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Leprosy is a chronic disease caused by Mycobacterium leprae. Worldwide, more than 200,000 new patients are affected by leprosy annually, making it the second most common mycobacterial disease after tuberculosis. The MHC/HLA region has been consistently identified as carrying major leprosy susceptibility variants in different populations at times with inconsistent results. To establish the unambiguous molecular identity of classical HLA class I and class II leprosy susceptibility factors, we applied next-generation sequencing to genotype with high-resolution 11 HLA class I and class II genes in 1,155 individuals from a Vietnamese leprosy case-control sample. HLA alleles belonging to an extended haplotype from HLA-A to HLA-DPB1 were associated with risk to leprosy. This susceptibility signal could be reduced to the HLA-DRB1*10:01~ HLA-DQA1*01:05 alleles which were in complete linkage disequilibrium (LD). In addition, haplotypes containing HLA-DRB3~ HLA-DRB1*12:02 and HLA-C*07:06~ HLA-B*44:03~ HLA-DRB1*07:01 alleles were found as two independent protective factors for leprosy. Moreover, we replicated the previously associated HLA-DRB1*15:01 as leprosy risk factor and HLA-DRB1*04:05~HLA-DQA1*03:03 as protective alleles. When we narrowed the analysis to the single amino acid level, we found that the associations of the HLA alleles were largely captured by four independent amino acids at HLA-DRβ1 positions 57 (D) and 13 (F), HLA-B position 63 (E) and HLA-A position 19 (K). Hence, analyses at the amino acid level circumvented the ambiguity caused by strong LD of leprosy susceptibility HLA alleles and identified four distinct leprosy susceptibility factors., Author summary Despite global efforts to eliminate leprosy over the past 25 years, more than 200,000 new cases are reported annually, and leprosy still represents a major public health problem in endemic regions. Leprosy presents a strong link with the host genetic background. The most significant susceptibility factors are located in the MHC region and likely involve classical HLA genes. However, the molecular identity of the HLA class I/II-leprosy risk factor(s) has been a matter of longstanding scientific dispute. By conducting a comprehensive sequenced-based analysis of HLA class I and class II genes, we are able to provide a unifying view of the complex relationship of leprosy susceptibility and HLA alleles. In addition, we show that four amino acid polymorphisms in HLA-DRβ1, HLA-B and HLA-A are sufficient to explain the majority of leprosy-HLA associations which opens the way for select protein-HLA peptide binding studies.

Leprosy is the second most prevalent mycobacterial disease globally. Despite the existence of an effective therapy, leprosy incidence has consistently remained above 200,000 cases per year since 2010. Numerous host genetic factors have been identified for leprosy that contribute to the persistently high case numbers. In the past decade, genetic epidemiology approaches, including genome-wide association studies (GWAS), identified more than 30 loci contributing to leprosy susceptibility. However, GWAS loci commonly encompass multiple genes, which poses a challenge to define causal candidates for each locus. To address this problem, we hypothesized that genes contributing to leprosy susceptibility differ in their frequencies of rare protein-altering variants between cases and controls. Using deep resequencing we assessed protein-coding variants for 34 genes located in GWAS or linkage loci in 555 Vietnamese leprosy cases and 500 healthy controls. We observed 234 nonsynonymous mutations in the targeted genes. A significant depletion of protein-altering variants was detected for the IL18R1 and BCL10 genes in leprosy cases. The IL18R1 gene is clustered with IL18RAP and IL1RL1 in the leprosy GWAS locus on chromosome 2q12.1. Moreover, in a recent GWAS we identified an HLA-independent signal of association with leprosy on chromosome 6p21. Here, we report amino acid changes in the CDSN and PSORS1C2 genes depleted in leprosy cases, indicating them as candidate genes in the chromosome 6p21 locus. Our results show that deep resequencing can identify leprosy candidate susceptibility genes that had been missed by classic linkage and association approaches., Author summary Leprosy is an infectious disease caused by the Mycobacterium leprae. The development of an effective treatment in the 1980s contributed to the decline of leprosy prevalence. Still, around 200 thousand new leprosy cases are detected every year. In the past two decades, multiple studies have identified genetic variants increasing the risk of developing leprosy, particularly using genome-wide association studies (GWAS). However, GWAS loci commonly encompass multiple genes, which poses a challenge in identifying the best candidates contributing to susceptibility. This limits the usefulness of the genetic findings for understanding leprosy pathogenesis. Our study tested if the accumulation of rare protein-altering variants in genes located in GWAS loci could distinguish the best functional candidates. We found that the burden of amino acid changes in IL18R1 and BCL10 was significantly different between leprosy cases and healthy controls, suggesting these genes as functional candidates in leprosy pathogenesis.

