Update on mesenchymal stem cell-based therapy in lupus and scleroderma

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Update on mesenchymal stem cell-based therapy in lupus and scleroderma

Audrey Cras12*, Dominique Farge3*, Thierry Carmoi4, Jean-Jacques Lataillade5, Dan Dan Wang6 and Lingyun Sun6

Author Affiliations

1 Assistance Publique-Hôpitaux de Paris, Saint-Louis Hospital, Cell Therapy Unit, Cord blood Bank and CIC-BT501, 1 avenue Claude Vellefaux, Paris, 75010, France

2 INSERM UMRS 1140, Paris Descartes, Faculté de Pharmacie, 4 avenue de l’observatoire, Paris, 75004, France

3 Assistance Publique-Hôpitaux de Paris, Saint-Louis Hospital, Internal Medicine and Vascular Disease Unit, CIC-BT501, INSERM UMRS 1160, Paris 7 Diderot University, Sorbonne Paris Cité, 1 avenue Claude Vellefaux, Paris, 75010, France

4 Hôpital du Val de Grace, Internal Medecine Unit, 74 boulevard de Port Royal, Paris, 75005, France

5 Percy Military Hospital, Department of Research and Cell Therapy, 101 Avenue Henri Barbusse, Clamart, 92140, France

6 Department of Immunology, The affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhong Shan Road, Nanjing 210008, China

For all author emails, please log on.

Arthritis Research & Therapy 2015, 17:301  doi:10.1186/s13075-015-0819-7
Audrey Cras, Dominique Farge, Dan Dan Wang and Lingyun Sun contributed equally to this work.

The electronic version of this article is the complete one and can be found online at: http://arthritis-research.com/content/17/1/301

Published: 3 November 2015

© 2015 Cras et al.

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Abstract

Current systemic therapies are rarely curative for patients with severe life-threatening forms of autoimmune diseases (ADs). During the past 15 years, autologous hematopoietic stem cell transplantation has been demonstrated to cure some patients with severe AD refractory to all other available therapies. As a consequence, ADs such as lupus and scleroderma have become an emerging indication for cell therapy. Multipotent mesenchymal stem cells (MSCs), isolated from bone marrow and other sites, display specific immunomodulation and anti-inflammatory properties and appear as ideal tools to treat such diseases. The present update aims at summarizing recent knowledge acquired in the field of MSC-based therapies for lupus and scleroderma.

Introduction

Autoimmune diseases (ADs) are a group of heterogeneous conditions characterized by aberrant activation of the immune system with failure of the immune regulation to maintain adapted tolerance. They are traditionally classified as “organ-specific AD”, where the consequences of organ failure can be improved by a replacement opotherapy or an organ transplantation, and as “diffuse or systemic AD”, notably including systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). However, progressive identification of the genetic background of each AD type [1] and elucidation of the mechanisms associated with self-directed tissue inflammation, unrelated to T- or B-cell abnormalities, revealed the important differences between autoimmunity and autoinflammation [2]. SLE, type 1 diabetes, and autoimmune thyroiditis are polygenic ADs with a predominant autoimmune component, whereas other polygenic ADs, such as Crohn’s disease, are characterized by a predominant autoinflammatory component. Therefore, the optimal treatment of AD should be discussed in light of this specific pathological continuum between autoimmunity and autoinflammation, which variably interacts in each AD phenotypic expression. Indeed, chronic immunosuppression is responsible for high treatment-related morbidity and still is associated with significant disease- and treatment-related mortality, notably in patients with severe inflammatory SLE or refractory SSc and with kidney, heart-lung, or brain damage. With a view to developing innovative therapies for AD, mesenchymal stem cell (MSC)-based therapies theoretically appear as ideal tools to target the respective autoinflammatory and autoimmune components of such diseases, and this update aims at summarizing recent knowledge acquired in the field.

A need for innovative stem cell therapies in severe or refractory forms of systemic lupus erythematosus and systemic sclerosis

SLE, with a prevalence of 40 to 50 out of 100,000 people, is a heterogeneous chronic multisystemic autoimmune inflammatory disorder whose original flare can be controlled by conventional immunosuppressive therapy. However, definitive cure is rarely achieved by this therapy and life-long immunosuppression is often required. Response rates vary from 20 to 100 % at 6 months according to the definition of response or improvement, the extent of visceral damage, the ethnic origin, and the socioeconomic profile. First-line validated standard therapies used to induce remission within the first 6 to 9 months of disease flare are the corticosteroids in combination with either (a) cyclophosphamide (CY), using the classic National Institutes of Health regimen or lower doses for shorter duration over the course of 3 months with a similar efficacy, according to the Eurolupus regimen [3], [4], or (b) mycophenolate mofetil, with good efficacy and tolerability [5], [6]. Other monoclonal antibodies against the T- or B-cell receptors, such as rituximab as an anti-CD20, or against the adhesion molecules involved in the T- or B-cell interaction and their co-stimulatory signals, have been used despite the paucity of validated therapeutic targets and the failure to demonstrate the efficacy of rituximab in renal and extra-renal manifestations of SLE [7]. In 2011, a monoclonal antibody against B cell-activating factor of the tumor necrosis factor family (BAFF), belimumab anti-Blys, was the first targeted therapy to demonstrate its efficacy in mild to moderate SLE by a randomized clinical trial [8]. Despite early diagnosis and treatment with immunosuppressive agents as well as a tight control of hypertension and infections, there is still a subgroup of patients with SLE that does not respond to the treatment and that has 10-year mortality of 10 % [9]. In addition, early death from rapidly progressive atherosclerosis in SLE suggests that, despite apparent reasonable disease control, subclinical inflammatory disease promotes endothelial damage and plaque formation and that prolonged exposure to corticosteroids and immunosuppressive drugs leads to further damage beyond the SLE itself.

SSc, which has a prevalence of 5 to 50 per 100,000, is a rare AD characterized by early vascular endothelium damage with consequent activation of the immune response and enhanced collagen synthesis, leading to progressive fibrosis of the skin and internal organs. Both antigen stimulation and genetic susceptibility may contribute to autoimmunity, with consequent early T-cell infiltration as well as B-cell and fibroblast activation, by pro-fibrotic cytokines, mainly transforming growth factor-beta (TGF-β) and connective tissue growth factor. Most patients progress, and the overall 10-year survival is only 66 %, and there is significant morbidity and altered quality of life among survivors. In rapidly progressive SSc, mortality rates reach 30 to 50 % in the first 5 years after disease onset, according to the extent of skin, cardiopulmonary, and renal involvement [10]. No treatment has ever shown any benefit in this severe disease, except autologous hematopoietic stem cell transplantation (HSCT), whose efficacy was recently established by a unique international multi-center, open-label phase III, ASTIS (Autologous Stem cell Transplantation International Scleroderma) trial [11] that enrolled 156 patients over the course of 10 years with early diffuse cutaneous SSc, showing that HSCT confers a significantly better long-term survival rate than 12 monthly intravenous pulses of CY.

In this context, new therapeutic approaches with fewer long-term side effects are warranted. Bone marrow (BM) stromal cells or MSCs, which can also be obtained from other human tissues, have recently enlarged the therapeutic tool set for SLE and SSc. Because MSCs display specific immunomodulation and immunosuppressive properties as well as regenerative potential, there is a strong rationale for MSC-based therapy in SLE and SSc to treat their respective autoimmune and autoinflammatory components at a certain time point of each disease evolution.

Biology of mesenchymal stem cells

Definition, isolation, and characterization of mesenchymal stem cells

MSCs were originally identified in BM by Friedenstein in 1976 as a fibroblast-like cellular population capable of generating osteogenic precursors [12]. Since then, these cells have been extensively investigated and given various names, until 1991 when Caplan proposed the definition ‘mesenchymal stem cells’ (MSCs) [13], which after consensus of the Mesenchymal and Tissue Stem Cell Committee of International Society for Cellular Therapy (ISCT) was changed to “multipotent mesenchymal stromal cells”. ISCT has provided three minimal criteria to define MSCs [14]: (a) plastic adherence in standard culture conditions; (b) differentiation into osteoblasts, adipocytes, and chondroblasts under specific conditions in vitro; and (c) expression of nonspecific markers CD105, CD90, and CD73 along with the lack of expression of hematopoietic markers such as CD34, CD45, CD14 or CD11b, CD79a, or CD19. MSCs show intermediate levels of major histocompatibility complex (MHC) class I molecules on their cell surface and have no detectable levels of MHC class II, mainly HLA-DR, and co-stimulatory molecules (CD40, CD80, and CD86), which allow their transplantation across MHC barriers [15]. Therefore, their privileged immunological phenotype makes them an appropriate stem cell source for allogeneic transplantation. They can also synthesize trophic mediators, such as growth factors and cytokines—macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-11, IL-15, stem cell factor, and vascular endothelial growth factor—involved in hematopoiesis regulation, cell signaling, and modulation of the immune response [16].

BM-MSCs were discovered first, and the BM was considered the main source of MSCs. BM-MSCs are classically expanded in vitro by consecutive passages in fibroblast growth factor-supplemented cell culture medium from the plastic-adherent BM cell population. Subsequently, MSCs, facilitated by their ability to adhere to plastic, have been isolated from various other sources such as skeletal muscle, adipose tissue, dental tissue, synovial membranes, placenta, cord blood, and Wharton’s jelly by using enzymatic tissue digestion and density gradient centrifugation methods [17]. These alternative sources are very attractive because BM harvesting is rather invasive and painful and is associated with potential donor-site morbidity. Moreover, because of the rarity of MSCs in the BM, where they represent 1 in 10,000 nucleated cells, tissues such as umbilical cord (UC) or adipose tissue (AT) represent promising sources. Indeed, MSCs can be more easily isolated from these tissues and considerably larger amounts of UC- or AT-derived MSCs can be obtained, compared with the BM. MSCs from these different sources share many biological features, although studies reported some differences in their immunophenotype, proliferative capacity, differentiation potential, or gene expression profile [18], [19].

Immunomodulatory properties of mesenchymal stem cells: evidence from in vitro data

Compared with other stem cell sources, such as hematopoietic stem cells (HSCs), MSCs appear as a promising source for overcoming autoimmunity because of their immunosuppressive properties [20]. MSCs modulate the immunological activity of different cell populations as shown by in vitro data. Their most important effects are T-cell proliferation and dendritic cell (DC) differentiation inhibition, which are key activating factors of autoimmune disorders. MSCs are effective in inhibiting proliferation of CD4 and CD8 T cells as well as memory and naïve T cells [21]. This mechanism relies on both cell–cell contact and several specific mediators, produced by MSCs, such as TGF-β1, prostaglandin E 2 , and indoleamine 2,3 deoxygenase [22]. The ability to suppress T-cell responses to mitogenic and antigenic signals is explained by a complex mechanism of induction of “division arrest anergy”, responsible for maintaining T lymphocytes in a quiescent state. Thus, the MSCs trigger the inhibition of cyclin D2 expression, thus arresting cells in the G 0 /G 1 phase of the cell cycle [23]. MSCs also inhibit the production of interferon-gamma (IFN-γ) and increase the production of IL-4 by T helper 2 cells. This indicates a shift in T cells from a pro-inflammatory state to an anti-inflammatory state [24], [25]. MSCs also stimulate the production of CD4 + CD25 + regulatory T cells, which inhibit lymphocyte proliferation in allogeneic transplantation [26]. In addition, MSCs inhibit B-cell proliferation through arrest at the G 0 /G 1 phase of the cell cycle and production of IgM, IgA, and IgG as well as their chemotactic abilities [27], [28]. A recent study demonstrated that this effect of MSCs on B cells is mediated by T cells [29]. However, some contradictory data showed that, in some culture conditions, IgG secretion and B-cell proliferation can be induced and B-cell survival sustained, and this effect does not depend on the presence of IFN-γ in the culture [30], [31]. MSCs have been demonstrated to interfere with DC differentiation, maturation, and function [32]–[34]. MSCs obtained from healthy human donors can indirectly reduce T-cell activation by inhibiting DC differentiation (mainly DC type I) from monocytes [35].

