit. Lee et al.37 devised a new classification system, named the Basic and Specific (BASP) classification (Fig. 3), which is comprehensive and systematic regardless of population or gender. The BASP classification37 was based on observed patterns of hair loss. The basic (BA) types represent the shape of the anterior hairline, and the specific types (SP) represent the density of hair on distinct areas (frontal and vertex). There are four basic types (L, M, C, and U) and two specific types (F and V). The final type is assigned according to a combination of the assigned BA and SP types.

Fig. 3

The BASP classification system includes four basic types (L, M, C, and U) and two specific types (V and F). The basic types represent the shape of the anterior hairline, and the specific types represent the density of hair on specific areas (frontal and

A total of 2,213 Korean subjects, comprised of 1,768 males and 445 females, were classified according to the BASP classification37. According to the severity of the phenotype, both the basic and specific types were subclassified into subtypes in order to generate a more stepwise and systematic classification. It is possible to describe patterns of hair loss in detail using the BASP method, and, thus estimate the further extent of hair loss and therapeutic response to a certain therapy. For both sexes, the majority of patients enrolled in the study were in the third and fourth decades of life (65.1% of males and 56.68% of females). In males, the older as well as the younger group were more likely to have little recession of the frontal hairline (classified as type M1-2) and diffuse thinning over the top of the scalp (type F1-2). The women in the study developed typical female AGA.

In men, regardless of age, 1,434 of the 1,768 males were classified as type M, accounting for 81.1% of cases. Among the subtypes and according to the severity of baldness, the majority of subjects below 50 years of age were classified as type M1, whereas most subjects over the age of 50 were classified as type M2. The incidence of Type L (9.3%) tended to decrease with age, but those of types C (5.8%) and U (3.8%) tended to increase. In women, type L showed the highest frequency in all age groups, accounting for 210 (47.2%) of 445 female subjects. Regardless of age, types M, C, and U were the next most common in order, observed in 121 women (27.2%), 111 women (25.0%), and 3 women (0.6%) of the 445 subjects, respectively. Type C0 was the second most common subtype in female subjects between the second and fourth decade of life, and its incidence decreased with age. In men, type F, which is identical to FPHL in the Ludwig classification, was observed in 42.4% (749/1,768) of male subjects, and type V was observed in 19.8% (350/1768). The grades of both types increased slightly with age. In women, type F was observed in 70.6% (314/445) of female subjects with AGA.

The BASP classification is a stepwise, systematic, and universal classification system for AGA, regardless of race or sex. It is an easily available comprehensive classification system. The BASP classification may prove particularly useful in communicating the exact amount and distribution of hair loss in those with AGA37. For these reasons, we use the basic and specific (BASP) classification in this review.

FAMILIAL FACTORS IN AGA

Family history plays an important role in the onset of AGA, which is believed to be influenced by genetic factors. However, the exact mode of inheritance has not been well characterized. Although there are some reports regarding the prevalence of AGA in male paternal family members, reports regarding the maternal side are rare. The AGA prevalence in male family members of patients (30.3%) was higher than those of controls (8.5%)38. AGA prevalence on the paternal side was greater than on the maternal side. However, no differences were found between paternal and maternal AGA prevalence, analyzed according to the age of onset and severity of AGA. These results suggest that AGA expression might be influenced by familial AGA prevalence and that paternal AGA prevalence has a greater effect in general on AGA expression than maternal AGA prevalence38.

In another Korean study, a family history of baldness was present in 48.5% of men and 45.2% of women with AGA8. In a Chinese study, a family history of AGA was present in 55.8% of men and 32.4% of women with AGA10. In contrast to the Korean study, the proportion of Chinese men with a positive family history was higher, suggesting that genetic background is important for determining the prevalence of AGA in Chinese men, and confirming that subjects with positive family histories are at greater risk of developing severe AGA9. The proportion of Chinese women with positive family histories was lower, further indicating that AGA is a polygenetic hereditary disease. In a Singaporean study, a positive family history of AGA was recorded in 58.9% (151/256) of subjects. Male patients tend to be more likely to have a father or male sibling with a similar problem, whereas female patients tend to be more likely to have a mother or female sibling with AGA39. In an Indian study of 150 subjects, positive family histories were found in 127 (85%) of subjects, paternal in 101 (67%), maternal in seven (5%) and both in 19 (13%). In 23 (15%) patients, no family history could be elicited.

In a Taiwanese study, an association was detected between moderate or severe AGA and family history of AGA from paternal relatives, whereas there was no corresponding association with maternal relatives9. Moderate or severe AGA was associated with a family history of AGA among first degree and second degree relatives but not among third degree relatives after adjusting for age. In addition, family histories of AGA among paternal relatives were predictive of moderate or severe AGA after adjusting for age. These findings do not support an association between moderate or severe AGA and a family history of AGA among maternal relatives. Moreover, a family history of AGA is associated with the risk of early onset AGA. This implies that those with a family history of AGA may have a higher risk of early onset AGA and a higher risk of developing severe AGA. Most importantly, early onset AGA showed a dose dependent association with AGA grade after adjusting for age and family history. From a clinical point of view, this suggests that patients with early onset AGA should receive early advice to prevent further deterioration9. These results of various epidemiologic familial studies suggest that AGA expression is influenced by familial AGA prevalence and, particularly, that paternal AGA prevalence has more effect on AGA expression than maternal AGA prevalence.

A recent study by Lee et al.40 using the BASP classification revealed that familial factors affecting the morphology of AGA in Asians differ between males and females, and for each BASP subtype. Parental influences on anterior hairline shape in men were predominantly from the paternal side, whereas these effects were less notable in women. In patients without family histories of AGA, a higher frequency of early-onset AGA than late-onset AGA was identified in men but not in women. Basic types of hair loss had a higher degree of heritability from the paternal side of the family, regardless of the specific type. This study provides detailed information indicating that each hair loss pattern according to the BASP classification has different familial factors in Asians40. Therefore, we can provide appropriate information to patients if we obtain careful personal and familial histories of AGA.

MANAGEMENT OF AGA IN ASIAN

General consideration

AGA is often related to poor self-image and low selfrespect. The problem must be viewed in perspective; an emphatic approach is important, as different people are affected in various ways when they lose hair. Patients should avoid hair-care products likely to injure the scalp and/or hair. Patients should maintain adequate diet, especially with adequate protein. The National Institutes of Health of the United States recommended daily allowance for protein is 0.8 g/kg41. Topical medications act only where the medication is applied; therefore, the whole area at risk of hair loss (the top of the scalp) should be treated with a given topical agent. If possible, any drugs that could negatively affect hair growth should be stopped and alternative substitutes used. Any underlying scalp disorders, such as seborrheic dermatitis or scalp psoriasis, should be treated as these conditions can affect the ability to use topical treatments for hair loss without irritation.

The typical man with MPHL who seeks treatment has significant concerns about the condition and has already engaged in considerable efforts to obtain information and at times even resorted to self-medication. Individualized consideration of attitudes, concerns, self-treating efforts, and expectations is crucial for effective management of men seeking medical treatment for MPHL42. Research has shown that most men and women who have unwanted hair loss have distressing experiences that diminish their body image43. Because of the psychological impact of hair loss, patients may seek inappropriate and unproven therapies. However, they must also appreciate the real goals and true limitations of each form of therapy. It is important that misconceptions should also be corrected. Some patients mistakenly think that their hormone levels are too high. Others erroneously place too many restrictions on their hair and grooming (e.g., hair styling, teasing, hair spray, washing frequency, hair color or permanents)44.

Knowledge and understanding of the genetic and physiological basis of AGA may help allay misconceptions and anxiety about its occurrence, and indirectly influence patient willingness to seek treatment for this condition45. Clinicians should follow the progress of their patients periodically to identify problems, utilizing photographic records of treatment results.

Medical treatment

A reduction in hair loss is usually seen after 3~6 months of medical treatment, and visible hair regrowth is observed after 6~12 months. Continuous treatment is needed to ensure sustained benefits. Unfortunately, available medical treatments are not curative. Ensuring that patients understand the limitations of these treatments is an important aspect of the management of AGA. Patients should be counseled that treatment for AGA will not restore hair growth to its prepubertal density and that the main aim is to prevent further progression of hair loss. Currently there are two agents, topical minoxidil and oral finasteride (only for males), approved by the United States Food and Drug Association (FDA) for the treatment of AGA.

1) Male

(1) Topical minoxidil solution

Topical minoxidil solution is administered at a dosage of 1 ml twice daily. Its mechanism of action is unknown. However, the main benefit appears to be a prolongation of the anagen phase and hair shaft diameter, irrespective of the underlying cause of baldness. It is well established that 5% minoxidil is more effective than 2% or 3% solution. Patients should be warned that during the initial 2~8 weeks, a temporary telogen effluvium may occur in some patients, which is self-limiting and subsides when subsequent anagen regrowth begins, and should not be a cause for treatment cessation46.

A recent advancement in the use of minoxidil as a hair loss treatment is the development of a 5% topical foam. Placebo controlled, double-blind trials have demonstrated that the hydroalcoholic foam is efficacious, safe, and well accepted cosmetically by patients14.

(2) Oral finasteride

Oral finasteride, a potent type II 5α-reductase inhibitor, should be administered at a daily dosage of 1 mg. In clinical trials over a 2-year period in men aged 18~41 years, the number of responding hairs was established after 1 year and continued treatment increased the length, diameter, and pigmentation of these hairs so that the coverage of the scalp increased over time. On stopping finasteride, the balding process resumed. An extension of the above study to 5 years showed that finasteride 1 mg/day was well tolerated, and led to durable improvements in scalp hair growth47.

Finasteride is generally well tolerated, side effects are typically mild and do not require discontinuation of therapy. Rare side effects may include some loss of libido and erectile function. At present, there is no proven benefit for finasteride in women. A placebo-controlled study in postmenopausal women with AGA given finasteride 1 mg/day over 1 year showed no significant benefit47.