Leprosy is a chronic infectious disease of the skin and peripheral nerves with a strong genetic predisposition. Recent genome-wide approaches have identified numerous common variants associated with leprosy, almost all in the Chinese population. We conducted the first family-based genome-wide association study of leprosy in 622 affected offspring from Vietnam, followed by replication in an independent sample of 1181 leprosy cases and 668 controls of the same ethnic origin. The most significant results were observed within the HLA region, in which six SNPs displayed genome-wide significant associations, all of which were replicated in the independent case/control sample. We investigated the signal in the HLA region in more detail, by conducting a multivariate analysis on the case/control sample of 319 GWAS-suggestive HLA hits for which evidence for replication was obtained. We identified three independently associated SNPs, two located in the HLA class I region (rs1265048: OR = 0.69 [0.58–0.80], combined p-value = 5.53x10-11; and rs114598080: OR = 1.47 [1.46–1.48], combined p-value = 8.77x10-13), and one located in the HLA class II region (rs3187964 (OR = 1.67 [1.55–1.80], combined p-value = 8.35x10-16). We also validated two previously identified risk factors for leprosy: the missense variant rs3764147 in the LACC1 gene (OR = 1.52 [1.41–1.63], combined p-value = 5.06x10-14), and the intergenic variant rs6871626 located close to the IL12B gene (OR = 0.73 [0.61–0.84], combined p-value = 6.44x10-8). These results shed new light on the genetic control of leprosy, by dissecting the influence of HLA SNPs, and validating the independent role of two additional variants in a large Vietnamese sample., Author summary Due to its extreme reductive evolution Mycobacterium leprae is a rare example of an essentially clonal bacterial pathogen that affects humans. However, exposed individuals display a wide diversity of symptoms reflecting the interactions between the host immune response and the bacterium. There is now accumulating evidence, in particular from genome-wide association study (GWAS), that common genetic variants play a role explaining this variability. Previous GWAS were based on case control design and almost all of them were conducted in the Chinese population. We conducted the first family-based GWAS of leprosy in the Vietnamese population and identified several genome-wide significant association signals within the HLA region. By performing a multivariate analysis in an independent case-control sample of the same ethnic origin we were able to decipher the HLA association signal and to reduce it to three independent SNPs, two in the class I and one in the class II region. We also validated two previously identified risk factors for leprosy, a missense variant in the LACC1 gene, and an intergenic variant located close to the IL12B gene. These results shed new light on the genetic control of leprosy and, in particular, on the influence of HLA SNPs in a large Vietnamese sample.

Leprosy is a human infectious disease caused by Mycobacterium leprae. A strong host genetic contribution to leprosy susceptibility is well established. However, the modulation of the transcriptional response to infection and the mechanism(s) of disease control are poorly understood. To address this gap in knowledge of leprosy pathogenicity, we conducted a genome-wide search for expression quantitative trait loci (eQTL) that are associated with transcript variation before and after stimulation with M. leprae sonicate in whole blood cells. We show that M. leprae antigen stimulation mainly triggered the upregulation of immune related genes and that a substantial proportion of the differential gene expression is genetically controlled. Indeed, using stringent criteria, we identified 318 genes displaying cis-eQTL at an FDR of 0.01, including 66 genes displaying response-eQTL (reQTL), i.e. cis-eQTL that showed significant evidence for interaction with the M. leprae stimulus. Such reQTL correspond to regulatory variations that affect the interaction between human whole blood cells and M. leprae sonicate and, thus, likely between the human host and M. leprae bacilli. We found that reQTL were significantly enriched among binding sites of transcription factors that are activated in response to infection, and that they were enriched among single nucleotide polymorphisms (SNPs) associated with susceptibility to leprosy per se and Type-I Reaction, and seven of them have been targeted by recent positive selection. Our study suggested that natural selection shaped our genomic diversity to face pathogen exposure including M. leprae infection., Author summary Each year, 200,000 new leprosy cases are reported worldwide. While there is unambiguous evidence for a role of host genetics in leprosy pathogenesis, the mechanisms by which the human host fights the infection are poorly understood. Here, we highlight the search for naturally occurring genetic variations that modulate gene expression levels following exposure to sonicate of Mycobacterium leprae, the bacterium causing the disease. Because M. leprae is not cultivable and the genuine immune cells involved in the host response during infection are still unknown, we performed a genome-wide search for such genetic variations after stimulation of whole-blood from leprosy patients with M. leprae sonicate. This design allowed to provide a general framework for the genetic control of host responses to M. leprae and outlined the contribution of host genetics to leprosy pathogenesis. Among the M. leprae-dependent genetic regulators of gene expression levels there was an enrichment of variants (i) associated with leprosy, (ii) located in transcription factor binding sites and (iii) targeted by recent positive selection.