Although the majority of data dealing with the immunomodulatory effects of MSCs are derived from BM-MSCs, some of these effects have also been described for MSCs from other sources. Results from studies comparing the immunomodulatory effects of various tissue-derived MSCs are controversial. Some studies concluded that BM- and UC-MSCs show similar effects, whereas others demonstrated that UC-MSCs have a higher capacity of inhibiting T-cell proliferation than adult MSCs [36], [37]. Some studies also indicate that AT-MSCs can be more effective suppressors of immune response compared with BM-MSCs. Indeed, AT-MSCs modulate mitogen-stimulated B-cell immunoglobulin production in vitro to a much greater extent than BM-MSCs. Also, in comparison with BM-MSCs, they inhibit, significantly more, the differentiation of blood monocytes into DCs and the expression of functionally important co-stimulatory molecules on the surface of mature monocyte-derived DCs [38], [39]. It may be postulated that MSCs express a different set of molecules depending of their tissue of origin, resulting in different immunosuppressive activities. Taken together, these in vitro data demonstrate that MSCs modulate the action of the various cells that are involved in immune response and preferentially inhibit T-cell proliferation and differentiation of DCs. However, it would be important to further investigate the molecular mechanisms that underlie the immunomodulatory properties of various tissue-derived MSCs since these differences may have functional relevance to the therapeutic use of these cells.

Mesenchymal stem cell-based therapy in animal models

Animal models of AD can be divided into two categories. The hereditary and spontaneous AD models, such as murine (BXSB) lupus, are characterized by autoimmune manifestations that affect the majority of the animals of a susceptible line and by a strong genetic predisposition displayed by the HSCs and manifested by anomalies of thymic development and/or function of lymphocytes B or T or antigen-presenting cells such as macrophages. Other experimental models, such as arthritis adjuvant and experimental acute encephalomyelitis [40], use active immunization by exposure to a foreign antigen to induce the AD. The rationale for using MSCs for the treatment of autoimmunity was first demonstrated in experimental acute encephalomyelitis, a model for multiple sclerosis [25]. Subsequently, several preclinical studies evaluating MSC injection in a collagen-induced arthritis model [41] or in an autoimmune type 1 diabetes model [42] provided support for the potential therapy of other ADs, including SLE and SSc.

Animal models of systemic lupus erythematosus

Both Fas mutated MRL/lpr mice and NZB/W F1 mice are widely used as genetically prone lupus models, which demonstrate progressive nephritis, elevated serum autoimmune antibodies, and immune abnormalities. The role of BM-MSC transplantation in SLE and its efficacy compared with conventional CY treatment has been investigated in MRL/lpr mice as an SLE mouse model [43], [44]. MSC injection resulted in a significant reduction in serum levels of anti-double-stranded DNA (anti-dsDNA) antibodies IgG and IgM, ANA, and immunoglobulins IgG1, IgG2a, IgG2b, and IgM as well as an increased serum albumin level. When compared with MSCs, conventional CY treatment partially reduced the levels of serum autoantibodies and immunoglobulin IgG2a, restored albumin level, and failed to reduce circulating immunoglobulins IgG1, IgG2b, and IgM. MSC treatment improved renal disorders, specifically restoring kidney glomerular structure and reducing C3 and glomerular IgG deposition. Although CY treatment could reduce glomerular IgG deposition, it did not restore the glomerular structure and C3 accumulation. MSC treatment, but not CY treatment, was able to completely restore renal function, shown as normalization of serum and urine creatinine levels in MRL/lpr mice, in comparison with disease-free control mice. In their study, Ma et al. determined that murine BM-MSC transplantation improved nephritis in MRL/lpr mice by suppressing the excessive activation of B cells via inhibition of BAFF production [45]. Nevertheless, in a similar study conducted in a different SLE mouse model (NZB/W), systemic MSC administration did not provide any beneficial effect and in fact worsened the disease [46], [47]. To resolve these conflicting results, Gu et al. assessed the differential effects of allogeneic versus syngeneic MSC transplantation on lupus-like disease in both mice models [48]. They showed that, in MRL/lpr and NZB/W mice, both normal MSCs and lupus MSCs from young mice ameliorated SLE-like disease and reduced splenic T and B lymphocyte levels. However, lupus MSCs from older NZB/W mice did not significantly reduce spleen weights, glomerular IgG deposits, renal pathology, interstitial inflammation, or T or B lymphocyte levels. This study suggests that allogeneic MSCs may be preferred over syngeneic lupus-derived MSCs given the decreased overall effectiveness of post-lupus-derived MSCs, which is partly triggered by the disease and is not exclusively an intrinsic defect of the MSCs themselves. The same group reported that human lupus BM-MSCs are not as effective as human healthy BM-MSCs and umbilical cord-derived MSCs (UC-MSCs) in ameliorating disease in MRL/lpr mice [49]. Moreover, in vitro assessments of immunomodulatory functions detected a reduced capacity of lupus BM-MSCs to inhibit IFN-γ production and CD19 + B-cell proliferation, although inhibition of CD3 + proliferation and IFN-γ licensing results were indicative of immune activity by lupus BM-MSCs. Although these studies showed that lupus MSCs are not yet a suitable source of MSCs for cell therapy, it is important to continue to define differences in MSCs because it appears that donors and the origin of the MSCs impact their function.

Some studies evaluated the effectiveness of MSCs derived from sources other than BM. Sun’s team had showed that UC-MSCs alleviated lupus nephritis in MRL/lpr mice in a dose-dependent manner [50]. Both single and multiple treatments with UC-MSCs were able to decrease the levels of 24-h proteinuria, serum creatinine, anti-dsDNA antibodies, and the extent of renal injury, such as crescent formation. Further studies dealing with the underlying mechanisms showed that UC-MSC treatment inhibited renal expression of monocyte chemotactic protein 1 and high-mobility group box 1 expression but that it upregulated Foxp3 + regulatory T cells. Moreover, carboxyfluorescein diacetate succinimidyl ester-labeled UC-MSCs could be found in the lungs and kidneys after infusion [50]. Using NZB/W F1 mice, Chang et al. showed that human UC-MSC transplantation significantly delayed the onset of proteinuria, decreased anti-dsDNA, alleviated renal injury, and prolonged the life span [51]. Subsequent studies looking at the mechanisms showed that the treatment effect was not due to a direct engraftment and differentiation into renal tissue but rather to the inhibition of lymphocytes, the induced polarization of T helper 2 cytokines, and the inhibition of the synthesis of pro-inflammatory cytokines. Choi et al. showed that long-term repeated administration of human AT-MSCs ameliorated SLE in NZB/W F1 mice [52]. Compared with the control group, the AT-MSC-treated group had a higher survival rate, decreased histological and serological abnormalities, improved immunological function, and a decreased incidence of proteinuria. Transplantation of AT-MSCs led, on the one hand, to significant decreased levels of antibodies targeting dsDNA and blood urea nitrogen levels. On the other hand, it significantly increased serum levels of granulocyte-macrophage colony-stimulating factor, IL-4, and IL-10. A significant increase in the proportion of CD4 + FoxP3 + cells with a marked restoration of their capacity to produce cytokines was observed in spleens from the AT-MSC-treated group.

Animal models of systemic sclerosis

Among the various experimental models aiming at reproducing the SSc (genetic models, such as tight skin (TSK) Tsk1 and Tsk2 mice, UCD-200 chicken, Fra-2 mice, TGFβRIIΔκ, or inducible models using injections of bleomycin or vinyl chloride or graft-versus-host disease (GVHD) mice), none displayed exactly the three components of scleroderma in humans [53]. Indeed, two forms of SSc are defined in humans. The first one is characterized by extensive skin fibrosis (proximal and distal), common pulmonary fibrosis, and the presence of antibody directed against DNA topoisomerase 1. In regard to the second form, referred to as the “limited cutaneous” form, the skin disease is limited to the distal limbs and lung symptoms are rare. The autoantibodies detected in this second form are against centromere (the main target being the centromeric protein CENP-B) and not against DNA topoisomerase 1. The TSK mouse model is characterized mainly by skin lesions, which do not reach the dermis; others use the mismatch transplant BM or spleen cells in mice sublethally irradiated. A scleroderma-like syndrome associated with chronic GVHD was induced with skin and lung fibrosis and was associated with signs of autoimmunity. Finally, induction of fibrosis by bleomycin injection could be used. But none reproduced a true picture of scleroderma. The role of free radicals in the development of SSc was studied and this helped to develop a mouse model of scleroderma, based on repeated injection of hypochlorous acid [54]. This model mimics the diffuse form of the human disease (cutaneous sclerosis, pulmonary fibrosis, renal disease, and anti-topoisomerase antibodies) and is a more satisfactory way to test new therapeutic approaches than other models. Despite the lack of perfectly reproducible models of SSc, the effect of MSCs on fibrosis is known and has been studied in the model of fibrosis induced by bleomycin [55]–[57]. Injection of MSCs allowed investigators to limit the pro-inflammatory and pro-fibrotic bleomycin effect through a mechanism involving IL-1RA [58]. Even though this model only partially reproduces SSc disease, all of the in vitro and in vivo data suggest that MSCs may have a beneficial effect in SSc.

Characteristics of mesenchymal stem cells derived from patients with systemic lupus erythematosus and systemic sclerosis

Because the majority of pathogenic autoreactive cells are the progeny of HSCs, it is conceivable that HSCs are involved in the AD process. BM-MSCs are key components of the hematopoietic microenvironment and provide support to hematopoiesis and modulate the immune system. Little is known about how MSCs are involved in immunological disorders. However, evidence has suggested that BM-MSCs from animal models and from patients with SLE and SSc exhibited impaired capacities of proliferation, differentiation, secretion of cytokines, and immune modulation. These alterations might be the consequence of the disease or play a fundamental role in the pathogenesis of SLE and SSc.

Mesenchymal stem cells derived from patients with systemic lupus erythematosus

BM-MSCs from patients with SLE have impaired hematopoietic function [59] and show significantly decreased bone-forming capacity and impaired reconstruction of BM osteoblastic niche in vivo [43]. Moreover, BM-MSCs from patients with SLE seem larger and flatter in appearance during in vitro culture and grow progressively slower compared with those from controls, thus demonstrating early signs of senescence [60], [61]. This senescent state is associated with differences in gene expression profile of BM-MSCs between SLE patients and controls, resulting in abnormalities in actin cytoskeleton, cell cycling regulation, BMP/TGF-β, and MAPK signaling pathways in BM-MSCs from patients with SLE [62]. In their study, Gu et al. found that senescent BM-MSCs from patients with SLE display reduced ability to upregulate regulatory T cells [63]. An increased p16INK4A expression plays a major role in this cellular senescence process by regulating cytokine secretion as well as the ERK1/2 signaling pathway. Wnt/b-catenin signaling also plays a critical role in the senescence of SLE BM-MSCs through the p53/p21 pathway [64]. Finally, SLE BM-MSCs exhibit an increased apoptosis rate, as reflected by downregulation of Bcl-2 and upregulation of cytochrome C in the cytoplasm, and display an enhanced aging process as shown by the overproduction of intracellular reactive oxygen species, which might be linked with the upregulation of p-FoxO3 and its upstream gene AKT [65].