2) Female

(1) Topical minoxidil solution

Topical minoxidil solution is administered at a dosage of 1 ml twice daily. The 5% solution was compared with the 2% solution in 2 studies involving 493 women. On the basis of hair-count data, the 5% solution was not significantly more effective than the 2% solution48. Patients should be warned that in the initial 2~8 weeks, a temporary telogen effluvium may occur in some patients, which is self-limiting and subsides when subsequent anagen regrowth begins, and should not be a cause for treatment cessation46. Side effects include hypertrichosis which occurs in 6% of women using 2% minoxidil, and 14% among those using the 5% solution46. This occurs on the face and resolves within 1~6 months after drug discontinuation. However, hypertrichosis diminishes or disappears after about 1 year, even with continued use of minoxidil.

(2) Oral antiandrogens

Cyproterone acetate, spironolactone and flutamide can be used as alternatives to minoxidil, but most of the antiandrogen therapies have not been rigorously studied in FPHL49. In general, better results are seen in women with hyperandrogenism. Side effects are generally greater with cyproterone acetate and spironolactone41.

Surgical management

Despite advances in medical therapy, hair transplantation remains the only means of permanent hair restoration in cases of severe AGA. It is contraindicated in patients with systemic diseases such as hypertension, cardiac disease, and diabetes mellitus, all of which must be controlled before hair transplantation. Local diseases such as cutaneous lupus erythematosus, morphea, alopecia areata, and scalp folliculitis must be quiescent for at least 6 months before hair transplantation. Complications of hair transplantation include ingrown hairs and foreign body reactions, infections, cobblestoning, graft depression, epidermal cysts, bleeding, headaches, scarring (keloid and hypertrophic scars), poor hair growth, arteriovenous fistula, osteomyelitis, wound dehiscence, telogen effluvium, accelerated hair loss, delayed temporary marked thinning, curly, lusterless hair, chronic mild folliculitis, and patient dissatisfaction.

After 4~6 months, the skin surfaces of the grafts have usually blended in perfectly with the surrounding scalp. In some patients, the grafts may be a shade lighter in color until they are “aged” by sun exposure50-52.

Other alternative medical therapies

Dutasteride is a dual type I and type II 5a-reductase inhibitor. In clinical trials, oral dutasteride showed significantly greater efficacy than placebo according to phototrichometric hair count, subject self-assessment, and investigator and panel photographic assessment53. Dutasteride is generally well tolerated, with rare side effects that may include some loss of libido and erectile function. Dutasteride is only approved by the Korean FDA for the treatment of AGA.

Topical alfatradiol may be an alternative, though reports of its efficacy have variable results to treat AGA54. Under the influence of 17alpha-estradiol (alfatradiol), an increased conversion of testosterone to 17beta-estradiol and androstendione to estrone improves hair growth55. Topical alfatradiol is available in Europe, South America, and Korea. Kim et al.56 reported single center, open-label, non-comparative, phase IV study of the efficacy and safety of alfatradiol (17α-estradiol) solution on female pattern hair loss in Korean women. Hair counts and diameter from baseline to 4 and 8 months after treatment were significantly increased in treated patients.

Bimatoprost and latanoprost, which are prostaglandin (PG) analogues, demonstrate stimulatory effects on hair growth of eyebrows and eyelashes and pigmentation in a high numbers of patients57. Currently bimatoprost is approved as eyelash growth enhancer. It might be used for the treatment of AGA off-label. The expressions of PG receptors were examined in mouse skin hair follicles, and mRNA was identified in dermal papilla and outer root sheath follicular structures during the anagen phase. In addition, other studies have demonstrated the ability of PG to stimulate movement from telogen to anagen in mice.

Ketoconazole might also be used for the treatment of AGA. The mechanism of ketaconazole is unknown, but may involve inhibition of inflammation, or anti-androgenic properties58. There is some evidence, both in humans and in rodents, that this agent may stimulate hair growth14. Prostaglandin analogues and ketoconazole are not approved for AGA treatment and further studies are needed to investigate the therapeutic effect on AGA.

Other devices and non-medical aesthetic aids

Devices can be used as alternative tools for the treatment of AGA. Laser hair comb (Low-level laser therapy)59,60 and Fractional photothermolysis laser61 have been tried to treat AGA. However, these treatments cannot be substituted for the medical and surgical approaches previously mentioned.

Non-medical approaches can provide cosmetic relief to both men and women with thinning hair, if medical treatments are not indicated, not effective, or not desired by the patient. Non-medical aesthetic aids include wigs, hairpieces, hair extensions, and topical powder makeup. They can also be used as adjuvant tools to medical or surgical treatments41.

CONCLUSION

Although the clinical aspects of AGA are recognized in both men and women and the role of DHT is well documented, much remains to be determined regarding the most appropriate treatments for AGA based on genetics and pathophysiology. AGA is a disconcerting experience for both sexes, but it may be substantially more distressing for women. Therefore, dermatologists should take into account the psychological well-being of patients with AGA, which can lead to the choice of an appropriate treatment. Moreover, most of the previously published studies of AGA were conducted among only patients of European descent. There are effective treatments, medical or surgical, currently available for some men and women with AGA. Compared to other populations, Asian patients with AGA have different types of hair loss and family histories, which may influence treatment response. This review of AGA in Asians may be practical for informing dermatologists regarding their approaches to understand, diagnose and treat Asian patients with AGA in clinical practice.

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Clinical Features of Bacterial Vaginosis in a Murine Model of Vaginal Infection with Gardnerella vaginalis

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Adam J. Ratner, Editor
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 author and source are credited.

Abstract

Bacterial vaginosis (BV) is a dysbiosis of the vaginal flora characterized by a shift from a Lactobacillus-dominant environment to a polymicrobial mixture including Actinobacteria and Gram-negative bacilli. BV is a common vaginal condition in women and is associated with increased risk of sexually transmitted infection and adverse pregnancy outcomes such as preterm birth. Gardnerella vaginalis is one of the most frequently isolated bacterial species in BV. However, there has been much debate in the literature concerning the contribution of G. vaginalis to the etiology of BV, since it is also present in a significant proportion of healthy women. Here we present a new murine vaginal infection model with a clinical isolate of G. vaginalis. Our data demonstrate that this model displays key features used clinically to diagnose BV, including the presence of sialidase activity and exfoliated epithelial cells with adherent bacteria (reminiscent of clue cells). G. vaginalis was capable of ascending uterine infection, which correlated with the degree of vaginal infection and level of vaginal sialidase activity. The host response to G. vaginalis infection was characterized by robust vaginal epithelial cell exfoliation in the absence of histological inflammation. Our analyses of clinical specimens from women with BV revealed a measureable epithelial exfoliation response compared to women with normal flora, a phenotype that, to our knowledge, is measured here for the first time. The results of this study demonstrate that G. vaginalis is sufficient to cause BV phenotypes and suggest that this organism may contribute to BV etiology and associated complications. This is the first time vaginal infection by a BV associated bacterium in an animal has been shown to parallel the human disease with regard to clinical diagnostic features. Future studies with this model should facilitate investigation of important questions regarding BV etiology, pathogenesis and associated complications.

Introduction

One in three women in the U.S. have bacterial vaginosis (BV) [1], a microbial imbalance of the vaginal flora characterized by the absence of normally dominant lactobacilli and overgrowth of complex communities dominated by Gram-negative bacteria and Actinobacteria [2], [3]. BV can be asymptomatic, maybe even part of a spectrum of ‘normal’ from the patient perspective, but often displays characteristic clinical features, including “thinning” of vaginal fluid secretions, increased pH (>4.5), a fishy odor upon potassium hydroxide treatment, and the presence of clue cells (epithelial cells studded with bacteria) in wet mounts. An additional defining feature of BV is the presence of vaginal sialidase [4][9], an enzyme that cleaves terminal sialic acid residues from complex glycans, which are abundant on host cell surfaces and secreted mucus proteins [10][12]. Women with BV are at increased risk of pelvic inflammatory disease, infections following surgery or other routine gynecologic procedures, sexually transmitted infections including HIV, and serious pregnancy complications such as intrauterine infection and preterm birth [13][22].

Unlike most common infectious diseases, BV appears to be polymicrobial in nature. Recent genomic studies have illustrated the complexity and heterogeneity of BV, which can vary in bacterial composition from day to day and from one individual to another [2], [3], [23][27]. Although more than a dozen bacterial species have been associated with BV, the potential causal contributions of each to the biochemical, cellular, and clinical features of BV remain elusive. Gardnerella vaginalis was the first bacterium implicated in the pathogenesis of BV and continues to be associated with the disease [28]. However, there has been much debate in the literature concerning the contribution of G. vaginalis to the development and pathogenesis of BV. G. vaginalis can be isolated/detected from asymptomatic women that do not meet the criteria for BV diagnosis at the time of detection [2], [27], [29][31], causing some to question its potential role in BV. However, consistent with the notion of G. vaginalis as a potential pathogen, strains identified as G. vaginalis have been isolated from invasive perinatal infections [32][34]. Moreover, several investigations have described the pathogenic potential of some G. vaginalis isolates in cell adhesion and entry, cytolytic toxin production, biofilm formation, and other phenotypes that may reflect virulence [26], [35][37].

One important diagnostic feature of BV is the presence of clue cells, which are thought to be exfoliated epithelial cells coated with bacteria. G. vaginalis, among other BV-associated bacteria, has been shown to interact with vaginal epithelial cells in culture [37], [38], and clinical studies have shown that vaginal specimens from women with BV have adhered bacteria on their surfaces [36], [39][42]. However, experimental investigation of the potential role of Gardnerella vaginalis in generating clue cells in vivo requires an animal model with features of human BV.

Upon infectious challenge, epithelial cells within the urinary and genital tracts can undergo a process termed exfoliation, in which superficial cells appear to be actively shed from the epithelial surface. In some cases, this is beneficial to the host, flushing potential pathogens from the mucosa [43], [44], while in other cases, potential pathogens can take advantage of their access to underlying mucosal tissue [45], [46]. Despite much speculation in the medical literature regarding the potential causes of epithelial exfoliation in BV, including enzymes such as sialidase and prolidase or the combination of amines and organic acids produced by anaerobes [37], [47][50], the degree of vaginal epithelial shedding among women with BV or normal flora does not appear to have been directly measured or reported in the clinical literature. Distinguishing whether epithelial exfoliation is actively induced in BV is necessary for establishing the pathophysiology of the disease and may be important for understanding why women with BV are at increased risk of secondary urogenital and intrauterine infections.