Leprosy Type-1 Reactions (T1Rs) are pathological inflammatory responses that afflict a sub-group of leprosy patients and result in peripheral nerve damage. Here, we employed a family-based GWAS in 221 families with 229 T1R-affect offspring with stepwise replication to identify risk factors for T1R. We discovered, replicated and validated T1R-specific associations with SNPs located in chromosome region 10p21.2. Combined analysis across the three independent samples resulted in strong evidence of association of rs1875147 with T1R (p = 4.5x10-8; OR = 1.54, 95% CI = 1.32–1.80). The T1R-risk locus was restricted to a lncRNA-encoding genomic interval with rs1875147 being an eQTL for the lncRNA. Since a genetic overlap between leprosy and inflammatory bowel disease (IBD) has been detected, we evaluated if the shared genetic control could be traced to the T1R endophenotype. Employing the results of a recent IBD GWAS meta-analysis we found that 10.6% of IBD SNPs available in our dataset shared a common risk-allele with T1R (p = 2.4x10-4). This finding points to a substantial overlap in the genetic control of clinically diverse inflammatory disorders., Author summary Leprosy still affects approximately 200,000 new victims each year. A major challenge of leprosy control is the prevention of permanent disability due to nerve damage. Nerve damage occurs if leprosy remains undiagnosed for extended periods or when patients undergo pathological inflammatory responses termed Type-1 Reactions (T1R). T1R is a rare example where beneficial inflammatory responses are temporal separated from host pathological responses. There is strong experimental evidence that supports a role of host genetic factors in T1R susceptibility. Here, we employed a genome-wide association study (GWAS) to investigate susceptibility factors for T1R in Vietnamese families. We followed up the initial GWAS findings in independent population samples from Vietnam and Brazil and identified a set of cis-eQTL genetic variants for the ENSG00000235140 lncRNA as global risk factors for T1R. To test our proposal that T1R is a strong model for pathological inflammatory responses we evaluated if inflammatory bowel disease (IBD) genetic risk-factors were enriched among T1R risk factors. We observed that more than 10% of IBD-risk loci were nominally associated with risk for T1R suggesting a shared mechanism of excessive inflammatory response in the both disease etiologies.

Background. Type 1 reactions (T1R) affect a considerable proportion of patients with leprosy. In those with T1R, the host immune response pathologically overcompensates for the actual infectious threat, resulting in nerve damage and permanent disability. Based on the results of a genome-wide association study of leprosy per se, we investigated the TNFSF15 chromosomal region for a possible contribution to susceptibility to T1R. Methods. We performed a high-resolution association scan of the TNFSF15 locus to evaluate the association with T1R in 2 geographically and ethnically distinct populations: a family-based sample from Vietnam and a case-control sample from Brazil, comprising a total of 1768 subjects. Results. In the Vietnamese sample, 47 single-nucleotide polymorphisms (SNPs) overlapping TNFSF15 and the adjacent TNFSF8 gene were associated with T1R but not with leprosy. Of the 47 SNPs, 39 were cis-expression quantitative trait loci (cis-eQTL) for TNFSF8 including SNPs located within the TNFSF15 gene. In the Brazilian sample, 18 of these cis-eQTL SNPs overlapping the TNFSF8 gene were validated for association with T1R. Conclusions. Taken together, these results indicate TNFSF8 and not TNFSF15 as an important T1R susceptibility gene. Our data support the need for infection genetics to go beyond genes for pathogen control to explore genes involved in a commensurate host response.