Mesenchymal stem cells derived from patients with systemic sclerosis

Studies on BM-MSCs from patients with SSc are more limited. In patients with SSc, the osteogenic and adipogenic differentiation potentials of MSCs appear to be altered when they are isolated from the BM by direct selection of nerve growth factor receptor (CD271)-positive cells and not by the conventional technique of adhesion [66]. In these patients, the ability of MSCs to differentiate into endothelial progenitor cells appear reduced, and the endothelial progenitor cells obtained have a reduced ability to migrate and a lower pro-angiogenic potential [67]. Cipriani et al. showed that although BM-MSCs from SSc patients undergo premature senescence, they maintain considerable immunosuppressive functions and a normal ability to generate functional regulatory T cells [68]. In our study, we showed that the SSc BM-MSCs have fibroblast colony-forming units ability with a phenotype and a frequency similar to those of MSCs derived from healthy donors [69]. They differentiate into adipose and osteogenic cells with variabilities similar to those observed within the BM-MSCs from healthy controls. In regard to the immunoregulatory activity of MSCs in SSc, we reported that MSCs from patients were capable of supporting normal hematopoiesis and retained their immunosuppressive properties on T cells, thus confirming the data published by Bocelli-Tyndall et al. [69], [70]. We have recently shown a significant increase of the level of receptor type II TGF-β in MSCs from SSc patients compared with MSCs from healthy donors, associated with an activation of the TGF-β signaling pathway, leading to an increase in the synthesis of target genes, including the gene encoding collagen type 1 [71]. This activation of MSCs in response to stimulation by TGF-β, known for its major role in the pathogenesis of the disease, obviously limits their clinical use and justifies the use of allogeneic MSCs in these patients.

All of these findings suggest that BM-MSCs from patients with SLE or SSc are defective in regard to certain functions. Therefore, we can speculate that an allogeneic rather than an autologous MSC-based therapy might be preferable for treatment. Even though some data bring their early senescence to light, MSCs maintain some immunosuppressive properties that support the potential autologous clinical application. These data emphasize the necessity for a better understanding of the MSC involvement in the pathogenesis and the underlying MSC-immunomodulatory mechanisms.

Hematopoietic stem cell-based and mesenchymal stem cell-based therapy in patients with systemic lupus erythematosus and systemic sclerosis

Use of hematopoietic stem cell transplantation in systemic lupus erythematosus or systemic sclerosis

The use of HSCT in patients with AD to induce tolerance by resetting the immune responses is supported by both experimental data and clinical evidence. The direct relationship between the hematopoietic system and AD was evidenced in 1985 by Ikehara et al., who first demonstrated that AD originated from defects in the HSCs [72]. Thereafter, data from genetically prone and immunized animal models of AD treated with allogeneic, syngeneic, and autologous BM transplantation (BMT) showed that allogeneic BMT (but not syngeneic or autologous) could be used to treat AD-prone mice [73]. Conversely, the AD transfer was possible in normal mice after allograft from a mouse lupus nephritis showing that it was in fact a stem cell disorder. Consensus indications for the use of transplantation of BM-derived or peripheral HSCs to treat severe ADs were first elaborated in 1997 [74] and were updated in 2012 [75]. Today, more than 3500 patients worldwide have received an HSCT for an AD alone; approximately 200 autologous HSCTs were for refractory SLE and 500 were for severe SSc. This allowed a sustained and prolonged remission with qualitative immunological changes not seen with any other forms of treatment. In SLE, these beneficial effects were limited by the increased short-term mortality underlying the need to develop new strategies. In severe SSc, adequate prospective trials allowed investigators to ensure the safety of non-myeloablative autologous HSCT for SSc when careful patient selection, follow-up, and center effect are considered, to avoid misleading use of CY when it is unlikely to be clinically meaningfully effective. In case of allogeneic transplantation, more data suggest preclinical and clinical evidence for a graft versus autoimmunity effect in replacement of a dysfunctional immune system by allogeneic HSCT, which also represents an attractive prospect. In this setting, analysis of the regenerating adaptive immune system showed normalization of the restricted T-cell repertoire, with sustained shifts in T- and B-cell subpopulations from memory to naïve cell dominance supporting a thymic reprocessing and re-education of the reconstituting immune system [76], [77]. Disappearance of circulating plasmablasts and restoration of normal or raised levels of CD4+ and CD8 + FoxP3+ regulatory T cells were shown in SLE following autologous HSCT. This normalization was accompanied by complete inhibition of pathogenic T-cell response to autoepitopes from histones in nucleosomes [78], [79]. This has never been shown previously after the use of conventional immunosuppressive therapies. Such clinical and immunological results allowed investigators to take into account the non-specific immunosuppressive changes, which can be observed both in blood and in tissues after cytotoxic therapy [76], [80], and immune re-educative changes supporting immune tolerance [81]. Therefore, for the first time in AD treatment, the interruption of the vicious circle of autoimmunity allowed the emergence of normal regulatory mechanisms and the eradication of the last auto-reactive T cell, which is one of the proposed mechanisms for using HSCs in the treatment of SLE and SSc.

Mesenchymal stem cell-based therapy in systemic lupus erythematosus and systemic sclerosis

Discovery and identification of MSCs within the BM content and of their therapeutic properties have led us and others to use MSCs derived from various tissues to treat AD. Indeed, the supportive function for HSCs in the BM niche and the immunomodulatory capacities of MSCs suggest their potential use for cell therapy. Allogeneic donor-derived BM-MSCs have already been used in several phase I and II and very few phase III clinical trials for the treatment of acute GVHD following allogeneic HSCT for leukemia or hematological malignancies [82]. With a better understanding of the combined components of autoimmunity and autoinflammmation in each AD, there is a rationale to propose combined therapies with different tools.

BM-MSCs and UC-MSCs have been transplanted in patients with severe SLE, who were not responsive to conventional therapies. The 4-year follow-up demonstrated that about 50 % of the patients entered clinical remission after transplantation, although 23 % of the patients relapsed [83]. MSC infusion induced disease remission for lupus nephritis [84], diffuse alveolar hemorrhage [85], and refractory cytopenia [86]. The multi-center clinical study showed that 32.5 % of patients achieved major clinical response (13 out of 40) and 27.5 % of patients achieved partial clinical response (11 out of 40) during a 12-month follow-up, respectively. However, 7 (17.5 %) out of 40 patients experienced a disease relapse after 6 months of follow-up, after a prior clinical response, which indicated that another MSC infusion would be necessary after 6 months [87].

Few data are available about MSC-based therapy in patients with SSc. A patient with severe refractory SSc received an intravenous injection of allogeneic MSCs [88]. Three months after injection of MSCs, a significant decrease in the number of digital ulcers was observed. At 6 months, blood flow to the hands and fingers seemed significantly improved, and transcutaneous partial pressure of oxygen was increased. Rodnan skin score dropped from 25 to 11. The titer of anti-Scl-70 antibody, however, remained high, and enumeration of lymphocytes T, B, and natural killer cells did not change. This first observations were supplemented by four other cases reported by the same German team using allogeneic MSCs to treat severe forms of SSc, without major side effects or specific abnormalities observed after respective follow-ups of 44, 24, 6, 23, and 18 months [89]. The first two patients received fresh MSCs, whereas the three others received cryopreserved allogeneic MSCs. No conclusion about the efficacy of the MSC transplantation can be drawn from these clinical cases, although skin improvement was noted in three out of five cases and these patients did not have a detailed immunomonitoring.

Although further studies are necessary, preclinical and clinical data underline the therapeutic potential of MSCs in patients with SLE and SSc. Now it is important to design a controlled study to further investigate the clinical efficacy of MSC transplantation, compared with conventional immunosuppressive therapies, or the efficacy of MSC transplantation combined with immunosuppressive drug treatment compared with drugs alone. Careful patient selection and performance are crucial for the proper use of this therapy.

Note

This article is part of a thematic series on Biology and clinical applications of stem cells for autoimmune and musculoskeletal disorders, edited by Christian Jorgensen and Anthony Hollander. Other articles in this series can be found at http://www.biomedcentral.com/series/MSC

Abbreviations

AD: Autoimmune disease

AT: Adipose tissue

BAFF: B-cell-activating factor of the tumor necrosis factor family

BM: Bone marrow

BM-MSC: Bone marrow-derived mesenchymal stem cell

BMT: Bone marrow transplantation

CY: Cyclophosphamide

DC: Dendritic cell

dsDNA: Double-stranded DNA

GVHD: Graft-versus-host disease

HSC: Hematopoietic stem cell

HSCT: Hematopoietic stem cell transplantation

IFN-γ: Interferon-gamma

IL: Interleukin

ISCT: International Society for Cellular Therapy

MHC: Major histocompatibility complex

MSC: Mesenchymal stem cell

SLE: Systemic lupus erythematosus

SSc: Systemic sclerosis

TGF-β: Transforming growth factor-beta

TSK: Tight skin

UC: Umbilical cord

UC-MSC: Umbilical cord-derived mesenchymal stem cell

Competing interests

The authors declare that they have no competing interests.

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Autologous immuno magnetically selected CD133+ stem cells in the treatment of no-option critical limb ischemia: clinical and contrast enhanced ultrasound assessed results in eight patients

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Autologous immuno magnetically selected CD133+ stem cells in the treatment of no-option critical limb ischemia: clinical and contrast enhanced ultrasound assessed results in eight patients

Vittorio Arici1, Cesare Perotti2, Calliada Fabrizio3, Claudia Del Fante2, Franco Ragni1, Francesco Alessandrino3, Gianluca Viarengo2, Michele Pagani4, Alessia Moia1, Carmine Tinelli5 and Antonio Bozzani1*

Author Affiliations

1 Vascular Surgery Unit, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Piazzale Golgi 19, Pavia, 27100, Italy

2 Haemotransfusional Service, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy

3 Radiology Service, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy

4 Anesthesiology and Intensive Care Unit 2, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy

5 Statistics and Epidemiology Service, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy

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Journal of Translational Medicine 2015, 13:342  doi:10.1186/s12967-015-0697-4

The electronic version of this article is the complete one and can be found online at: http://www.translational-medicine.com/content/13/1/342

Received: 5 April 2015
Accepted: 14 October 2015
Published: 3 November 2015

© 2015 Arici et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Objectives

Demonstrate the safety and effectiveness of highly purified CD133+ autologous stem cells in critical limb ischemia (CLI).

Design

Prospective single-center not randomized. Clinicaltrials.gov identifier: NCT01595776

Methods

Eight patients with a history of stable CLI were enrolled in a period of 2 years. After bone marrow stimulation and single leukapheresis collection, CD133+ immunomagnetic cell selection was performed. CD133+ cells in buffer phosphate suspension was administered intramuscularly. Muscular and arterial contrast enhanced ultra sound (CEUS), lesion evolution and pain management were assessed preoperatively and 3, 6 and 12 months after the implant.

Results

No patient had early or late complications related to the procedure. Two patients (25 %) didn’t get any relief from the treatment and underwent major amputation. Six patients (75 %) had a complete healing of the wounds, rest pain cessation and walking recovery. An increase in CEUS values was shown in all eight patients at 6 months and in the six clinical healed patients at 12 months and had statistical relevance.

Conclusions

Highly purified autologous CD133+ cells can stimulate neo-angiogenesis, as based on clinical and CEUS data.