Defining a role for G. vaginalis (or other associated bacteria) in BV pathogenesis has been hampered by the absence of robust small animal models that displays phenotypes seen in human BV. Here we describe the development of a new murine model of G. vaginalis vaginal infection. This model displays several key features of BV, including presence of vaginal sialidase activity, and the presence of epithelial cells with attached bacteria (reminiscent of clue cells). Additionally, we provide the first quantitative evaluation of vaginal epithelial exfoliation in BV, demonstrating increased shedding of epithelial cells in both G. vaginalis infected mice and in clinical specimens from women with BV compared to mock-infected mice or women with normal flora respectively. This epithelial response is contrasted by an absence of inflammatory cell infiltrate, consistent with the lack of vaginal inflammation found in human BV throughout the literature [51][53]. The results from this murine model suggest that G. vaginalis alone is sufficient to yield BV phenotypes and provide further justification for considering G. vaginalis as a contributor to the causes and complications associated with BV.

Results

Murine Vaginal and Ascending Uterine Infection by G. vaginalis Occurs in the Absence of an Overt Inflammatory Infiltrate

To establish a G. vaginalis murine vaginal infection model, we used a sialidase-positive G. vaginalis strain (JCP8151B), a clinical isolate from a woman with BV. From our initial experiments we found that the majority of β-estradiol-treated C57/Bl6 mice contained vaginal flora producing large, mucoid colonies on Gardnerella semi-selective media that occluded the smaller G. vaginalis colonies. These ‘contaminating’ vaginal flora colonies were resistant to the addition of a combination of sulfamethoxazole, trimethoprim sulfate, gentamicin and perfloxacin to selection plates. Previous vaginal models of G. vaginalis did not acknowledge the presence of endogenous flora or describe methods by which G. vaginalis was distinguished and measured [54], [55]. We performed sequencing of genes encoding 16S rRNA from isolated colonies of the murine vaginal flora and identified several isolates to be species of Enterococcus (faecalis and gallinarum). To circumvent this issue and enable accurate determination of G. vaginalis titers in the murine reproductive tract, we isolated a spontaneous streptomycin resistant (SmR) mutant of JCP8151B. We confirmed that the SmR isolate contained the canonical mutation in the Rpsl gene (K43N) and displayed logarithmic growth rate and sialidase activity indistinguishable from the parent strain (data not shown). Using this new JCP8151B-SmR strain, we inoculated G. vaginalis vaginally into C57/Bl6 mice and determined CFU levels in vaginal washes and vaginal homogenates at 24 and 72 hours post infection (hpi) (Fig. 1A–B). Colonies presumed to be G. vaginalis due to growth on streptomycin-containing plates were also confirmed by PCR using primers reported to be specific for G. vaginalis [56] (data not shown).

Figure 1

Vaginal infection by G. vaginalis results in minimal histological inflammation.

BV has been characterized clinically as a microbial condition that most often lacks obvious signs of inflammation in vaginal tissue [57]. To determine the extent of histological inflammation (edema, neutrophil infiltrate) in our murine model, we performed hematoxylin and eosin (H&E) staining on vaginal tissues collected at 24 and 72 hpi. Similar to clinical observations of BV, G. vaginalis infection did not result in marked tissue inflammation, edema, or polymorphonuclear (PMN) cell infiltrate (Fig. 1C). The mean CFU levels in both vaginal washes and homogenates decreased significantly from 24 hpi to 72 hpi (Fig. 1A–B), suggesting clearance of the bacteria is occurring by PMN-independent mechanisms.

There was a strong positive correlation between CFU determined from vaginal washes and vaginal homogenates (Fig. 2A). Additional experiments determined that G. vaginalis could persist in the murine vagina of ∼50% of mice for as long as 8 days post infection (data not shown). G. vaginalis was also found at low levels (mean 69.5 CFU/g) in the uterine horns of 55% and 45% of mice at 24 and 72 hpi, respectively. As might be expected, animals with higher titers of vaginal bacteria were more likely to exhibit ascending infections of the uterine horns (Fig. 2B–C).

Figure 2

G. vaginalis titers in vaginal washes reflect titers in vaginal tissue and correlate directly with titers of G. vaginalis in uterine horns.

G. vaginalis Leads to Vaginal Sialidase Activity in vivo

A hallmark feature of bacterial vaginosis is the presence of high levels of sialidase activity in vaginal fluid compared to specimens from women with normal flora. Our JCP8151B-SmR4 isolate produces robust sialidase activity in culture. To determine whether JCP8151B-SmR4 expresses sialidase activity during vaginal infection, we performed sialidase activity assays on vaginal washes collected above. Sialidase activity was present in washes from 67% of G. vaginalis infected mice at 24 hpi, while the majority (86%) of mock-infected mice contained no detectable sialidase activity (Fig. 3A). Isolation of vaginal bacteria from the few mock-infected animals with vaginal sialidase activity demonstrated that these mice were colonized with sialidase-positive Eubacteria consortium or Enterococcus spp. (data not shown). The level of sialidase activity present in washes of infected animals correlated positively with G. vaginalis CFU levels in vaginal washes and homogenized vaginal tissue, strongly suggesting that the observed sialidase activity in infected animals is in fact produced by G. vaginalis (Fig. 3B and C). Together these results strongly suggest that G. vaginalis expresses sialidase in the murine vagina and for the first time establish a prominent biochemical feature of BV in a murine infection model.

Figure 3

Vaginal sialidase activity correlates with G. vaginalis vaginal infection and is associated with ascending uterine horn infection.

A greater percentage of mice displaying vaginal sialidase activity also had G. vaginalis in their uterine horns than those mice that were sialidase negative (Fig. 3D). These data are consistent with the observation that higher vaginal titers increase the propensity for ascending infection.

G. vaginalis Interacts with Murine Vaginal Epithelial Cells in vitro and in vivo

Another feature of BV, and a component of the Amsel criteria for BV diagnosis, is the presence of epithelial cells with adherent bacteria, termed clue cells. G. vaginalis has been shown to interact with cultured human vaginal epithelial cells and experiments using vaginal biopsy [42] and vaginal fluid [58] samples suggests that bacteria may form an adherent biofilm on epithelial cells in the human vagina. Vaginal washes from G. vaginalis infected mice often contained clumps of epithelial cells with apparent attached bacteria (Fig. 4A, panel b, arrowheads).

Figure 4

G. vaginalis adheres to murine vaginal epithelial cells in vitro and in vivo.

Additionally, abundant hematoxylin-rich puncta, indicative of adherent bacteria, were also apparent in some samples upon H&E staining of vaginal sections (Fig. 4A, panel d). These results suggested that G. vaginalis may interact with murine vaginal epithelial cells; however, we could not definitively distinguish G. vaginalis from murine vaginal flora using histology. Previously used antibodies for immunofluorescence [36] were unavailable. Therefore, to provide further confidence that G. vaginalis could interact with murine vaginal epithelial cells we performed infection assays with fluorescently labeled G. vaginalis. First we assessed adherence ex vivo using epithelial cells present in vaginal washes from uninfected, β-estradiol treated mice. Epithelial cells were washed extensively to remove endogenous flora, then infected with fluorescently labeled G. vaginalis and visualized by fluorescent confocal microscopy. G. vaginalis adhered to mouse vaginal epithelial cells, appearing as distinct fluorescent puncta or biofilm-like collections decorating the epithelial cell surface (Fig. 4B). Similar puncta were not observed on epithelial cells inoculated with a “mock” preparation (containing the fluorescent label but no bacteria), demonstrating that this pattern of fluorescence was specific to G. vaginalis and not an artifact of the introduction of the RBITC label. Next we determined whether we could observe such interactions during in vivo infection. Following vaginal inoculation with fluorescently labeled G. vaginalis, similar fluorescent puncta were detected on epithelial cells in vaginal washes from infected mice at 4 hpi. Together, these results demonstrate that G. vaginalis adheres to murine vaginal epithelial cells, similar to what has been seen for human-derived cultured epithelial cells [37], [38].

G. vaginalis Infection Results in Robust Epithelial Exfoliation

While G. vaginalis did not elicit a robust inflammatory response, we observed that the epithelial surfaces of G. vaginalis infected mice displayed evidence of epithelial cell exfoliation (see Fig. 1C). To gain a semi-quantitative perspective of this phenotype, we scored the degree of histological exfoliation in slides that were blinded to the observer, with 0 being none and 3 being very robust (see Fig. 5B for representative images). Vaginal sections from G. vaginalis-infected mice had significantly higher exfoliation scores compared to mock-infected controls (Fig. 5A). Additionally, G. vaginalis infection resulted in increased thickness of the transitional epithelium (Fig. 5C), which correlated positively with exfoliation score (Fig. 5D), suggesting that there may be increased epithelial proliferation in response to the surface exfoliation. Inoculation of heat-killed G. vaginalis did not result in a significant increase in either epithelial exfoliation score or thickness (Fig. 5A&C), demonstrating that live bacteria are required to induce this response.

Figure 5

G. vaginalis induces a robust histological epithelial exfoliation and proliferation response.

As an additional assessment of this apparent exfoliation response, we examined vaginal washes by wet mount light microscopy. Consistent with our observations of H&E stained vaginal sections, vaginal washes from both mock and G. vaginalis infected mice contained predominantly epithelial cells with very limited, if any, leukocytes (representative images shown in Fig. 6A). We enumerated epithelial cells, again in a blinded manner, and found that G. vaginalis infected mice had significantly higher numbers of epithelial cells in vaginal washes compared to mock-infected animals or those exposed to heat-killed G. vaginalis (Fig. 6B). Finally, the degree of epithelial exfoliation in mice infected with G. vaginalis correlated positively with both vaginal wash CFU and sialidase activity (Fig. 6C–D), consistent with this response being relative to infectious burden. Together these results show that G. vaginalis JCP8151B induces a robust vaginal epithelial exfoliation response in a murine vaginal infection model.