After sustained exposure to Mycobacterium leprae, only a subset of exposed individuals develops clinical leprosy. Moreover, leprosy patients show a wide spectrum of clinical manifestations that extend from the paucibacillary (PB) to the multibacillary (MB) form of the disease. This “polarization” of leprosy has long been a major focus of investigation for immunologists because of the different immune response in these two forms. But while leprosy per se has been shown to be under tight human genetic control, few epidemiological or genetic studies have focused on leprosy subtypes. Using PubMed, we collected available data in English on the epidemiology of leprosy polarization and the possible role of human genetics in its pathophysiology until September 2015. At the genetic level, we assembled a list of 28 genes from the literature that are associated with leprosy subtypes or implicated in the polarization process. Our bibliographical search revealed that improved study designs are needed to identify genes associated with leprosy polarization. Future investigations should not be restricted to a subanalysis of leprosy per se studies but should instead contrast MB to PB individuals. We show the latter approach to be the most powerful design for the identification of genetic polarization determinants. Finally, we bring to light the important resource represented by the nine-banded armadillo model, a unique animal model for leprosy.

Background Depending on the epidemiological setting, a variable proportion of leprosy patients will suffer from excessive pro-inflammatory responses, termed type-1 reactions (T1R). The LRRK2 gene encodes a multi-functional protein that has been shown to modulate pro-inflammatory responses. Variants near the LRRK2 gene have been associated with leprosy in some but not in other studies. We hypothesized that LRRK2 was a T1R susceptibility gene and that inconsistent association results might reflect different proportions of patients with T1R in the different sample settings. Hence, we evaluated the association of LRRK2 variants with T1R susceptibility. Methodology An association scan of the LRRK2 locus was performed using 156 single-nucleotide polymorphisms (SNPs). Evidence of association was evaluated in two family-based samples: A set of T1R-affected and a second set of T1R-free families. Only SNPs significant for T1R-affected families with significant evidence of heterogeneity relative to T1R-free families were considered T1R-specific. An expression quantitative trait locus (eQTL) analysis was applied to evaluate the impact of T1R-specific SNPs on LRRK2 gene transcriptional levels. Principal Findings A total of 18 T1R-specific variants organized in four bins were detected. The core SNP capturing the T1R association was the LRRK2 missense variant M2397T (rs3761863) that affects LRRK2 protein turnover. Additionally, a bin of nine SNPs associated with T1R were eQTLs for LRRK2 in unstimulated whole blood cells but not after exposure to Mycobacterium leprae antigen. Significance The results support a preferential association of LRRK2 variants with T1R. LRRK2 involvement in T1R is likely due to a pathological pro-inflammatory loop modulated by LRRK2 availability. Interestingly, the M2397T variant was reported in association with Crohn’s disease with the same risk allele as in T1R suggesting common inflammatory mechanism in these two distinct diseases., Author Summary A major challenge of current leprosy control is the management of host pathological immune responses coined Type-1 Reactions (T1R). T1R are characterized by acute inflammatory episodes whereby cellular immune responses are directed against host peripheral nerve cells. T1R affects up half of all leprosy patients and are a major cause of leprosy-associated disabilities. Since there is evidence that host genetic factors predispose leprosy patients to T1R, we have conducted a candidate gene study to test if LRRK2 gene variants are T1R risk factors. The choice of LRRK2 was motivated by the fact that LRRK2 was associated with leprosy per se in some but not in other studies. We reasoned that this may reflect different proportions of leprosy patients with T1R in the different samples and that LRRK2 may in truth be a T1R susceptibility gene. Here, we show that variants overlapping the LRRK2 gene, reported as suggestive leprosy per se susceptibility factors in a previous genome-wide association study, are preferentially associated with T1R. The main SNP carrying most of the association signal is the amino-acid change M2397T (rs3761863) which is known to impact LRRK2 turnover. Interestingly, eQTL SNPs counterbalanced the effect of the M2397T variant but this compensatory mechanism was abrogated by Mycobacterium leprae antigen stimulation.

The Mitsuda reaction, a delayed granulomatous skin reaction elicited by the intradermal injection of heat-killed Mycobacterium leprae, is an in vivo test reflecting the ability to generate an immune granuloma after sensitization by diverse mycobacterial infections. Accumulating evidence for the genetic control of the Mitsuda reaction has been reported. We performed a genomewide linkage scan for the quantitative Mitsuda reaction in 19 large families from Vietnam with a history of leprosy (114 offspring). Suggestive linkage was found at chromosomal regions 2q35 (P = 9 x 10(-4) at the SLC11A1 locus) and 17q21-25 (P = 8 x 10(-4)). Interestingly, these 2 regions have been previously linked to mycobacterial infection and other granulomatous diseases.