Keywords:

Peripheral arterial disease; Critical limb ischemia; Stem cell therapy; Contrast enhanced ultrasound

Background

Symptomatic peripheral arterial disease (PAD) has a prevalence of 3 % in a population aged 40 years and above and of 6 % in patients over 60. Critical Limb Ischemia (CLI) is the worst and terminal clinical picture of PAD often preceding gangrene and amputation: typical symptoms are rest pain refractory to analgesics lasting more than 2 weeks and ischemic lesions (Fontaine classification stage 3–4 and Rutherford classification stage 4–6). CLI diagnosis is confirmed instrumentally by calf arterial pressure <50 mmHg, Ankle/Brachial Index (ABI) <0.5 and Transcutaneous PO 2 (TcPO 2 ) <30 mmHg. The Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II) [1] stresses the absolute indication to revascularization for these patients either by surgical or endovascular treatment. Nevertheless in some cases, mostly due to lack of distal run off, revascularization is not feasible or shows very low success rates. For the above mentioned reasons, the prognosis of these CLI patients is poor, with a major amputation rate of about 50 % at 1 year.

Many non-interventional treatments have been proposed for these “no-options” patients: spinal cord stimulation, prostanoids and prostaglandins administration, hyperbaric oxygen therapy and so on. More recently some authors have focused the attention on the local administration of stem cells, and particularly Endothelial Progenitor Cells (EPCs). EPCs local administration effectiveness in post-ischemic myocardial damage has been demonstrated in animal models and in humans [2]–[5]. Many observations lead to argue that the EPCs play a basic role in re-endothelization and neo-vascularization processes: actually the EPCs number may be reduced in peripheral blood in cardiovascular diseases, diabetes and rheumatoid arthritis and under the action of exogenous (as smoke habit) or endogenous (as high C-reactive protein levels) factors. Otherwise EPCs number may be increased by factors as physical exercise, estrogens, erythropoietin, statins and several cytokines or secreting proangiogenic factors like hepatic growth factor (HGF), insulin like growth factor (IGF-1) and the vascular endothelial growth factor (VEGF). These observations persuaded some authors to apply this cell therapy approach to CLI [6].

Our starting experience in the treatment of no options CLI with EPCs in three patients has been previously reported [7]. The bench marks of our study are the use of a selected EPCs population to investigate more precisely the cellular mechanisms, and to assess whether patients with PAD treated with EPCs show variation in muscle perfusion as displayed by contrast enhanced ultrasound (CEUS).

According to the current guidelines, reliable diagnostic tools for the management of patients with PAD, are ankle-brachial index (ABI), duplex ultrasound, transcutaneous oxygen pressure (TcPO 2 ), magnetic resonance (MR), contrast computer tomography (CT scan) and conventional angiography (CA). Among imaging modalities under development, micro bubble—based CEUS is a real-time, high spatial-resolution imaging technique that can easily be applied. Its high sensitivity, which provides detection of extremely low concentrations of micro bubbles, combined with the true blood pool distribution of contrast agent, gives CEUS the potential to help visualize and quantify the vasculature in vivo. CEUS was recently proposed as a valuable method to detect perfusion deficit and collateralization in patients with PAD [8], [9]. This method has been also validated for the detection of impaired microcirculation in patients with diabetes mellitus [10].

We present our experience in eight no-options CLI patients transplanted with peripheral immune-selected apheresis-derived autologous CD133+ cells and followed up with CEUS for 12 months.

Patients and methods

Type of the study

Prospective single centre not randomized.

Clinicaltrials.gov identifier: NCT01595776

Aim of the study

To assess the safety, feasibility and efficacy of local intramuscular administration of autologous immuno-selected CD133+ cells in patients suffering from CLI. The protocol started at our institution in July 2011 with the local Ethical Committee (EC) approval (number CHVAS-01-08-10/03/08). The investigation conforms with the principles outlined in the Declaration of Helsinki.

Enrolment criteria

All the patients enrolled were suffering from clinical stable CLI according to the TASC 2 definitions [1] and had no revascularization option, on the basis of contrast CT scan, RM or angiography imaging and the evaluation of our vascular and endovascular team. A detailed informed consent, approved by our EC, had been required.

Exclusion criteria

Patients under 18 and over 70 years of age. Elderly patients have been excluded because of expected bone marrow low responsiveness. Clinical unsteadiness of CLI (such as gangrene requiring major amputation) and poor life expectancy are exclusion criteria for supposed latency of the EPCs action. Severe systemic illness was judged to increase the risk of bone marrow stimulation. Complete inclusion and exclusion criteria are depicted in Table 1.

Table 1. Inclusion and exclusion criteria

Patients

Between September 2011 and September 2013 we enrolled eight patients with a history of Rutherford stage 4 (rest pain) or 5 (small ischemic lesions) PAD. All patients had previous vascular imaging (contrast CT or MR or angiography) excluding revascularization options, either endovascular and surgical and encountered inclusion criteria. Every patient underwent routine physical and instrumental examination including electrocardiogram, chest X-ray and blood sample analysis. The patients’ features are summarized in Table 2. The median age was 46.8 (SD 11.8, range 37–69 years), with a M:F ratio of 3:1. Younger patients met Buerger disease criteria (n = 4, 50 %), whereas others had pure atherosclerotic lesions. Only one patient (ID 7, female) had diabetes mellitus. Six patients out of eight had ischemic lesions on the forefoot (Rutherford stage 5) with poor healing and a long history of wound treatment. All patients complained moderate/severe pain and took high doses of analgesics (slow release opiates in two cases).

Table 2. Patients baseline

CEUS imaging protocol

Two operators who were blinded to treatment performed CEUS for all patients. The imaging criteria were: (1) reduced transmit power, at approximately 7–10 frames per second and one focus well below the level of the target to ensure a more uniform pressure field. (2) dual-mode presentation of a grayscale image side-by-side with the contrast image facilitating real-time identification of anatomic structures and region of interest (ROI) selection. (3) Image loops of approximately 60 s. (4) Uniform gain across the image and avoid gain saturation. (5) The time gain compensation (TGC) set such that before contrast arrival a uniform black image was shown.

A vial of contrast agent (SonoVue BR1; Bracco, Milan, Italy) was prepared at a concentration of about 2 × 10 8 sulfur hexafluoride—filled micro bubbles per milliliter, according to the manufacturer’s recommendations. The position of the probe was recorded for each patient in order to maintain the same position during follow up. The injection was made with the patient supine and after 10 min of rest to avoid exercise related micro-vascular dilatation. The radiologist maintained a constant image plane with the aid of the tissue (fundamental image) of the “Contrast Side/Side” imaging mode.

Image analysis

The main image analysis tasks were: (1) identification of anterior tibialis artery (ATA) area, (2) selection of a representative region of normal anterior tibialis muscle (ATM) and (3) formulation of time-intensity curves (TIC). Two manually defined ROI, 2 and 4 cm sided-squares, were placed, respectively, over the ATM with no evidence of arterial branches, over ATA and over a small tibialis arterial branch. The ROIs were placed in the same anatomical position for each patient to avoid unwanted differences during follow up examinations. One TIC was obtained for each ROI. The image loops were transferred to a personal computer for further analysis. From the analysis of TIC, we computed regional blood flow (RBF) and regional blood volume (RBV). TIC were extracted using commercial quantification software (QontraXt v.3.60, AMID, Rome, Italy). This software allows manual ROI selection, measurement of the selected ROI area and provides linear data for the TIC. For the ROI in the normal ATM, effort was made to place the region in an area without large vessels. The ATA ROI was a 2 cm square area and the ATM ROI was a 4 cm square area. TICs were obtained by computing the mean intensity of pixels comprised within the ROI at each time point. For each image loop were calculated:

RBV which consists in the total amount of contrast media within the selected ROI, in a period of time. Due to the characteristics of US contrast media, it reflects the quantity of blood in a defined region. It is directly related to the area under the curve (AUC).

RBF consists in the contrast media flow (related to the blood flow) in a selected ROI. It is related to the mean transit time.

Bone marrow stimulation

Human recombinant granulocyte colony-stimulating factor (rhGCSF) was administered subcutaneously for 4–5 consecutive days at a dosage of 10 µg/kg daily, split in two doses. Starting from the third day of stem cells mobilization, the CD34+/133+ cells count was monitored daily by cytofluorimetric analysis. The minimum CD34+/133+ cells count acceptable for leukapheresis collection was 20 and 10/µl, respectively. Patients were monitored for any G-CSF related side effects.

Leukapheresis (LKF) collection

A single LKF collection was planned for each patient using a third generation cell separator device (Spectra Cobe, Lakewood, CO, USA), processing at least 2.5 blood volumes according to our internal protocol for stem cell collection. Immediately after the LKF collection, a sample from patient’s peripheral blood was taken for haemocytometric analysis to evaluate platelet count and haemoglobin levels. Each LKF collection was diluted with 10 % acid citrate dextrose (ACD-A) and maintained overnight at 4 °C degrees before immuno-magnetic cell selection.

Immunomagnetic cell selection

CD133+ immunomagnetic cell selection (ICS) was performed the day after LKF collection using the Clini-MACS (Miltenyi Biotec) device according to the manufacturer’s standard protocol.

Quality controls

A sample taken from the CD133 cell positive fraction was seeded for short term (14 days) clonogenic assays to evaluate the quality of immunoselected stem cells in terms of proliferative capacity. A standard mixture of methylcellulose plus recombinant human growth factors was employed (Stem Cell Technologies, Vancouver, BC, Canada; MACS Media, Miltenyi Biotec GmbH, Bergisch Gladbach,Germany). Microbial cultures on the waste bag containing the negative fraction were carried out to detect aerobic-anaerobic bacteria and fungal contamination. A sample of 10 ml was inoculated in the culture medium (Bact/Alert FA and BacT/Alert FN, Organon Teknika Corp., Durham, NC) and incubated for 10 days at 37 °C.

Cytofluorimetric analysis

Samples obtained from peripheral blood before mobilization with G-CSF, at time of LKF and after immunomagnetic cell selection were analyzed by flow cytometry to evaluate the expression of specific stem cell and endothelial antigens. Becton–Dickinson FACSCanto was employed for all flow cytometric analysis with a lyse no-wash technique, using the following monoclonal antibodies: anti-CD45 fluorescein isothiocyanate (FITC) (Becton–Dickinson, San Jose, CA, USA), anti-CD34 Peridinin-chlorophyll-protein complex (PerCP) (8G12 clone, Becton–Dickinson), anti-CD133 phycoerythrin (PE) (AC133 clone, Miltenyi Biotec) and anti-VEGF-R2 allophycocyanin (APC) (R&D systems), following the manufacturer instructions.

Each sample was acquired with BD FACSCanto recording 100.000 events inside the lymphocyte plus monocyte gate. Data files were analyzed with FACS Diva 6.1 software. Viability was assessed using 7-amino-actinomycin D (7-AAD) (Molecular Probes, Eugene, OR, USA).

Implant procedure

After loco-regional anesthesia and below the knee cutaneous disinfection, 45–48 ml of autologous CD133+ cell in buffer phosphate (Miltenyi Biotec) suspension was administered intramuscularly with 1 ml deep injections through a 18G needle. The injections were so allocated: 10 ml in the anterior compartment of leg, 10 ml in the superficial posterior compartment, 10 ml in the deep posterior compartment, 10 ml in the lateral compartment and the remaining part in the foot (Additional file 1: Figure S1).

Baseline assessment and follow up

Pain assessment was carried out with a personal scale of 3 degrees (mild, moderate and severe) and the pain killing drugs use monitored. Ischemic lesions were treated weekly by a wound management skilled nurse. CEUS, lesion evolution and pain management were assessed at baseline and 3, 6 and 12 months after the implant.

Statistical analysis

All quantitative variables were normally distributed (Shapiro–Wilk test) and so the results were expressed as mean values and standard deviation (SD); qualitative variables were summarized as counts and percentages. Pearson’s r coefficient was used to test correlation between two study variables. Linear regression models for repeated measure were used to assess the increase over time of the CEUS parameters. Data analysis was performed with STATA statistical package (release 11.1, 2010, Stata Corporation, College Station, TX, USA).