Figure 6

G. vaginalis-induced exfoliation is evident in vaginal washes and correlates with vaginal titers and sialidase activity.

Epithelial Exfoliation as a Clinical Feature of Bacterial Vaginosis

Pathogen induction and blockade of host epithelial exfoliation responses has been described in other murine urogenital infection models. For example, uropathogenic E. coli (UPEC) induces exfoliation of superficial umbrella cells lining the bladder, which is thought to be consistent with the progression of acute UTI in humans [59][62]. However, N. gonorrhea has been shown to block vaginal epithelial exfoliation through interactions with human specific receptors (carcinoembryonic antigen-related cell adhesion molecules, CEACAMs) [63], [64]. Although the presence of epithelial clue cells is a well-established clinical feature of BV, we found no examples in the literature measuring whether the relative number of epithelial cells in clinical specimens is increased in women with BV compared to those with normal flora. To determine whether the increased number of epithelial cells seen in our murine model is a verifiable feature of human BV, we performed microscopic enumeration of vaginal epithelial cells on slides prepared from human vaginal swabs from women with (+) and without (−) BV as defined by Nugent score (7–10 and 0–3 respectively) (Fig. 7). Consistent with the murine model, significantly higher levels of epithelial cells were observed on slides prepared from specimens of women with BV compared to women with normal flora (Fig. 7). These results strongly suggest that an epithelial exfoliation host response occurs in the clinical setting of BV.

Figure 7

Human clinical samples display evidence of epithelial exfoliation associated with bacterial vaginosis.

Discussion

Bacterial vaginosis (BV) is a common vaginal condition in women [1], [13] and is associated with increased risk of sexually transmitted infection and adverse pregnancy outcomes, including preterm birth [13][22]. Despite its prevalence, the etiolog(ies) of BV symptoms and complications are poorly understood. There is an obvious and dramatic shift in the overall vaginal flora from a Lactobacillus-dominant state to one overrun by high titers of Gram-negative anaerobes and Actinobacteria. A handful of bacteria have come to be known as BV-associated bacteria, including G. vaginalis. However the contribution of these BV-associated bacteria to the overall disease state is largely undefined. A significant contributing factor to this dearth of understanding is the absence of relevant small animal models.

We found a few recent reports in the literature describing murine vaginal inoculation with G. vaginalis. Two of these papers investigated the effects of Lactobacillus probiotic strains on G. vaginalis colonization in an outbred mouse strain [54], [55]. However, the methods of isolation and enumeration of G. vaginalis vaginal titers in these papers were only loosely described. For example, G. vaginalis recovered from infected mice were reported as a percentage of the no probiotic control group rather than an absolute enumeration of recovered colony forming units, hampering assessment of the overall bacterial load. Although possibly explained by a difference in mouse strain used or conditions of the housing or breeding facility, there was also no reference to the incidence of contaminating vaginal flora, which we found invariably present in our mouse model. In addition to these two studies, there is one report of vaginal G. vaginalis infection in gnotobiotic mice [65], which naturally circumvents the issue of contaminating flora.

Here we describe a murine vaginal infection model with G. vaginalis that is, to our knowledge, the first to recapitulate key BV phenotypes. Technically speaking, this model is rather straightforward and very similar to infection models utilized for other vaginal pathogens. However, the model described here is distinguishable by the fact that previous studies of G. vaginalis infection in mice did not investigate BV-related phenotypes in vivo. Furthermore, we took multiple measures to ensure accuracy of our G. vaginalis titer enumeration, including 1) generating a streptomycin-resistant (SmR) isolate, and 2) confirming by PCR that bacteria re-isolated from mouse vaginal washes were G. vaginalis. Vaginal inoculation with G. vaginalis was sufficient to yield 1) vaginal sialidase activity; 2) epithelial cells with adherent bacteria (reminiscent of clue cells); 3) epithelial exfoliation in the absence of an inflammatory infiltrate. Together, results from this model demonstrate that G. vaginalis likely plays a role in generating these clinical features observed in humans and each is discussed in further detail below.

Sialidase Activity

Sialidase activity is rarely detected in women with normal flora [19], [66], [67]. In pregnant women, sialidase activity has also been independently correlated with increased risk of chorioamnionitis and PTB [19], [68]. Production of sialidase by isolated BV-associated bacteria grown in culture strongly suggests that BV-associated sialidases are bacterial in origin [69], [70]. Bacterial sialidases have been characterized as virulence factors in bacterial infections of various mucosal sites [71]. Our previous in vitro studies have highlighted the potential role of G. vaginalis sialidase in the deglycosylation and degradation of host glycoproteins [72]. In our murine model, the level of sialidase activity correlates with vaginal G. vaginalis titers and sialidase positive mice are more likely to develop uterine horn infection. These data suggest that higher vaginal titers are more likely to result in ascending infection. Although further studies are required, these results may also suggest potential role for sialidase in the mechanisms facilitating ascending infection. Consistent with this idea, sialidase activity levels in pregnant women correlated directly with increased risk of chorioamnionitis and preterm birth [7], [68].

G. vaginalis and the Formation of Clue Cells

It has long been established that BV bacteria interact with epithelial cells. In fact, the presence of exfoliated clue cells is a qualitative diagnostic feature of the disease [73], [74]. It has previously been suggested that G. vaginalis may be responsible for clue cell formation, since it was detected on the surface of exfoliated vaginal epithelial cells more frequently and at higher levels than the BV-associated anaerobes Mobiluncus, Bacteroides, and Fusobacterium [39]. A more recent high-resolution phylogenetic study examining correlations between different species of BV bacteria and clinical features revealed that G. vaginalis is positively associated with the presence of clue cells [75]. G. vaginalis has also been shown to interact with a vaginal epithelial cell line [36] and epithelial cells present in human clinical samples [40], [76]. We demonstrated, using fluorescently labeled bacteria, that G. vaginalis interacts with murine vaginal epithelial cells, forming clue-like cells in vitro. Labeled G. vaginalis was also detected on the surface of epithelial cells recovered from vaginal washes of mice following infection with G. vaginalis. Together these data provide evidence that our murine model of G. vaginalis infection displays this important feature of BV and provides an avenue for investigating clue cell formation in vivo.

G. vaginalis Induces Epithelial Exfoliation in the Absence of Overt Inflammation

BV is characterized by a heavy overgrowth of Actinobacteria and Gram-negative anaerobes but a surprising absence of the type of inflammatory infiltrate seen in other urogenital infections such as gonococcal infection [77], [78] or urinary tract infection [46], [79], [80]. Epithelial shedding, or exfoliation, appears to be a common mechanism of protection employed by mucosal surfaces [81], [82]. G. vaginalis vaginal infection in mice produced a robust exfoliation response that correlated directly with vaginal titers and vaginal sialidase activity.

Consistent with the lack of overt inflammation in women with BV [83], our histological and wet mount microscopy analyses of vaginal specimens from mice infected with G. vaginalis displayed no signs of polymorphonuclear leukocyte (PMN) recruitment. Even though estradiol treatment suppresses the influx of PMNs that naturally occurs in mice after ovulation [84], literature evidence has shown that PMN recruitment to the vagina can still occur upon infectious challenge in C57/Bl6 mice [85]. Although no quantitative data was given, Teixeira et al. reported the presence of ‘inflammatory lesions’ in the vaginas of gnotobiotic mice infected with G. vaginalis [65]. It is reasonable to suspect that indigenous microflora in the vagina may contribute to host innate immune responses, as has been shown in the gut [86], [87]. This may influence inflammatory responses to G. vaginalis in mice lacking endogenous flora. Further studies are required to provide a better understanding of the role of G. vaginalis in the apparent suppression of inflammatory responses that occurs during BV. Recent biochemical and genomic investigations have revealed that G. vaginalis isolates can be remarkably diverse [88][91], perhaps allowing different host responses to various strains of G. vaginalis.

Epithelial Exfoliation as a Measurable Clinical Feature: Implications for Understanding BV

Epithelial exfoliation has long been discussed in the BV literature, most often with regard to clue cells. Although there are countless references to “increased epithelial exfoliation” in BV, we found no examples of quantitative analysis in a defined experimental or clinical setting. We present evidence that an increased vaginal epithelial exfoliation response, a robust feature of the G. vaginalis animal model described here, is also apparent in Gram-stained images of vaginal fluids from women with BV (Nugent 7–10) compared to normal controls (Nugent 0–3). Our analysis of clinical samples provides the first concrete evidence to classify an exfoliation response as a BV phenotype.

The modest but significant increase in the number of exfoliated epithelial cells in women with BV may be a beneficial response if it removes adherent potential pathogens and when surface epithelial layers can be replenished. However, excessive exfoliation may promote access to underlying tissue, which may facilitate the establishment of BV-associated bacteria and increase the risk of secondary infection. In fact, BV is known to be associated with increased risk of certain sexually-transmitted infections (STI) [18], [92], [93] and some vaginal pathogens can take advantage of the exfoliation process to facilitate access to underlying tissue. For example, T. vaginalis causes contact-dependent cytotoxicity upon adherence to vaginal epithelial cells, thereby leading to exfoliation and erosion of the epithelium. It has been suggested that this may allow trichomonads into extracellular matrix and basement membrane sites within the vaginal tissue [45]. It is possible that removal of the outer epithelial cell layer by G. vaginalis provides a niche for formation of an adherent biofilm, which has been observed in vaginal biopsies from women diagnosed with BV [42]. Interestingly, BV correlates with T. vaginalis infection, therefore it is conceivable that exfoliation induced by G. vaginalis and T. vaginalis could be mutually beneficial and may also impact other vaginal organisms. Interestingly, some vaginal microbicides have been shown to have paradoxical effects, actually increasing susceptibility to HIV and other sexually-transmitted pathogens. This increased susceptibility was found to be coincident with rapid exfoliation and re-growth of epithelial cell layers [94]. These previous findings are consistent with the idea that epithelial exfoliation in BV may contribute to increased STI risk. Future studies should examine whether epithelial exfoliation may contribute to the overall risk of secondary infections associated with BV.