Results

Patient’s mobilization and LKF

No relevant side effects related to G-CSF administration were registered. A single LKF collection per patient was performed. No side effects were registered. Patients did not require any red blood cells or platelet transfusion after LKF procedures. Total nucleated cells (TNC) content and viability in peripheral blood of the eight patients enrolled are depicted in Table 3.

Table 3. Cytofluorimetric analysis

Immunomagnetic cell selection

The immunomagnetic cell selection was carried out the day after LKF collection. Almost all cells expressing CD133 antigen also expressed CD34 antigen. The mean CD133+ cell recovery was 46.1 % (SD 21.5, range 9.9–81.7). The mean purity was 85.4 % (SD 19.6, range, 37.2–96.5). TNC viability was always >90 %. Results of cytofluorimetric analysis performed on samples obtained from the positive fraction are detailed in Table 3. The mean number of CD133+ infused cell per limb kilogram was 25.2 × 10 6 (SD 13.8; range 2.6–42.2). Clonogenic assays demonstrated the maintained proliferative capacity of immunoselected stem cells. The result of microbial cultures was always negative.

Clinical results

No patient had early or late complications related to the procedure. Two patients (number 3 and 7, 25 %) didn’t get any relief from the treatment and underwent major amputation. Patient number 3 had lesion and pain worsening and was amputated below the knee after 5 months. Patient number 7 had pain worsening and final gangrene of the foot and underwent above the knee amputation after 7 months; this patient had diabetes and a heavy smoking habit (40 cigarettes/day) persisting in spite of physician’s indication. Five patients (number 1, 2, 4, 5 and 6, 62.5 %) had a complete healing of the wounds, complete rest pain cessation and walking recovery or increased pain free walking distance. One patient (number 8, 12.5 %) had rest pain cessation, and a mild improvement in pain free walking distance. None statistical correlation has been found between the number of infused CD133+ cells and the clinical results.

CEUS

An increase in RBF and RBV was shown in all eight patients at 3 and 6 months and in the six clinical healed patients at 12 months and has statistical relevance: at 12 months mean increasing for TAM-RBV was 48.8 % (p = 0.018), for TAM-RBF 59.4 % (p = 0.0016), for ATA-RBV 52.8 % (p = 0.017) and for ATA-RBF 48.6 (p = 0.007). The trends in increasing values at 3, 6 and 12 months for RBV and RBF, both in artery and in muscle, are depicted in Fig. 1. No statistical correlation between RBF and RBV value, and CD133+/CD34+ infused cells was found at 6 and at 12 months. Additional file 2: Figure S2 shows the correlation between clinical healing and CEUS values improvement in patient 1. Table 4 shows a synopsis of the results.

thumbnailFig. 1. The diagrams show the percentage increase of both RBV and RBF both in muscle and in anterior tibial artery. RBF regional blood flow, RBV regional blood volume. In the x axis are months of follow-up. In the y-axis is the percentage of increase of the values of RBV and RBF compared to the baseline. Dotted lines represent single patient values. Continue line represents the mean percentage increase with the statistical significance. P values compare the value of mean increase to the baseline

Table 4. Results

Discussion

CLI is a manifestation of PAD that includes patients with typical chronic ischemic pain at rest or patients with ischemic skin lesions, either ulcers or gangrene. The term CLI should only be used in relation to patients with chronic ischemic disease, defined as symptoms lasting more than 2 weeks. The diagnosis of CLI should be confirmed by ABI, toe systolic pressure or transcutaneous oxygen tension. Ischemic rest pain most commonly occurs below an ankle pressure of 50 mmHg or a toe pressure less than 30 mmHg [1].

The first large report on the use of bone marrow-derived mononuclear cells (BM-MNC) in limb ischemia was the therapeutic angiogenesis by cell transplantation (TACT) study by Tateishi-Yuyama et al. At 4 weeks, ankle-brachial index (ABI) was significantly improved in legs injected with BM-MNC and similar improvements were seen for transcutaneous oxygen pressure. They concluded that autologous implantation of BM-MNC could be safe and effective for achievement of therapeutic angiogenesis, because of the natural ability of marrow cells to supply endothelial progenitor cells and to secrete various angiogenic factors or cytokines [11]. Since then, several trials were published, both using BM-MNC, and mobilized peripheral blood mononuclear cells (PBMNC) with intra-arterial or intra muscular administration, well reviewed by Lawall et al. [12].

In their review, Lawall et al. sustain that the role of “EPCs” in human angiogenesis in the setting of peripheral vascular obstruction remains doubtful, and the translation of a truly “EPC” based endothelial repair into clinic practice has not been achieved so far. The well substantiated concept of arteriogenesis strengthens the importance of several different bone marrow cell types, however all sharing a monocytic phenotype. They migrate to the perivascular space of sprouting collaterals and induce collateral artery growth by the release of angiogenic growth factors. A growing body of evidence strongly suggests that these secreted molecules mediate a number of protective mechanisms including cell survival, neo-vascularization, remodeling, and proliferation. The regulatory system governing paracrine factor release appears to be complex and dependent on spatiotemporal parameters. [13].

Based on available data, cell therapies in PAD based on the application of whole BM-MNC or on whole stimulated PBMNCs are more successful than methods which use subfractionated cell preparations [12], e.g. CD 133+ [14] or highly purified CD 34+ cells from peripheral blood after granulocyte- colony stimulating factor (G-CSF) mobilisation only [15]. Nevertheless available data are very scarce about efficacy of a specific subset cells population versus the entire pool of mononuclear cells, because great majority of the previous and ongoing studies employ BM-MNC or PBMNCs, due to both lower costs and relative simplicity of the method. Except the case report of Canizo et al., the only previous study employing autologous selected CD 133+ cells is by Burt et al.: they treated nine patients with positive results in 7 [16].

Previous EPCs in CLI studies considered only data measured with ABI and TcPO 2 . As a matter of fact both ABI and TCpO2 have an intra and inter patient variability due to detection method, operator experience, vasodilatation state and emotional stress, that make standardization difficult. Moreover, in case of very low velocity flow, as in peripheral blood circulation in CLI, ABI detection variability increases [17]. CEUS offers a reliable method to measure peripheral blood flow, and is a valid alternative: it’s equally a non-invasive method, because the medium contrast hasn’t got any contraindication, except hypersensitivity, and the assessment method is not operator, but dedicated software related. It has only a minimal intra patient variability, when the detection method is accurate.

The aims of our study are (1) to demonstrate that a highly purified autologous stem cells population can induce neo-angiogenesis in a safe, feasible and effective way and (2) to assess neo-angiogenesis with a non-invasive method as most objective and reproducible as possible. In consideration of the end stage disease character of CLI, we didn’t considered ethical randomizing eligible patients. The possible advantages in studying a specific cells population in order to understand the mechanisms of neo-angiogenesis are to avoid the overlapping effects of entire MNC populations infused and as second step to better identify the cytokines pattern, derived from a single cell type, given the hypothesis of a paracrine mechanism.

Stem cells mobilization with G-CSF administration induce a high white blood cells count (WBCc) and a subsequent theoretical blood hyper viscosity. In patients with CLI blood hyper viscosity can be an issue. In our series the mean WBC count at the 4th day after mobilization was 48.1 × 10 6 /ml (range 26.6–77). These values are similar to these observed in healthy donors mobilized for allogeneic hematopoietic stem cell transplant. Even in the patient (ID = 7) with the highest WBCc (77.0 × 10 6 /ml) we didn’t observe any significant side effect related to blood hyper viscosity. However, we administered a prophylactic dose of low molecular weight heparin (3800/4000 UI/daily) considering the pro thrombotic risk related to G-CSF administration and patient immobilization. Remarkably also the CD34+ and CD133+ mobilization in these patients is comparable to healthy donors showing that their stem cell reservoir is not depleted. Stem cell collection was performed following our internal protocol, processing 2.5 blood volumes without any relevant side effects. On the whole we can argue that patients with compromised peripheral circulation can tolerate very well both mobilization and LKF. The wide range of CD133+ cell recovery after the ICS may be related to the different antigen expression on the stem cell surface. All the immunoselected CD133+ cell samples showed an high in vitro clonogenic potential (similarly to hematologic field) demonstrating the good quality of the product infused. Nevertheless the purity was always very high (≥90 %), except in one case (ID = 5), showing that every patient, but one, was treated with a single cell population. From this point of view, we obtained results comparable with the study of Burt et al. [16].

Altogether, 6 out of 8 patients (75 %) had clinical improvement, with ulcer healing, cessation of rest pain, increased walk pain free distance and above all, avoided amputation and maintained their improvement for a long period of time. Indeed they had the longest follow up so far in literature, 12 months with CEUS and at least 18 months from a clinical point of view. The graphics in Fig. 1 show the increase (in percentage) from baseline of RBV and RBF, both for ATM and ATA: mean percentage increase (continuous line) is always positive and statistically significant (see also Table 4 for p values). Clinical improvement is consistent with instrumental data showing an increased blood flow in the limb, reasonably related to an induced neo-angiogenesis. Nevertheless other important local factors may be involved both in initiating and maintaining angiogenesis, like resident cells and chemokines and cytokines environment. Data demonstrate that the resident progenitor cells could differentiate into a variety of cell types in response to different culture conditions. However, collective data were obtained mostly from in vitro culture assays and phenotypic marker studies. There are many unanswered questions concerning the mechanism of cell differentiation and the functional role of these cells in vascular repair and the pathogenesis of vascular disease [18].

Patient 3, despite the maximum values of cell dose infused (42.2 × 10 6 /limb kg) showed a complete lack of responsiveness both clinical, and instrumental (Table 4). Conversely, Patient 5 received the lowest dose of CD133+ cells with the lowest purity (Table 3): however she showed a satisfactory clinical response (ulcer healing, rest pain relief and increased pain free walk distance) and TAM-RBV/RBF increasing (+28.6 and +42.9, respectively). We can speculate that these opposite results are related not exclusively to the cell dose infused, but also to responsiveness of the resident stem cells. Indeed we didn’t find any statistical correlation between the number of infused CD133+ cells (10 6 per limb kg) and the clinical and CEUS results.

Patient 7 showed apparently conflicting results: at 6 months TAM-RBV/RBF were increased around 77 % with a slightly improvement of clinical condition. Unfortunately the following month she developed a sudden worsening in the limb ischemia, with subsequent above the knee amputation. It’s crucial to emphasize that the patient had insulin dependent diabetes mellitus (IDDM) and a heavy smoking habit (around 40 cigarettes daily). We can suppose a negative role of IDDM and a precipitating role of the smoke habit during the transient neo-angiogenesis process, as assessed by CEUS values increasing. However impaired angiogenesis in diabetes has been already demonstrated both in animal models and in humans [19], [20].

In conclusion, our study shows interesting perspective and issues. Highly purified autologous CD133+ cells, routinely employed in transplant for oncohematologic diseases, can stimulate neo-angiogenesis, either directly or through a paracrine effect, as based on clinical (ulcer healing, rest pain cessation, increasing pain free walk distance and limb salvage) and CEUS data. Our study gives an instrumental demonstration of neo-angiogenesis. The limits of the study are the lack of randomization, which we judged unethical for this kind of end-stage disease, and the low number of patients, mainly due to the restrictive inclusion and exclusion criteria. Goals for future studies are to enrol a major number of patients including also less advanced stages of PAD, and to consider other powerful stem cell sources as cord blood derived or mesenchymal stem cells.

Additional files

. Additional file 1: Figure S1. The procedure of implant through multiple intramuscular 1 ml injection of CD133+ cells suspension.