We found a single reference in the literature that performed quantitative analysis of vaginal epithelial shedding (by counting epithelial cells present in vaginal lavage samples). Interestingly, this study uncovered a link between vaginal epithelial exfoliation and smoking [95], a behavior that has been shown to be associated with BV [96]. If exfoliation promotes vaginal colonization by BV bacteria, it is tempting to speculate that the link between smoking and BV could be explained, at least in part, by the initiation of epithelial exfoliation that occurs in smokers. Ultimately, the downstream ramifications of epithelial exfoliation for the overall pathophysiology of BV remain to be explored and the new murine model presented here provides a valuable tool for these investigations.

G. vaginalis as a Pathogen

Since it was first described, there has been vigorous debate in the literature regarding the role of G. vaginalis in BV. G. vaginalis is one of the most frequently isolated bacterial species from women with BV. Consistent with the notion of G. vaginalis as a potential pathogen, strains identified as G. vaginalis have been isolated from placenta, amniotic fluid, and blood [32][34]. G. vaginalis has also been implicated in uterine infections and development of endometritis [97]. Results from a comparison of epithelial adhesion, cytotoxicity and biofilm formation between several BV associated bacteria suggested that G. vaginalis may be more virulent than other species associated with the disease [37]. The observation that G. vaginalis produces BV phenotypes in our murine model, in the absence of other BV-associated bacteria, emphasizes its likely role in BV etiology. However, the main controversy appears to lie in the fact that G. vaginalis can also be detected from women with normal flora [2], [27], [29][31]. We argue that the presence of G. vaginalis in healthy individuals does not constitute a basis for disregarding this bacterium in the causes and complications of BV. Just as “healthy” people can be asymptomatic carriers of such pathogens as Streptococcus pneumoniae [98], Group A Streptococcus [99], [100], Haemophilus influenzae [98] or Clostridium difficile [101], carrier states may also exist for G. vaginalis. Indeed, recent genomic and phenotypic studies support the hypothesis that variations in bacterial strain virulence, titers, and/or windows of host susceptibility may bring a colonization state to a state of pathogenesis [38], [88], [91], [102], [103].

In summary, our results demonstrate for the first time that G. vaginalis is sufficient to yield key BV phenotypes in an animal model. The quantitative experimental methods described here show that infection with G. vaginalis leads to vaginal sialidase activity, bacterial adherence to vaginal epithelial cells, and a robust epithelial exfoliation response that we demonstrate as a relevant clinical feature of BV in quantitative, controlled experiments. These data provide strong evidence that G. vaginalis can play an active role in generating clinical features associated with BV. The phenotypic parallels to human BV displayed in the murine system provide a new experimental tool with great potential to expand our understanding of G. vaginalis-host interactions in the vagina.

Materials and Methods

Ethics Statement

Vaginal swabs were collected as part of the Contraceptive CHOICE project [104] according to protocols approved by the Washington University Institutional Review Board (IRB ID# 201108155). Mouse experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Studies Committee of Washington University School of Medicine (Protocol Number: 20110149).

Bacterial Strains and Growth Conditions

Gardnerella vaginalis clinical isolate JCP8151B (GenBank JX860320) was obtained from a vaginal swab from a woman with BV (based on Nugent score) obtained in accordance with IRB-approved protocols in collaboration with the Washington University Contraceptive CHOICE Project (IRB ID# 201108155). The vaginal swab was transported from the clinic to the lab using Port-A-Cul™ pre-reduced anaerobic transport media tubes (BD). Tubes were brought into a Vinyl anaerobic airlock chamber (Coy Products) under an atmosphere maintained at approximately 1% hydrogen and 0 ppm oxygen. Within 24 hours and the swab was used to inoculate “Gardnerella semi-selective media,” (agar plates with 5% defibrinated sheep blood, 10 mg/L colistin, 10 mg/L nalidixic acid, and 4 mg/L amphotericin B). Plates were pre-incubated in the chamber for at least 16 hours for equilibration to anaerobic conditions and were incubated anaerobically post-inoculation at 37°C for 24–48 hours. Translucent pinpoint colonies were isolated, and candidate G. vaginalis strains were tested by diagnostic PCR using primers previously reported to be specific for G. vaginalis (forward primer GGGCGGGCTAGAGTGCA and reverse primer GAACCCGTGGAATGGGCC) [56]. Additional validation of the G. vaginalis identity was obtained by sequencing 16S rDNA. Full details of this strain are being reported in a separate manuscript.

A spontaneous streptomycin resistant mutant, JCP8151B-SmR #4, was isolated by plating JCP8151B (concentrated from a 2 day, 25 mL NYC-III culture) on Gardnerella semi-selective media +1 mg/mL streptomycin and selecting resistant colonies after incubating anaerobically at 37°C for 72 hours. We confirmed the streptomycin resistance in this isolate was due to point mutation of the Rpsl gene (as that reported for other bacteria) by amplifying G. vaginalis Rpsl with primers rpsL F2 (CATGGTTTAAGGTGTGCTG) and rpsL R (GTTAATCAACTGAGCCACG) and sequencing using rpsL F2. To confirm that the point mutation did not result in any apparent growth defects or changes in sialidase activity, JCP8151B and JCP8151B-SmR #4 were grown anaerobically overnight in 5 ml NYC-III medium at 37°C, then diluted to OD600 of 0.1 in 5 mL NYC-III medium. A 25 µL aliquot of each bacterial suspension was analyzed for sialidase activity as described below. The remaining bacterial suspension was incubated anaerobically at 37°C for 24 hr to monitor growth. Aliquots were removed at 0, 1, 3, 6 and 24 h, serial diluted and plated on Gardnerella semi-selective media to enumerate colonies. Results for sialidase activity and growth curves were indistinguishable between JCP8151B and JCP8151B-SmR. The JCP8151B-SmR isolate was used for murine infection model experiments described below. Control experiments (as dictated in figures and figure legends) treated a parallel group of mice infected with JCP8151B-SmR that was heat-killed by incubation of the bacterial inoculum at 80°C for 10 min.

Murine Vaginal Infection Model

Female C57/Bl6 mice (6–8 weeks) were injected intraperitoneally with 0.5 mg β-estradiol in 100 µL filter-sterilized sesame oil three days prior to and on the day of inoculation. Mice were anaesthetized with isofluorane and inoculated vaginally with ∼5×107 CFU G. vaginalis in 20 µL sterile PBS (OD600 = 5.0). Vaginal washes were collected by flushing vaginas with 50 µL sterile PBS using a P200 pipet (GeneMate), pipetting up and down 10x, followed by rinsing into an additional 10 µL PBS in a sterile 1.5 mL Eppendorf tube. G. vaginalis titers were determined from washes by preparing 10-fold serial dilutions in PBS (in the anaerobic chamber) and spotting 5 µL of each dilution in quadruplicate onto 1 mg/mL streptomycin selection plates (either Gardnerella semi-selective media or NYC-III agar). Colonies were then enumerated and reported as recovered colony forming units (CFU) per mL of vaginal fluid. Vaginal washes were also analyzed for sialidase activity and epithelial exfoliation as described below.

Mice were sacrificed at 24 hpi or 72 hpi to harvest vaginas and uterine horns. One uterine horn and half of the vagina (bisected longitudinally) from each mouse was homogenized followed by serial dilution and plating as for vaginal washes. Colonies were enumerated and reported as CFU per gram of tissue. The remaining vaginal tissue and uterine horn from each mouse were fixed in 10% buffered formalin phosphate at room temperature followed by paraffin embedding. Histological slide preparation and H&E staining were performed by the Washington University School of Medicine Histology Core.

Sialidase Activity Assays

Vaginal wash samples collected as described above (25 µL) were diluted 1[ratio]2 with 100 mM sodium acetate pH 5.5 containing 300 µM 4-methylumbelliferyl-Neu5Ac (50 µL). Substrate hydrolysis was monitored using a Tecan M200 plate reader.

G. vaginalis Interaction with Murine Vaginal Epithelial Cells in vitro and in vivo

G. vaginalis JCP8151B was fluorescently labeled with Rhodamine B isothiocyanate (RBITC; Aldrich 283924). RBITC was prepared fresh at 0.2 mg/mL in 20 mM HCl and 5 µL of this stock was added to 500 µL G. vaginalis JCP8151B in sterile PBS (prepared as described for mouse experiments above). The bacteria were then incubated anaerobically at 37°C for 30 min, centrifuged, resuspended in 500 µL NYC-III and allowed to recover with an additional 30 min, 37°C anaerobic incubation. Finally, the labeled bacteria were washed twice with PBS and resuspended in 500 µL PBS for inoculation of epithelial cells, as described below. For each experiment a “mock” labeled sample, lacking bacteria, was prepared in parallel.

While G. vaginalis was being labeled, vaginal washes were collected from β-estradiol-treated mice, as described above. Washes were pooled and spun at 300 g for 5 min to collect epithelial cells. Epithelial cells were then washed 3 times with sterile PBS to remove the majority of endogenous flora and then distributed in 50 µL aliquots into 1.5 mL eppendorf tubes. RBITC-labeled G. vaginalis or the “mock” labeled sample (5 µL) were added to the epithelial cells and the samples were rotated at 37°C for 3 hours. Finally, epithelial cells were washed twice to remove unassociated bacteria and then visualized on an Olympus BX61 confocal fluorescent microscope using SlideBook 5.0 software.

For in vivo analyses, G. vaginalis was labeled with RBITC as described for in vitro experiments and then inoculated vaginally into β-estradiol-treated mice as described above. An additional group of mice was inoculated with a “mock”-label preparation containing no bacteria. At 4 hpi, mouse vaginas were washed with 50 µL PBS and epithelial cells were visualized by confocal fluorescent microscopy as described above.