Format: TIFF Size: 2.9MB Download fileOpen Data

. Additional file 2: Figure S2. The complete healing of a deep and painful ischemic lesion with bone exposure in patient 1. Beside the corresponding change in RBV and RBF measurements.

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Authors’ contributions

Concept and design: CP, VA, CDF, AB. Supervision: VA, CP. CEUS: FC, FA. Cell management: CP, CDF, GV. Patient management: VA, AB, AM, MP, FR. Data collection: VA, CP, AB, FR, FC, FA,GV, MP, AM. Manuscript draft: all the authors. Manuscript revision and approbation: all the authors. Statistical analysis: CT.

Funding

This study was entirely supported by “Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy”.

Competing interests

The authors declare that they have no competing interests.

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Time series analysis of reported cases of hand, foot, and mouth disease from 2010 to 2013 in Wuhan, China

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Time series analysis of reported cases of hand, foot, and mouth disease from 2010 to 2013 in Wuhan, China

Banghua Chen1, Ayako Sumi2*, Shin’ichi Toyoda3, Quan Hu4*, Dunjin Zhou4, Keiji Mise5, Junchan Zhao6 and Nobumichi Kobayashi2

Author Affiliations

1 Department of Infectious Diseases Prevention and Control, Wuhan Centers for Disease Control and Prevention, Wuhan, Hubei, China

2 Department of Hygiene, Sapporo Medical University School of Medicine, S-1, W-17, Chuo-ku, Sapporo 060-8556, Hokkaido, Japan

3 Department of Information Engineering, College of Industrial Technology, Hyogo, Japan

4 Wuhan Centers for Disease Control and Prevention, 24 Jianghanbei Road, Wuhan 430000, Hubei, China

5 Department of Admission, Center of Medical Education, Sapporo Medical University, Hokkaido, Japan

6 School of Mathematics and Statistics, Hunan University of Commerce, Changsha, Hunan, China

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BMC Infectious Diseases 2015, 15:495  doi:10.1186/s12879-015-1233-0

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2334/15/495

Received: 2 February 2015
Accepted: 19 October 2015
Published: 3 November 2015

© 2015 Chen et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Background

Hand, foot, and mouth disease (HFMD) is an infectious disease caused by a group of enteroviruses, including Coxsackievirus A16 (CVA16) and Enterovirus A71 (EV-A71). In recent decades, Asian countries have experienced frequent and widespread HFMD outbreaks, with deaths predominantly among children. In several Asian countries, epidemics usually peak in the late spring/early summer, with a second small peak in late autumn/early winter. We investigated the possible underlying association between the seasonality of HFMD epidemics and meteorological variables, which could improve our ability to predict HFMD epidemics.

Methods

We used a time series analysis composed of a spectral analysis based on the maximum entropy method (MEM) in the frequency domain and the nonlinear least squares method in the time domain. The time series analysis was applied to three kinds of monthly time series data collected in Wuhan, China, where high-quality surveillance data for HFMD have been collected: (i) reported cases of HFMD, (ii) reported cases of EV-A71 and CVA16 detected in HFMD patients, and (iii) meteorological variables.

Results

In the power spectral densities for HFMD and EV-A71, the dominant spectral lines were observed at frequency positions corresponding to 1-year and 6-month cycles. The optimum least squares fitting (LSF) curves calculated for the 1-year and 6-month cycles reproduced the bimodal cycles that were clearly observed in the HFMD and EV-A71 data. The peak months on the LSF curves for the HFMD data were consistent with those for the EV-A71 data. The risk of infection was relatively high at 10 °C ≤ t < 15 °C (t, temperature [°C]) and 15 °C ≤ t < 20 °C, and peaked at 20 °C ≤ t < 25 °C.

Conclusion

In this study, the HFMD infections occurring in Wuhan showed two seasonal peaks, in summer (June) and winter (November or December). The results obtained with a time series analysis suggest that the bimodal seasonal peaks in HFMD epidemics are attributable to EV-A71 epidemics. Our results suggest that controlling the spread of EV-A71 infections when the temperature is approximately 20–25 °C should be considered to prevent HFMD infections in Wuhan, China.

Keywords:

Hand, foot, and mouth disease; Seasonality; Meteorological variable; Time series analysis; Spectral analysis

Background

Hand, foot, and mouth disease (HFMD) is an infectious disease that typically presents as vesicular exanthema of the oral mucosa and peripheral extremities. Enteroviruses, such as Coxsackievirus A16 (CVA16) and Enterovirus A71 (EV-A71), are most commonly isolated from HFMD patients [1]. Over the past decade, Asian countries have experienced enormous large-scale HFMD outbreaks, with deaths predominantly among children [2]–[6]. The epidemics in China have been particularly serious and HFMD has become one of the leading causes of child death in China and a public health priority [7]. In 2008–2012, 7,200,092 cases of HFMD, including 2457 fatal cases, were reported by the Chinese Center for Disease Control and Prevention [8]. However, no vaccine or effective curative treatment is currently available. The incidence of HFMD will be also significantly affected by the continued mutation of the virus and increasing climate change. Therefore, HFMD remains an important public health problem in China.

Many studies have reported the seasonality of HFMD epidemics in China, and understanding the seasonality of these epidemics may identify potentially modifiable risk factors. Epidemics in several regions of China peak in late spring/early summer, with a second smaller peak in late autumn/early winter [9]–[13]. Researchers have interpreted the seasonality of HFMD cases in terms of climate variables in specific regions. Meteorological parameters, such as temperature and relative humidity, may affect the transmission and frequency of HFMD. However, the effects of climate variables are not consistent across published studies, and these discrepancies could arise from various local climatic conditions, differences in socioeconomic status, and the demographic characteristics of different regions. Therefore, our understanding of the impact of seasonal and meteorological variables on disease transmission remains limited. Further research is required into the effects of climate variations on the incidence of HFMD.

Wuhan in Hubei Province is the largest mega-city in Central China, and has experienced a relatively high prevalence of HFMD in recent years. A better understanding of the temporal pattern of HFMD incidence might allow the appropriate allocation of health-care resources for better disease control and prevention. No study has yet examined the effects of meteorological variables on the occurrence of HFMD in Wuhan.

In this study, we investigated the association between the incidence of HFMD and its pathogens and several meteorological variables (including monthly average temperature, maximum temperature, minimum temperature, relative humidity, total rainfall, and wind velocity) in Wuhan, China, where high-quality surveillance data for HFMD have been collected. We used the time series analysis method “MemCalc” (Suwa-Trast, Tokyo, Japan) [14]–[16], which has been successfully used to investigate associations between the occurrence of infectious diseases, pathogens, and meteorological variables, including rotavirus in India [15], cholera in Bangladesh [17], and chickenpox in Japan [18]. Based on the result for the seasonality of HFMD, we conducted a prediction analysis for HFMD epidemics.

Methods

Study area

Figure 1 shows the location of Wuhan, China. Wuhan, the capital city of Hubei Province in central China, has a total area of 8494 km 2 and a population of 10.3 million. Wuhan is situated at a latitude of 30°34′N and a longitude of 114°16′E, in an area with a subtropical wet monsoonal climate, where the four seasons are very clearly defined. Based on the assumption that the seasons coincide with the weather and temperature patterns in Wuhan, the seasons were defined as spring (April), summer (May–September), autumn (October), and winter (November–March). The monsoon occurs in Wuhan from the middle of June to the middle of July (summer) every year.

thumbnailFig. 1. Location of Wuhan in China. Source: revised from “Chinese latitude and longitude map” (http://www.baidu.com)

Data

HFMD data

A probable case of HFMD is defined as a patient with papular or vesicular rash on the hands, feet, mouth, or buttocks, with or without fever. A confirmed case is defined as a probable case with laboratory evidence of enteroviral infection (including EV-A71, CVA16, or other non-EV-A71 or non-CVA16 enteroviruses) detected with reverse transcription—polymerase chain reaction (RT-PCR), real-time RT-PCR, or viral isolation [19]. Probable and confirmed cases are reported on-line to the China Information System for Disease Control and Prevention (CISDCP, http://www.cdpc.chinacdc.cn) by all the hospitals in Wuhan, using a standardized form. In this study, we analyzed the daily number of cases of HFMD reported in Wuhan between January 1, 2010 and December 31, 2014 (1825 data points). The data are available from the CISDCP website through the Wuhan Centers for Disease Control and Prevention. First, we investigated the associations between the incidence of HFMD and its pathogens and meteorological variables, using the HFMD data from between January 2010 and June 2013 (1276 data points), and then we conducted a prediction analysis using the HFMD data from between July 2013 and December 2014 (549 data points).

Pathogen data

According to the national guidelines [20], the samples were collected from the first five probable cases who presented to hospital outpatient departments each month in each of the 13 districts of Wuhan. The appropriate clinical specimens, including throat swabs, rectal swabs, fecal samples, vesicular fluid, and/or cerebrospinal fluid, were collected. The samples were identified with real-time PCR in biosafety level 2 facilities in the Wuhan Center for Disease Control and Prevention. The test results were classified into four categories: enterovirus negative, EV-A71 positive, CVA16 positive, or positive for another enterovirus without further serotype identification. All pathogen data were uploaded to CISDCP and were downloaded as monthly data. We accessed the relevant pathogen data for the study period from January 2010 and June 2013 (42 data points) from the CISDCP website and the case data for HFMD.

Meteorological data

Daily meteorological data, including average temperature, maximum temperature, minimum temperature, relative humidity, total rainfall, and wind velocity, were collected in the study region by the Meteorological Department, Wuhan, which received and managed real-time data from 116 meteorological surveillance sites widely distributed in Wuhan. The daily data were gathered for 1276 days from January 2010 to June 2013 (1276 data points).

The descriptive statistics for the monthly meteorological data are shown in Table 1. The mean monthly average values in Wuhan were: temperature 16.5 °C, maximum temperature 21.4 °C, minimum temperature 12.6 °C, relative humidity 78.2 %, total rainfall 111.3 mm, and wind velocity 2.1 m/s.

Table 1. Summary statistics for the monthly meteorological conditions in Wuhan, China

Time series analysis

The series of analyses used in the present study was composed of spectral analyses based on the maximum entropy method (MEM) in the frequency domain and the nonlinear least squares method (LSM) in the time domain. This method of analysis can be used for prediction analysis [21], [22].

Theoretical background[21]

We assumed that the time series data x(t) (where t = time) were composed of systematic and fluctuating parts [23]:

x(t)=systematicpart+fluctuatingpart.(1)

The systematic part in Eq. (1) is regarded as the underlying variation in the original time series, and the fluctuating part, including undeterministic components such as noise, was obtained as the residual time series when the underlying part was subtracted from the original time series. The estimation of the underlying variation is a key point.

The underlying variation in the original time series data x(t) is assumed to be described by the function X (t), as follows:

X(t)=A0+n=1NpAncos{2πfn(t+θn)},(2)

which is calculated using the LSM for x(t) with unknown parameters f n , A0 , and An (n = 1, 2, 3, …, N), where f n (=1/T n ; T n is the period) is the frequency of the nth component; A0 is a constant that indicates the average value of the time series data; A n and θ n are the amplitude and the phase of the nth component, respectively; and N p is the total number of components. The LSM using Eq. (2) must be nonlinear. Linearization of this nonlinearity is required to obtain the unique optimum values of these parameters. In the present study, linearization was achieved using the value of f n estimated with the MEM spectral analysis.

An outline of the analysis procedure is described as follows. The details of the procedure for the method are described in our previous work [14], [22].