Analysis of Murine Epithelial Cell Exfoliation

H&E stained mouse vaginal histology sections collected above were visualized on an Olympus BX61 microscope to assess the degree of inflammation (24 hpi and 72 hpi time points) and epithelial exfoliation (24 hpi time point). Images were captured and epithelial thickness was measured using StreamStart® software, with averages calculated from 5 measurements per vaginal section.

For assessment of epithelial exfoliation in mouse vaginal washes, wet mounts were prepared with 5 µL vaginal wash (from the 24 hpi time point) and visualized by phase contrast microscopy using an Olympus BX61 microscope. Five representative images were captured from each specimen (1 per mouse) and epithelial cells were counted from each image to determine an average.

Clinical Specimen Handling and Analysis of Epithelial Exfoliation

Vaginal swabs (Starplex) were collected as part of the Contraceptive CHOICE project [104)] according to protocols approved by the Washington University Institutional Review Board (IRB ID# 201108155) and underwent Nugent scoring using published methods as previously described [72], [105]. Gram stained slides (the same used for Nugent scoring) were analyzed for epithelial exfoliation by enumerating epithelial cells in three representative images as described for murine samples above.

Statistical Analysis

GraphPad Prism 5.0 software was used for all statistical analyses presented. The statistical tests used to analyze each set of data are indicated in the figure legends.

Acknowledgments

We thank Jeffrey Peipert, Jenifer Allsworth, Gina Secura, and Jennifer Bick for contributions of Contraceptive CHOICE project clinical specimens and confirmatory data. We recognize and thank Justin Perry and Lloyd Robinson for technical assistance. We are thankful for help from Fredrick Kraus with analyzing H&E stained histology slides and are grateful to Jennifer Lodge for use of her confocal microscope.

Funding Statement

This work was supported in part by a Basil O’ Connor award from the March of Dimes (to ALL), a Translational Research Pilot Grant funded by the Barnes-Jewish Hospital Foundation (to ALL and WGL), and a postdoctoral fellowship from the American Heart Association (to NMG). The study was also supported in part by NIH grant P50DK064540-11 (PI: Hultgren). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Recent mouse and rat methods for the study of experimental oral candidiasis

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Abstract

The Candida genus expresses virulence factors that, when combined with immunosuppression and other risk factors, can cause different manifestations of oral candidiasis. The treatment of mucosal infections caused by Candida and the elucidation of the disease process have proven challenging. Therefore, the study of experimentally induced oral candidiasis in rats and mice is useful to clarify the etiopathology of this condition, improve diagnosis, and search for new therapeutic options because the disease process in these animals is similar to that of human candidiasis lesions. Here, we describe and discuss new studies involving rat and mouse models of oral candidiasis with respect to methods for inducing experimental infection, methods for evaluating the development of experimental candidiasis, and new treatment strategies for oral candidiasis.

Keywords: Candida albicans, oral candidiasis, murine model, oral cavity

Introduction

Oral candidiasis is caused by Candida yeasts, which are present in the oral cavities of approximately 50% of healthy individuals. Candida albicans is the most virulent and prevalent species, followed by the non-albicans species C. tropicalis, C. glabrata, C. parapsilosis, C. krusei, C. dubliniensis, and others.1-7 C. albicans colonizes the oral surface and can cause damage through the expression of its virulence factors, including adherence to host cells, morphological transition, hydrophobicity and secretion of hydrolytic enzymes.8-10 A major virulence factor of C. albicans is its ability to adapt to a variety of different habitats and the consequent formation of surface-attached microbial communities known as biofilms.11 Initially, the biofilm is established by the adherence of yeast to host cells or abiotic surfaces, e.g., prostheses. These yeast form colonies, produce germ tubes and filaments, and secret extracellular polymeric substance (EPS) that contribute to the three-dimensional structure of the biofilm.12,13 The organization of the biofilm protects the C. albicans cells from host immune mechanisms and antifungal agents.12,14,15

C. albicans causes disease mainly in individuals with local or systemic risk factors,16 including denture use,17 steroid inhaler use,18,19 reduced salivary flow,20 a high-sugar diet,21 extreme age or youth,22 endocrine disorders,23 nutritional deficiencies,24 receipt of broad-spectrum antibiotics,25 and immunosuppression.5,7,26 In fact, oropharyngeal candidiasis (OPC) is the most frequent opportunistic infection encountered in HIV positive individuals. The disease occurs in up to 90% of patients during the course of HIV infection.27,28

Candida infections of the oral mucosa manifest in various guises and are subdivided into pseudomembranous, erythematous, and hyperplastic. The last form is chronic, whereas the first two are classified as acute lesions. Hyperplastic candidiasis is the least common of the triad of major clinical variants. Pseudomembranous candidiasis or thrush, which is characterized by white patches on the surface of the buccal mucosa, tongue, and the soft palate, occurs in patients using corticosteroids topically or by aerosol, in HIV-positive patients, and in other types of immunocompromised patients. Erythematous candidiasis is characterized by localized erythema of the oral mucosa that commonly occurs on the tongue and the palate and is associated with broad-spectrum antibiotics, corticosteroids, and HIV infection. In the tongue dorsum, erythematous candidiasis presents depapillated areas caused by the loss of filiform papillae. In addition to the three major variants of oral candidiasis, there are other clinical manifestations that are called “Candida-associated lesions”, since the yeast is not the sole etiologic agent. These lesions include denture stomatitis, angular cheilitis, and median rhomboid glossitis.29-34

The recognition that C. albicans is an important pathogen, especially in immunocompromised patients, has led to the development of a suitable experimental model that supplies a standard and easy tool to control and manipulate the assays required to obtain knowledge of the disease process.35,36 Thus, various animal models have been used to explore the host–Candida interaction during oral infections to assess the etiopathology, diagnose and manage the disease, and test therapeutic approaches.37-43 Therefore, the purpose of this review is to discuss new studies in the literature that describe experimental oral candidiasis in rats and mice and to compare different methods for the induction, development and treatment of oral candidiasis.

Structure of the Tongue Dorsum of Rats and Mice (Normal Aspects)

Since the tongue dorsum is the primary habitat of the yeast in the oral cavity of healthy and immunocompromised individuals,44 most studies of experimental oral candidiasis in rodent models were performed on the tongue dorsum. Therefore, the general morphological features observed in the tongue dorsum of rats and mice are described below.

The rat or mouse tongue is a muscular organ coated with keratinized stratified squamous epithelium that forms an upper region, the dorsum, and a lower region, the ventral. Under the epithelium, the tongue is formed of conjunctive tissue, which is rich in blood vessels, and striated muscle, in which the fibers are grouped in bundles in three planes. Among the muscle fibers of the posterior region, there are serous and mucous salivary glands.

The tongue dorsum presents a buccal section, which extends from the tip to the vallate papilla, and a pharyngeal section, which extends from the vallate papilla to the most posterior area of the tongue dorsum. Similar to the human tongue dorsum, the buccal section of these rat and mouse models is formed by several projections, namely filiform and fungiform papillae (Fig. 1). In rats and mice, filiform papillae are classified as simple conic papillae (located on the anterior two thirds of the tongue dorsum), true papillae (located on the intermolar tubercle), and giant papillae (located between the simple conic papillae and the true papillae). Among the simple conic papillae, there are a lower number of fungiform papillae, which are short and broad with a rounded base that contains the taste corpuscle in the epithelium of the superior surface (Fig. 2).

figure viru-4-391-g1

Figure 1. Morphological features of the murine tongue dorsum. (A) Macroscopic aspect of the mouse tongue dorsum. The vallate papilla (VP) shared the pharyngeal section (PS) from the buccal (___). In the buccal section, we can see the area of simple
figure viru-4-391-g2

Figure 2. Sagittal cut of the tongue dorsum of the rat. (A) The simple conic papillae are short and distant from each other with many interpapillar surfaces, HE: 200×. (B) The true papillae are more elongated and closer to each other,

Models of Experimental Oral Candidiasis in Rats

There are two varieties of rats, Sprague-Dawley and Wistar, that are commonly used to study experimental oral Candida infections. Rats have the advantages of a low maintenance cost, ease of breeding and handling, ready availability, and a sufficiently sized oral cavity that easily permits inoculation and sample collection.35,36 Moreover, the tongues of the rats are easily colonized by C. albicans, eliciting disease conditions such as erythematous candidiasis and median rhomboid glossitis.36,45 This model is useful for studying long-term candidal colonization and chronic infection.35 For the establishment of persistent oral candidiasis in rats, C. albicans infection has been induced by exclusion of the protective effects of normal salivary flow, alteration or manipulation of the oral cavity by the administration of antibiotics and treatment with immunosuppressive agents.35,46

It has already been established that in humans alterations in salivary glands caused by Sjögren syndrome, radiotherapy, advanced age, and some medications lead to a reduction in salivary flow and xerostomia, favoring the development of oral candidiasis by decreasing the mechanical removal and action of antimicrobial peptides present in saliva.47-49 This condition has been used by various researchers to induce oral candidiasis in rat models.38,47-50 Green et al.50 studied C. albicans’ expression of ALS genes (encoding large cell-surface glycoproteins that function in the process of adhesion to host surfaces) in the hyposalivatory Sprague-Dawley rat model after surgical removal of the salivary glands. At 5 d post-infection, disease progression produced more severe lesions in a defined hierarchy of lesion severity (tongue > mandible > buccal mucosa > palatal and pharyngeal mucosa) and a greater number of different ALS transcripts, such as ALS1–5 and ALS9. The expression of the ALS gene in the experimental candidiasis in rats were compared with clinical specimens collected from HIV-positive patients, demonstrating a similarity in expression patterns between the rat model and the human clinical specimens. This study suggests that the hyposalivatory rat model can be useful for studying gene expression in oral candidiasis.