(1) Setting up the time series data for analysis. The sampling intervals for the HFMD and meteorological data (daily) and pathogen data (monthly) differed. To analyze these three kinds of data together, it was necessary to choose equal sampling time intervals. Therefore, we calculated the monthly data for the HFMD cases and meteorological variables (42 data points) from the original daily data to conform to the monthly pathogen data. All the meteorological parameters studied and the values used for testing the associations are summarized in Additional file 1. For example, the monthly average maximum temperature was calculated by averaging the daily maximum temperature for a month, and the total rainfall was calculated by summing the amount of rainfall measured for the whole month. The monthly meteorological variables are described as follows: T{A}, average temperature (°C); T{M}, maximum temperature (°C); T{m}, minimum temperature (°C); RH, relative humidity (%); RF, total rainfall (mm); and WV, wind velocity (m/s).

(2) Determination of T n (spectral analysis). The value of T n was determined from the positions of the peaks in the MEM power spectral density (MEM-PSD). The MEM spectral analysis has a high degree of resolution and is useful for clarifying periodicities within short time series, such as the time series data examined in this study [21]. The MEM spectral analysis produces a power spectral density (PSD). The formulation of the MEM-PSD is described in an additional file (see Additional file 2).

(3) Determination of N p (assignment of the dominant periodic modes). The contribution of the dominant periodic modes to the underlying variation can be estimated easily from the trend in the standard deviations (SD) of the residual time series xR (t) (= x(t) – X(t)). The value of N p is then determined.

(4) Determination of A 0 , A n , and θ n (least-squares analysis). The optimum values for parameters A0 , A n , and θ n (n = 1, 2, 3, …, N p ), in Eq. (2), but not N p , were determined exactly from the optimum least squares fitting (LSF) curve calculated using the periodic function (Eq. (2)) with the MEM-estimated periods (T n ).

(5) Prediction analysis. The optimum LSF curve X(t) was extrapolated to predict the original time series because the optimum LSF curve is regarded as the predictable part [24]. For the HFMD data, we extended X(t) from the analysis range (January 2010–June 2013) to the prediction range (July 2013–December 2014).

Statistical calculations

All statistical analyses were performed with SPSS 17.0 J for Windows (SPSS Inc., Chicago, IL, USA), and Spearman’s rank correlation (ρ) was used. A two-tailed analysis was used for all statistical tests and a p value of ≤ 0.05 was considered the criterion for statistical significance.

Correlation between pathogen data and meteorological data

The average occurrence of EV-A71 infections and CVA16 infections in the different domains of average temperature (T{A}), T to T + ΔT, was calculated with the following formula [25]:

NT{A},j=inCi,jf(ti)inf(ti),j={E:EVA71C:CVA16(3)

where i is a sequence from 0 to n, t i is T{A} for the ith month period, C i,j is the total number of cases of pathogen j infection in the ith month, and f(t i ) is a function with the following values:

f(ti){=1whenTti<T+ΔT=0otherwise(4)

The numerator on the right side of Eq. (3) represents the sum of all C i,j comprising the 1-month average temperature (t i ) within the temperature domain of T to T + ΔT during the data period. The denominator is the total number of occasions upon which T < t i  < T + ΔT during the same data period.

Similarly, the average occurrences of pathogen infections in the different variable domains for maximum temperature (NT{M},E and N T{M},C ), minimum temperature (NT{m},E and N T{m},C ), relative humidity (NRH,E and NRH,C ), total rainfall (NRF,E and NRF,C ), and wind velocity (NWV,Eand NWV,C ) were determined. The variables t i , T, and ΔT for T{A} in Eq. (4) were replaced with tM i , TM, and ΔTM, respectively, for T{M}; with tm i , Tm, and ΔTm, respectively, for T{m}; with h i , H, and ΔH, respectively, for RH; with r i , R, and ΔR, respectively, for RF; and with w i , W, and ΔW, respectively, for WV.

In Fig. 2, we show the values for NT{A},E against Temp (Temp; temperature [°C]) when ΔT = 1, 3, and 5 °C. When ΔT = 1 °C and 3 °C, the curve of NT{A},E displays irregular variability, whereas when ΔT = 5 °C, the curve of NT{A},E becomes regular in shape. Therefore, we used ΔT = 5 °C for T{A} in the present study. Similarly, the values for ΔTM, ΔTm, ΔH, ΔF, and ΔW were determined as 5 °C, 5 °C, 5 %, 30 mm, and 0.1 m/s, respectively.

thumbnailFig. 2. Dependence of the occurrence of EV-A71 infection and average temperature (NT{A},E ) on temperature interval (ΔT). Dashed line, ΔT = 1 °C; dotted line, ΔT = 3 °C; solid line, ΔT = 5 °C

Results

Case description

From January 2010 to June 2013, 48,882 cases of HFMD were reported to the CISDCP, 4.5 % (2195 HFMD cases) of which were laboratory-confirmed. The age distribution of the reported cases is shown in Fig. 3. The number of reported cases varied greatly with age, with the highest proportion in children under 5 years. This age group contributed over 90 % of the reported cases during the study period.

thumbnailFig. 3. Age distribution of the reported HFMD cases at hospitals in the whole of Wuhan

Temporal variations in HFMD data, pathogen data, and meteorological data

The monthly time series data used in this study are illustrated in Fig. 4. For the HFMD data (Fig. 4a), two peaks occurred in a 1-year cycle, one in a summer month (June in 2010, July in 2011, and May in 2012) and the other in a winter month (December in 2011 and 2012). This bimodal cycle was also clearly observed in cases of EV-A71 infection in 2011 and 2012 (Fig. 4b), although it was not evident in 2010. Large peaks in the number of CVA16 infections (Fig. 4c) were observed in 2011 (November and December) and 2012 (April). The temporal patterns of T{A}, T{M}, and T{m} (Fig. 4d) showed large peaks in a summer month (August) in the annual cycle. The temporal pattern of RH (Fig. 4e) indicated a decreasing trend from approximately 85 % at the beginning of 2010 to approximately 65 % by May 2011. Thereafter, the temporal pattern of RH increased to approximately 80 %, and then remained relatively constant at around 80 %. The time series data for RF (Fig. 4f) indicated a large peak in the summer months of the annual cycle (June in 2010 and 2011, and May–July in 2012). However, as for RH (Fig. 4e), no seasonal pattern in the data for WV (Fig. 4g) was obvious at first glance.

thumbnailFig. 4. Monthly time series data for reported cases of HFMD, their pathogens, and meteorological variables. a HFMD, b EV-A71, c CVA16, d T{A}, T{M}, and T{m}, e RH, f RF, and g WV

Spectral analysis and LSF analysis

(i) MEM spectral analysis: The MEM-PSDs for the time series data (Fig. 4) are shown in Fig. 5. For all PSDs, except that of CVA16 (Fig. 5c), prominent spectral peaks were observed at f = 1.0 (= f1 ), corresponding to a 1.0-year period.

thumbnailFig. 5. Power spectral densities of the monthly time series data. a HFMD, b EV-A71, c CVA16, d T{A}, T{M}, and T{m}, e RH, f RF, and g WV

(ii) Assignment of fundamental modes: To obtain the optimum LSF curve, we assigned the fundamental modes constructing the underlying variation in Eq. (1). In the present study, we investigated the contributions of 10 MEM-estimated periods to the LSF curve. We then calculated the SD of the residual time series with the variation in N p . For the HFMD data, for example in Fig. 6, the values of SD were plotted against N p . The figure shows inflection points at 6–8 modes. We separated the contributions of the 10 periods into two parts: the underlying variation and the fluctuating part, as described in Eq. (1). For the HFMD data, we determined N p  = 5 and assigned the five periods as the fundamental modes (1.63, 1.01, 0.64, 0.51, and 0.37 years), which are listed in Table 2 with the corresponding periods and intensities (powers) of the spectral peaks. The fundamental modes for the pathogen data and the meteorological data were similarly assigned at N p  = 5 and are shown in Table 2. In Fig. 7, each LSF curve calculated with the fundamental modes reproduces the original time series data well. The good fit of each LSF curve to the original time series data was supported by the high values of ρ between the original data and the LSF curve: 0.93 for HFMD, 0.95 for EV-A71, 0.90 for CVA16, 0.99 for T{A}, T{M}, and T{m}, 0.94 for RH, 0.93 for RF, and 0.82 for WV. Thus, the fundamental modes assigned to the results of the MEM spectral analysis for each set of time series data (Fig. 5, Table 2) were confirmed as appropriate.

thumbnailFig. 6. Contributions of periodic modes to the LSF curve of HFMD data

Table 2. Characteristics of the fundamental modes of monthly data for HFMD cases, pathogens, and meteorological variables

thumbnailFig. 7. Comparison of the LSF curves calculated for the fundamental modes with the original data. LSF curve, solid line; original data, dashed line. a HFMD, b EV-A71, c CVA16, d T{A}, T{M}, and T{m}, e RH, f RF, and g WV

Prediction analysis

The optimum LSF curve for HFMD, calculated with the five fundamental modes (Table 2), was extended from the analysis range (January 2010–June 2013) to the prediction range (July 2013–December 2014) and the results are shown in Fig. 8. The LSF curve in the prediction range reproduced the position of the peak in autumn 2013 and that in spring 2014 fairly well. The LSF curve in the prediction range (July 2013–December 2014) lies within the 95 % confidence interval, reproducing the underlying variation in the original data well.

thumbnailFig. 8. Comparison of the optimum LSF curve for the HFMD data in the prediction range. LSF curve, solid line; original data, dashed line; gray lines, 95 % confidence

Bimodal cycles of HFMD data and pathogen data

It is noteworthy that the dominant spectral lines for HFMD, EV-A71, and CVA16 (Fig. 5a, b, and c, respectively) were observed at f = 0.5, corresponding to a 6-month period, resulting from the bimodal cycles observed in the HFMD and EV-A71 data in 2011 and 2012 (Fig. 4a and b, respectively) and in the CVA16 data in 2011–2012 (Fig. 4c).

The LSF curves for HFMD and EV-A71 were calculated with the 1-year and 6-month cycles, which were clearly observed in the PSDs (Fig. 5a and b, respectively). The LSF curves obtained were normalized in amplitude and overlapped, as shown in Fig. 9a. The peak months on the LSF curves for the HFMD and EV-A71 data during 2010–2012 were mutually consistent, whereas the peak month on the LSF curve for HFMD in 2013 (June) was delayed by 1 month relative to that for EV-A71 (May). The value of ρ between the LSF curve for HFMD and that for EV-A71 was high (0.90).

thumbnailFig. 9. Normalized LSF curves. a The curves for HFMD (solid line) and EV-A71 (dashed line) calculated with the 1-year and 6-month cycles, and (b) the curves for HFMD (solid line) and CVA16 (dashed line) calculated with the 6-month cycle

Similarly, the LSF curves for HFMD and CVA16 were calculated with the 6-month cycle clearly observed in the PSDs (Fig. 5a and c, respectively), and the results obtained are shown in Fig. 9b. The peak months on the LSF curve for HFMD in 2010 (June and December), 2011 (June), and 2013 (June) were delayed by 1 month relative to those for CVA16. The other peak months on both LSF curves were mutually consistent. The value of ρ between the LSF curve for HFMD and that for CVA16 was high (0.65).

Correlations between EV-A71 infection and meteorological variables

The values of ρ between the pathogen data (EV-A71 and CVA16) and the meteorological variables are listed in Table 3. EV-A71 infections were positively associated with T{A}, T{M}, T{m}, and RF, and negatively associated with RH and WV. Of these variables, T{A}, T{M}, T{m}, and RF showed strong mutual associations with high ρ values ranging from 0.33 to 0.37. In contrast, RH, RF, and WV were not significantly associated with EV-A71 infections.

Table 3. Spearman’s correlation coefficients for monthly data on pathogens and meteorological variables

Based on the results for EV-A71 shown in Table 3, we investigated NT{A},E , NT{M},E , NT{m},E , and NRF,E (Eq. (3)). The results obtained are shown in Fig. 10. In the case of T{A} (Fig. 10a), the value for NT{A},E was relatively high when 10 °C ≤ Temp < 15 °C and 15 °C ≤ Temp < 20 °C, and peaked when 20 °C ≤ Temp < 25 °C. The value of NT{A},E became small when 25 °C ≤ Temp < 30 °C, but increased again when 30 °C ≤ Temp < 35 °C.

thumbnailFig. 10. Occurrence of EV-A71 and CVA16 infections and meteorological variables. ad Average EV-A71 infection occurrence (NT{A},E , NT{M},E , NT{m},E , and NRF,E ) was defined as the average number of EV-A71 infections observed during a 1-month period for a given domain of T{A}, T{M}, T{m}, and RF. e The average CVA16 infection occurrence (NRH,C ) was defined as the average number of CVA16 infections observed during a 1-month period for a given domain of RH

The inverse V-shaped relationship between the NT{A},E values against Temp (Fig. 10a) was also observed for NT{M},E , with a peak when 25 °C ≤ Temp < 30 °C (Fig. 10b), and for NT{m},E , with a peak when 15 °C ≤ Temp < 20 °C (Fig. 10c), corresponding to before and after the peak of NT{A},E when 20 °C ≤ Temp < 25 °C (Fig. 10a). This result is consistent with the following two facts: (i) the time series data for T{A}, T{M}, and T{m} (Fig. 4d) oscillate in the same phase; and (ii) the differences between the mean values for T{A} (16.5 °C; Table 1) and T{M} (21.4 °C; Table 1) and between the mean values for T{A} (16.5 °C; Table 1) and T{m} (12.5 °C) are approximately 5.0 °C.

For the total rainfall (Fig. 10d), the value for NRF,E was relatively high when 150 mm ≤ r < 200 mm and 250 mm ≤ r < 400 mm (r, total rainfall [mm]).

Correlations between CVA16 and meteorological variables

RH was a strongly associated with CVA16 infections (ρ = 0.35; Table 3). Therefore, we investigated NRH,C , and the results are shown in Fig. 10e. The pattern shows a positive slope with respect to RH. However, T{A}, T{M}, T{m}, RF, and WV were not significantly associated with CVA16 infections (Table 3).

Discussion

In this study, we found that the HFMD infections occurring in Wuhan showed two seasonal peaks, in summer (June) and winter (November or December). The LSF curves shown in Fig. 7 suggest that the bimodal seasonal peaks in the HFMD epidemics are attributable to EV-A71 and CVA16 epidemics. The following factors may explain the bimodal seasonal peaks in the EV-A71 and CVA16 epidemics in Wuhan (Fig. 4b): (i) the association between EV-A71 and CVA16 infections and meteorological variables; and (ii) the environmental conditions in Wuhan.

(i) Association between EV-A71 and CVA16 infections and meteorological variables. The results shown in Fig. 10 support the results of Chang et al. [25], who found that cases of HFMD were reported in Taiwan at temperatures of 13–26 °C, the temperature range in which the EV-A71 virus is activated, and decreased at temperatures lower than 13 °C or higher than 26 °C. In Wuhan, where the temperature falls below 15 °C during autumn-winter and exceeds 25 °C in summer, the occurrence of HFMD epidemics is bimodal (Fig. 4a). This is similar to a previous finding in Guangzhou, China [12], where the association between the incidence of HFMD and temperature increased rapidly below 25 °C but flattened above 25 °C.

However, the results shown in Fig. 10a indicate that the low value of NT{A},E when 25 °C ≤ Temp < 30 °C returns to a high value when 30 °C ≤ Temp < 35 °C, which differs from the infections recorded in Taiwan [25] and Guangzhou, China [12]. This large value for NT{A},E in Wuhan when 30 °C ≤ Temp < 35 °C was recorded on only one isolated occasion in July 2010 (Fig. 4b). To understand the correlation between EV-A71 infection and temperature in Wuhan in more detail, further surveillance data for EV-A71 (including data on HFMD) and other pathogens will be required. The findings of this study show that when the temperature is between 15 and 25 °C in Wuhan, public-health authorities should prepare fully to respond to an epidemic of HFMD, including increasing access to health-care resources, the distribution of scientific knowledge to the public, medical staff and public health personnel, the availability of essential medical equipment, active disease surveillance, and the design of other more-specific control measures to mitigate the risk of disease transmission.

Our finding of a positive correlation between the reported cases of EV-A71 infections and rainfall (Fig. 10d) is supported by a previous study that demonstrated that some tropical and subtropical countries experienced more outbreaks in the rainy season [26]. The large values for NRF,E when 250 mm ≤ r < 400 mm (Fig. 10d) are consistent with the peak rainfall during the monsoon, which brought large amounts of rain in June 2010, June 2011, June 2012, and May–June 2013 (Fig. 4f). The large values of NRF,E when 150 mm ≤ r < 200 mm correspond to the relatively high values for rainfall before and after the monsoons in April–May and July–August (Fig. 4f).

(ii) Environmental conditions in Wuhan. The winter peak in HFMD, which occurs after the first peak in summer, is probably attributable to disease transmission from the patients who formed the first peak because EV-A71 persists in the environment [27]. EV-A71 can be found in an infected person’s feces for several weeks after the onset of symptoms, and possibly remains for days or weeks on materials in domestic and institutional environments [28], [29]. The high population density in Wuhan could also increase the disease transmission rate and the likelihood of outbreaks.

The strong correlation between RH and CVA16 infections (Fig. 10e) may explain the fairly large numbers of CVA16 infections in the winter of 2011 and the spring of 2012, with very few cases in other years (Fig. 4c), although there has been no convincing explanation of these annual fluctuations in CVA16 infections. The annual fluctuations in disease have been interpreted in terms of many factors, including meteorological factors, host susceptibility, and changing contact rates between susceptible and infectious individuals [30]. This organizational process has been investigated with the susceptible/exposed/infective/recovered (SEIR) model, which is described with nonlinear differential equations [31], [32], but no definite conclusions regarding CVA16 infections have yet been drawn.

We found no statistically significant association between WV and either EV-A71 or CVA16. This result is inconsistent with a Hong Kong study [33] for the period 1981–2010, when WV was reported to be 3.1 m/s, which was greater than the average wind speed in Wuhan during the present study period (2.1 m/s; Table 1). It is possible that there is a threshold effect of wind speed, which is not exceeded in Wuhan.

The prevalent month/week of the seasonal cycle of HFMD incidence has attracted the attention of researchers in the hope of predicting disease outbreaks [9]–[13]. To investigate the seasonality of the disease incidence, some studies have used time series analyses [9]–[12]. One of the important approaches used with time series is the autoregressive model, which is a special case of the linear filter model, and includes sophisticated versions, such as the autoregressive moving-average model and the seasonal autoregressive integrated moving-average model [9], [34]. In the present study, we applied our prediction analysis method to the HFMD data (Fig. 8). The present method is based on the most traditional method of prediction analysis, which uses an extrapolation curve corresponding to the underlying variations of the time series data, X(t) (Eq. (2)) in future. The reproducibility of the HFMD data is considered to arise because the fundamental modes constructing X(t) (Table 2) were well assigned by the MEM spectral analysis and reconstruct the periodic structure of the underlying variation in the data in the prediction range (Fig. 8). We anticipate that the present method of time series analysis using an MEM spectral analysis and LSM will allow the further development of prediction analyses for HFMD epidemics.

A limitation of this study was that we used monthly pathology data for EV-A71 and CVA16 rather than daily or weekly data, because monthly measures are the minimum unit of measurement released by the CISDCP. Further studies using daily or weekly data are required in the future. Another limitation was that the percentage of laboratory confirmation was low (< 5 %), because the purpose of testing samples from HFMD cases is to determine the predominant virus circulating in Wuhan, rather than to identify further patients with the disease.

Conclusion

The results of our study indicate that in Wuhan, EV-A71-based HFMD infections correlate strongly with the average, maximum, and minimum temperatures and total rainfall, and that CVA16-based HFMD infections correlate strongly with relative humidity.

The Intergovernmental Panel on Climate Change Third Assessment Report states that “changes in climate that will affect potential transmission of infectious diseases include temperature, humidity, altered rainfall, and sea-level rise” [35]. EV-A71 and CVA16 lack a thermostatic mechanism, and their reproduction and survival rates are strongly affected by fluctuations in temperature, as are those of other viruses, parasites, and bacteria [36], [37]. Therefore, the effects of meteorological variables on the epidemiology of EV-A71 and CVA16 must be investigated to control HFMD, as in this study.

Abbreviations

HFMD: Hand, foot, and mouth disease

EV-A71: Enterovirus A71

CVA16: Coxsackievirus A16

T{A}: Average temperature

T{M}: Maximum temperature

T{m}: Minimum temperature

RH: Relative humidity

RF: Rainfall

WV: Wind velocity

MEM: Maximum entropy method

LSF: Least squares fitting

LSM: Least squares method

PSD: Power spectral density

SD: Standard deviation

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

BC conceived the study, and managed and analyzed the HFMD incidence data. Data collection was administered and supervised by QH and DZ. AS, ST, and KM analyzed the data and AS drafted the manuscript. JZ partly analyzed the data. NK attracted funding. All authors contributed to writing the final version of this paper. All authors read and approved the final manuscript.

Additional files

Additional file 1:. Meteorological parameters used for examining the relationship with the monthly EV-A71 and CVA16 data. (XLSX 10 kb)

Format: XLSX Size: 10KB Download fileOpen Data

Additional file 2:. MEM spectral analysis. (DOCX 17 kb)

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Acknowledgments

This study was supported, in part, by Grants-in-Aid for Scientific Research from the Health and Family Planning Commission of Hubei Province of China (grant no. JX6B102), the Health and Family Planning Commission of Wuhan Municipality of China (grant no. WG13B02), the National Science Foundation of China (grant no. 61203159), and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant no. 25305022 and no. 25460769). The authors thank Edanz Group Ltd for their careful checking of the grammar and spelling of the manuscript.

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

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

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

Author Affiliations

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

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

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

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

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

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

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

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

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

For all author emails, please log on.

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

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2334/15/496

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

© 2015 Van Beckhoven et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Background

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

Methods

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

Results

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

Conclusions

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

Keywords:

HIV; Cascade; Continuum of care; Migrants; Belgium

Background

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

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

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

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

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

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

Methods

Data sources and setting

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

Definitions

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

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

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

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

Continuum of HIV care

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

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

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

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

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

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

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

Data management and statistics

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

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

Results

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Discussion

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Conclusions

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

Abbreviations

ARC: AIDS Reference Centre

ARL: AIDS Reference Laboratory

ART: Antiretroviral therapy

IDU: Injecting drug use

MSM: Men who have sex with men

PLHIV: People living with HIV

VL: Viral load

WIV-ISP: Scientific Institute of Public Health

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

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

Additional file

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

Format: DOCX Size: 15KB Download fileOpen Data

Acknowledgements

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

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

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Challenges to the management of curable sexually transmitted infections

Marcus Y Chen12 and Sepehr N Tabrizi345*

Author Affiliations

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

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BMC Infectious Diseases 2015, 15:337  doi:10.1186/s12879-015-1061-2 The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2334/15/337

Received: 15 June 2015
Accepted: 23 July 2015
Published: 1 December 2015
© 2015 Chen and Tabrizi. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

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

Editorial

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

Competing interests

The authors declared that they have no competing interests.

Authors’ contributions

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

Acknowledgments

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

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