Immunosuppression and administration of broad-spectrum antibiotics are also important risk factors for developing of oral candidiasis in humans.29,30 Thus, these predisposing factors have been widely used to induce oral candidiasis in rats.45,51,52 Chami et al.53 compared the effect of substances from a vegetal source (carvacrol and eugenol) to nystatin on experimental oral candidiasis induced in Wistar rats that were immunosuppressed with dexamethasone and treated with tetracycline in the drinking water. Microbiological analysis showed that treatment with carvacrol, eugenol, and nystatin reduced 94.5%, 76.9%, and 91.54% of the CFUs of C. albicans, respectively, compared with a control group (infected and untreated). Histologically, the control group showed extensive colonization of the epithelium of the tongue dorsum by numerous hyphae. For the group treated with carvacrol, no C. albicans were observed in the folds of the tongue mucosa. However, under eugenol and nystatin treatment, hyphae were found in some folds of the tongue mucosa. The authors reported that this model of experimental oral candidiasis in rats was a simple and highly reproducible method of studying the efficacy of antifungal agents.

Junqueira et al.52 induced oral candidiasis in Wistar rats while treating the animals with tetracycline only in the drinking water. A C. albicans suspension was dripped into the mouth and spread on the tongue with a swab that was previously soaked in the suspension. This study verified that the majority of the rats no longer showed C. albicans colonization after 8 d of infection, suggesting that during the development of experimental candidiasis, the yeast and hyphae are eliminated from the organism by the host’s immune system. However, all animals showed typical candidiasis lesions, which were characterized by numerous epithelial lesions, including epithelial hyperplasia, disorganization of the basal layer, exocytosis, spongiosis, and loss of filiform papillae. After the establishment of candidiasis lesions, the rats were treated with photodynamic therapy (PDT) using methylene blue and a laser. Subsequently, the action of PDT on oral candidiasis was evaluated by the quantification of the epithelial lesions and by the intensity of inflammatory infiltrate in the conjunctive tissue. The rats treated with photodynamic therapy exhibited fewer epithelial alterations and had a lower inflammatory response than the untreated rats, suggesting that PDT was effective for the treatment of oral candidiasis. Furthermore, the experimental oral candidiasis in rats was a useful model to assess the effects of in vivo PDT.

In addition to the studies relating to genes expression and new antifungal countermeasures, such as substances from a vegetal source and PDT, rat models have also been used to study the influence of physiological factors on oral candidiasis. The influence of ovarian hormone on the development of oral candidiasis was demonstrated in the literature by Junqueira et al.39 These authors inoculated the oral cavity of ovariectomized Wistar rats and sham-ovariectomized rats with C. albicans suspensions. The ovariectomized rats presented fewer occurrences of candidiasis than sham-ovariectomized rats (33% vs. 75%) and exhibited a faster recovery time (22 d vs. 67 d), suggesting that ovarian hormones have a significant influence on oral candidiasis.

Since psychological stress has been found to suppress cell-mediated immune responses that are important for limiting the proliferation of C. albicans, Núnez et al.54 evaluated the effects of stress on oral candidiasis in rats exposed to a repeated auditory stressor. The results showed that stress exacerbated C. albicans infection in the tongue dorsum of the rats. Significant increases in C. albicans counts, the percentage of the tongue’s surface covered with clinical lesions, the percentage of abnormal papillae, and the colonization of the epithelium by hyphae were found in stressed rats compared with the nonstressed rats.

Another advantage of oral candidiasis model in rats is that this model allows the placement of a prosthetic device in the palate to mimic human Candida denture stomatitis, the most common form of oral candidiasis in the elderly population denture wearers. Unlike mice, rats have a larger oral cavity that facilitates denture placement. Previous models of denture stomatitis in rats were pioneered in the 1970s and 1980s. However, research since then has been scarce, presumably due to the HIV epidemic and the concomitant rise in the incidence of oropharyngeal candidiasis.55 Recently, new studies using rat models for denture stomatits were developed with a focus on Candida biofilm formation.55,56 Nett et al.56 induced denture stomatitis in Sprague-Dawley rats by using acrylic denture material applied over a palate around the cheek teeth (8 by 10 mm), immunosuppression with cortisone, and subsequently administering C. albicans (108 cells/mL) on the palate. The analysis of the images revealed a biofilm composed of adherent cells, yeasts, hyphae, host cells encased in extracellular matrix, and associated bacteria. The mucosal histopathology was consistent with that of acute human denture stomatitis, as it exhibited fungal invasion and neutrophil infiltration. In this study, the authors demonstrated that the oral denture model in rats can be very useful for testing the impact of gene disruption on biofilm formation, studying anti-infectives agents, examining the biology of mixed Candida-oral bacterial flora biofilm infections, and characterizing the host immunologic response to the disease process.

Models of Experimental Oral Candidiasis in Mice

The major advantage of using mice instead of rats for experimental oral candidiasis is that the immunobiology of the healthy mouse mucosa has also been fairly well characterized, enabling the suitable evaluation of the humoral and cellular immune response in oral candidiasis.35,45,46 Additionally, mice are widely available, easy to handle and to inoculate, and inexpensive to maintain.35,46 The availability and ease of production of genetically modified animals is another advantage of mice.46,57 The development of transgenic and knockout mice with targeted immune defects has prompted a more closely focused assessment of the role of specific components of the immune response to C. albicans.58 According to Samaranayake and Samaranayake,35 the variants of the mouse model include gnotobiotes, athymic (nu/nu), euthymic (nu/+), beige (bg/bg), black (bg/+), beige athymic (bg/bg nu/nu), beige euthymic (bg/bg nu/+), severe combined immune deficiency (SCID), and murine acquired immune deficiency syndrome (MAIDS). Nevertheless, the mouse model has the disadvantage of a small oral cavity that interferes with the examination of the mucosa by the naked eye.

As in rat models, mice models require the use of immunosuppressive agents or other predisposing factors to establish persistent infection since these animals are not naturally colonized by C. albicans.35,38,46,59-61 The effect of xerostomia in sialoadenectomized mice prolonged oral colonization by C. albicans to 75 d post-infection vs. 30 d of colonization for the control animals.38 With 1 or 4 inoculations with a C. albicans suspension, the last procedure increased the permanence of C. albicans in the oral cavity of sialoadenectomized mice and produced lesions in the tongue that contained pseudohyphae inside the epithelium, acanthosis, and neutrophilic infiltrate forming intraepithelial microabscesses, with a higher prevalence of lesions in the true and simple conic papillae.59 This model favored the permanence of C. albicans in the oral cavity, and higher frequencies of yeast inoculation influenced the presence and extension of candidiasis lesions.

Rhaman et al.60 reported a new murine model of oral candidiasis using estrogen administration. BALB/c mice received a weekly intramuscular and subcutaneous 5 µg dose of estrogen, of which the intramuscular administration was more powerful for inducing oral colonization. After 2 and 3 weeks of C. albicans infection, fungal cells and hyphae were detected in the keratinized and upper prickle-cells layer of the epithelium at varying sites, namely, the tongue dorsum, the buccal mucosa, the mouth floor, the palate, and the lingual alveolar mucosa. By week 5, fungal cells and hyphae were still clearly present in the oral epithelium. In this study, the authors also evaluated immunization for C. albicans. The animals were immunized intranasally on two consecutive days with 5 μg purified Sap2 (secreted aspartyl proteinase 2) that is a putative virulence factor of C. albicans thought to contribute to human mucosal infections). On days 14 and 15 after the second immunization, mice were inoculated orally with C. albicans and exhibited a significant reduction of the fungal burden compared with the control group. According to the authors, this model of mucosal colonization can be used to assess potential vaccine candidates and permits the detailed analysis of host-fungal interactions during the natural state of Candida colonization.

Although predisposing factors, such as xerostomia and estrogen administration, have been used for inducing oral candidiasis in mice, most studies have been performed in immunosuppressed mice models.37,41,43,62-68 Takakura et al.37 developed a murine model for experimental candidiasis that combines immunosuppression and treatment with tetracycline for successful disease progression, allowing the study of fungal pathogenesis and new therapeutic options. These authors observed symptoms characteristic of oral thrush in the oral cavities of mice treated with prednisolone at a concentration of 100 mg/kg body weight and 0.83 mg/mL tetracycline in the drinking water 1 d prior to infection. At 3 and 7 d post-infection, 105–106 CFU were recovered from each mouse, and whitish, curd-like patches were observed on most of the tongues. Animals with symptoms of pseudomembranous candidiasis showed extensive colonization on the epithelium of the dorsal surface by numerous hyphae, as well as the severe destruction of the epithelial layers. Furthermore, this model was useful for verifying the therapeutic activity of fluconazole and amphotericin B, both of which reduced C. albicans and cured the lesions.

This murine model of experimental candidiasis, developed by Takakura et al.,37 inspired many other studies, which are described in Table 1.37,41,43,62-68 The advantage of this model is the development of thrush-type oral candidiasis that mimics the natural infection in humans and is useful for both symptomatological and mycological evaluation of the responsiveness to antifungal treatments. Therefore, several treatments for oral candidiasis were tested in this model including: salivary factors,62,63 quorum-sensing molecules,64 photodynamic therapy,43,66 natural plant extracts,41,67 and inhibitors of efflux pump.68

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Table 1. Studies of experimental candidiasis using the methodology developed by Takakura et al. (2003)26 for establishment of a mice model with local symptoms characteristic of oral thrush that mimics the natural infection in humans

In addition to the murine model developed by Takakura et al.,37 other models of experimental candidiasis in immunosuppressed mice have been published in the literature and are described below. Teichert et al.69 induced oral candidiasis in beige nude mice with severe combined immunodeficiency disease (SCID) by swabbing the oral cavity with C. albicans 3 times a week for a period of 4 weeks. The animals were treated with photodynamic therapy using methylene blue and diode laser light. The therapy exhibited antifungal activity, decreasing the number of fungal cells and increasing the inflammatory infiltrate and neutrophilic exocytosis, thereby demonstrating the capability of the SCID model to generate an immune response despite the absence of T cells and B cells. Solis and Filler61 described a simple method to induce oropharyngeal candidiasis by injecting cortisone acetate (220 mg/kg) subcutaneously before infection with C. albicans. The mice were inoculated sublingually for 75 min with a swab saturated with the C. albicans suspension. On day 5 after inoculation, 105–106 CFU were recovered from the oral tissue, and the histopathology showed characteristics of oropharyngeal candidiasis.

In summary, multiple experimental models of oral candidiasis using immunosuppressed mice have been developed and, regardless to the type of immunosuppression used, the animals developed typical lesion of pseudomembranous candidiasis that is seen in humans. These models have become important tools to study the virulence of C. albicans mutants, the host response to mucosal infection, and the efficacy of new antifungal agents.

Methods for Analysisof Experimental Oral Candidiasis

To estimate colonization by C. albicans in the rat or mouse oral cavity, CFU counting is the most useful method. Samples from the entire oral cavity or only from a specific site, e.g., the surface of the tongue, are collected with the aid of a swab for posterior CFU counting onto Sabouraud dextrose agar (SDA) with antibiotics. This method assesses whether the animals were colonized by C. albicans and the antifungal effects of the therapeutic modality tested.37,42,43,60,69 Additionally, the tongue and other oral tissue can be removed from the oral cavity, homogenized in saline solution, and later plated on SDA.61 After plating, the Petri dishes are incubated at 35–37 °C for 24 or 48 h.

Acute oral candidiasis can present as pseudomembranous or erythematous forms. These manifestations cause macroscopic lesions (Fig. 3) that can be quantitatively analyzed. Takakura et al.37 evaluated macroscopic lesions of pseudomembranous candidiasis by scoring lesions from 0 to 4 according to the extension and severity of whitish, curd-like patches on the tongue surface. The authors also described the appearance of lesions consisting of white patches on the tongue dorsum. This methodology has been used by many others authors to analyze the development of disease during their experiments.41,43,62,63,67,68

figure viru-4-391-g3

Figure 3. Macroscopic analysis of a mouse tongue with oral candidiasis presenting whitish regions (▲) and papillary atrophy (↓).

After euthanasia of the animals, the tissues are removed and prepared for histopathological study. The sections are stained with periodic acid-Schiff (PAS) reagent, which allows for the verification of the extent of fungal filamentation and the depth of tissue penetration.61 Some authors proposed a quantification of the amount of yeasts/hyphae by attributing scores to histological fields in the anteroposterior direction (Fig. 4).39 Through hematoxylin-eosin (HE) staining, many epithelial alterations and the presence of inflammatory cells are observed and can be quantified according to the methodology of Junqueira et al.,52 such as epithelial hyperplasia, disorganization of the basal cell layer, exocytosis, spongiosis, loss of filiform papillae, hyperkeratosis, development of intraepithelial microabscesses, and extension of the inflammatory infiltrate (Fig. 5). Histopathological data are important because from a histopathological and diagnostic point of view, most of the lesions described in animal models faithfully reproduce human candidal lesions.35

figure viru-4-391-g4

Figure 4. Sagittal section of mouse tongue dorsum showing yeasts and hyphae in keratin (↓) and intraepithelial microabscesses (▲). PAS: 400×.
figure viru-4-391-g5

Figure 5. Sagittal section of rat tongue dorsum showing tissue lesion characterized by epithelial hyperplasia, disorganized basal layer, exocytosis, spongiosis, loss of filiform papillae, and hyperparakeratosis. HE: 100×.

In addition to the histopathological study, the tissue, which is usually the tongue dorsum, can be analyzed by scanning electron microscopy (SEM) and laser confocal scanning microscopy (LCSM). In the SEM analysis, alterations and fungal elements are observed on tongue dorsum tissue from rats and mice infected with C. albicans. Analysis reveals hyphae penetrating perpendicularly into the tissue on the anterior surface of the papillae, desquamation and degradation of tissues, atrophy and destruction of the filiform papillae, and increased interpapillar surfaces (Fig. 6A and B).39

figure viru-4-391-g6

Figure 6. (A) SEM of the tongue dorsum of the mouse. The presence of hyphae penetrating perpendicularly into the tissue (↓) is verified. SEM: 3 700×. (B) SEM of the tongue dorsum of the rat. Hyphae (↓), keratin desquamation

The use of LCSM to analyze the oral candidiasis in mouse model was recently proposed by Dongari-Bagtzoglou et al.70 Oral candidiasis was induced in mice immunosuppressed by inoculation of a GFP-expressing strain of C. albicans. Confocal images followed by 3D reconstruction of C. albicans biofilms formed on the dorsum tongue revealed microanatomical epithelial variations of the lingual papillae, including “valleys”, higher “elevations” of stacked fungal cells, and dark areas suggestive of extracellular matrix. The presence of β-glucan was immunoaccessible throughout the biofilm mass and on the surfaces of both yeast and hyphal organisms. Neutrophils were visualized as forming “nests” within the biofilm mass. The biofilm formed on the tongue was a mixed biofilm composed of C. albicans and various bacteria that were identified as Enterococcus spp., Lactobacillus spp., and Staphylococcus spp., while some bacteria were observed invading the epithelium together with C. albicans. Therefore, LCSM analysis provided data about the architecture of the living tongue biofilm, epithelial microanatomical variations and the distribution of fungal cells in the structure of the biofilm, and gave an idea of the dispersion of the extracellular matrix. In addition, this study provides new insights related to potential synergistic relationship between C. albicans and the oral bacterial flora during the development of oral candidiasis.

Recently, a new bioluminescence mouse model for real-time monitoring of oral candidiasis was described by Mosci et al.71 Experimental oral candidiasis was induced in immunosuppressed mice by inoculation of a C. albicans strain that produces a genetically-engineered cell surface luciferase. At selected days post infection, mice were imaged in the IVIS-200TM Imaging system under s.c. anesthesia. The total photon emission from oral areas within the images of each mouse was quantified with Living ImageR software package. The authors obtained highly reproducible levels of infection, providing tools for longitudinal monitoring of the course of OPC in live animals. The advantages of this novel method compared with the conventional methods for analysis of experimental oral candidiasis include: (1) it gives an accurate estimation of the C. albicans load in the whole oral cavity, including the load of invasive hyphae; (2) it allows the longitudinal monitoring of oral candidiasis in individual animals, providing a unique possibility to determine the progression or resolution of OPC in real-time and avoiding individual variations when the time course of CFU is assessed; (3) it allows the monitoring of C. albicans dissemination from the oral cavity to the gastrointestinal tract; and (4) it decreases the need to sacrifice a large number of animals.

All of the methods described above are very important in analyzing the experimental oral candidiasis in rat or mouse and comparing results from different laboratories. In addition, a monitoring of the body weight of each animal can be performed to estimate the disease severity. The magnitude of weight loss caused by the infection and induction of predisposing factors indicate the time point to conclude the experiment, avoiding unnecessary suffering of rats and mice.61 Another important aspect to consider in the evaluation of experimental oral candidiasis is the use of a sedative agent in the animals for C. albicans inoculation. The inoculation of C. albicans cells in the oral cavity without any anesthetic drug impairs the colonization by Candida and subsequently the development of disease. According to Takakura et al.,37 the degree of severity of the infection is correlated with the length of the sedated period. The sedation avoids the intake of Candida by animals, decreasing the spread for esophagus and stomach, and promoting a better colonization and tissue invasion of C. albicans in the oral cavity.

Conclusions and Future Directions

The study of oral candidiasis in rats and mice constitutes an important tool for the understanding of the pathogenesis of Candida in the oral mucosa and the interference of predisposing factors in the disease process. These animal models have also been very useful in testing different therapeutic modalities for candidiasis lesions in the oral cavity.42,43,66-68 Furthermore, the elucidation of the virulence mechanisms of Candida spp. and of the host–pathogen interaction in experimental animal models can provide important information for the discovery of possible targets of new antifungal agents. For any type of research to be conducted, it is worth noting that the choice of animal model must be guided by the aim of the study and must follow standard protocols to obtain reliable and reproducible results.

In summary, rats and mice models that employ antibiotic therapy and hyposalivatory conditions to induce oral candidiasis develop lesions characterized by areas of papillary atrophy in the tongue dorsum without white patches, such as erythematous candidiasis and median rhomboid glossitis. The animals in these models have an intact immune system and represent what would be expected in individuals treated with broad-spectrum antibiotics or patients with reduced salivary flow caused by Sjögren syndrome, advanced age, or radiotherapy. In the other hand, the immunosuppression models induce candidiasis lesions with white patches that are comparable to the pseudomembrane observed in human thrush and reflect the oral conditions of AIDS patients.50 In addition, rat models have a sufficiently sized oral cavity for placement of a prosthetic device in the palate that induces candidiasis lesions similar to human Candida denture stomatitis. The advantages of using mice for experimental oral candidiasis is that the immunobiology of the healthy mouse mucosa has been well characterized and there are several genetically modified animals available to research.

According to the studies cited in this review, it is evident that the most studies of oral candidiasis in mice and rats were performed with C. albicans. However, other non-C. albicans species have arisen in recent years due to a growing population of immunosuppressed patients, and future studies must be conducted to investigate the interaction of Candida mixed biofilms in the oral cavity and their relation to recurrent episodes of oral candidiasis in these patients. Furthermore, the methods for the analysis of experimental oral candidiasis should be improved because we have new technologies available, such as the real-time monitoring of microbial infections using bioluminescent fungi described recently by Mosci et al.71 This new technology and many more under development will be very useful for the advancement of oral experimental candidiasis.

Acknowledgments

The authors thank the São Paulo Council of Research (FAPESP) for financial support (Grants 2010/18753-7 and 2010/19117-7) and scholarships (Processes 2010/00879-4 and 2011/21346-7).

Glossary

Abbreviations:

EPS
extracellular polymeric substances
CFU
colony-forming units
PDT
photodynamic therapy
SCID
severe combined immune deficiency
MAIDS
murine acquired immune deficiency syndrome
LCSM
laser confocal scanning microscopy
SDA
Sabouraud dextrose agar
HE
hematoxylin-eosin
PAS
periodic acid-Schiff
SEM
scanning electron microscope

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

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