Background. Reversal, or type 1, leprosy reactions (T1Rs) are acute immune episodes that occur in skin and/ or nerves and are the leading cause of neurological impairment in patients with leprosy. T1Rs occur mainly in patients with borderline or multibacillary leprosy, but little is known about additional risk factors. Methods. We enrolled 337 Vietnamese patients with leprosy in our study, including 169 subjects who presented with T1Rs and 168 subjects with no history of T1Rs. A multivariate analysis was used to determine risk factors for T1R occurrence, time to T1R onset after leprosy diagnosis, and T1R sequelae after treatment. Results. Prevalence of T1Rs was estimated to be 29.1%. Multivariate analysis identified 3 clinical features of leprosy associated with T1R occurrence. Borderline leprosy subtype (odds ratio, 6.3 [95% confidence interval, 2.9-13.7] vs. polar subtypes) was the major risk factor; 2 other risk factors were positive bacillary index and presence of>5 skin lesions. In addition, age at leprosy diagnosis was a strong independent risk factor for T1Rs (odds ratio, 2.4 [95% confidence interval, 1.3-4.4] for patients aged ≥15 years old vs.

Leprosy is a debilitating infectious disease of human skin and nerves. Genetic factors of the host play an important role in the manifestation of disease susceptibility. The human NRAMP1 gene is a leprosy susceptibility candidate locus since its murine homologue Nramp1 (formerly Lsh/Ity/Bcg) controls innate resistance to Mycobacterium lepraemurium. In this study, 168 members of 20 multiplex leprosy families were genotyped for NRAMP1 alleles and 4 closely linked polymorphic markers. Highly informative haplotypes overlapping the NRAMP1 gene were constructed, and the haplotype segregation into leprosy-affected offspring was analyzed. It was observed that the segregation of NRAMP1 haplotypes into affected siblings was significantly nonrandom. This finding is consistent with the hypothesis that NRAMP1 itself is a leprosy susceptibility locus.

Leprosy is a persistent infectious disease caused by Mycobacterium leprae that still affects over 200,000 new patients annually. The host genetic background is an important risk factor for leprosy susceptibility and the PARK2 gene is a replicated leprosy susceptibility candidate gene. The protein product of PARK2, Parkin, is an E3 ubiquitin ligase that is involved in the development of various forms of Parkinsonism. The human macrophage is both a natural host cell of M. leprae as well as a primary mediator of natural immune defenses, in part by secreting important pro-inflammatory cytokines and chemokines. Here, we report that down-regulation of Parkin in THP-1 macrophages, human monocyte-derived macrophages and human Schwann cells resulted in a consistent and specific decrease in interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1/CCL2) production in response to mycobacteria or LPS. Interestingly, production of IL-6 at 6 hours by THP-1 cells stimulated with live M. leprae and M. bovis BCG was dependent on pretreatment with 1,25-dihydroxyvitamin D3 (VD). Parkin knockdown in VD-treated cells blocked IL-6 induction by mycobacteria. However, IκB-α phosphorylation and levels of IκB-ξ, a nuclear protein required for IL-6 expression, were not affected by Parkin silencing. Phosphorylation of MAPK ERK1/2 and p38 was unaffected by Parkin silencing while JNK activation was promoted but did not explain the altered cytokine production. In a final set of experiments we found that genetic risk factors of leprosy located in the PARK2 promoter region were significantly correlated with M. leprae sonicate triggered CCL2 and IL6 transcript levels in whole blood assays. These results associated genetically controlled changes in the production of MCP-1/CCL2 and IL-6 with known leprosy susceptibility factors., Author Summary Leprosy is an infectious disease with a strong host genetic component. The identification of host genetic lesions predisposing to disease is a powerful approach for mapping key junctions in the host pathogen interplay. Genetic variants located in the promoter region of the PARK2 gene are replicated leprosy susceptibility factors. To better understand a possible contribution of PARK2 to host effector mechanisms in leprosy patients, we developed a cellular model to test the contribution of the PARK2 encoded parkin protein to host responses to mycobacterial antigens. We observed that parkin was a mediator of IL-6 production in response to mycobacterial antigen in both THP-1 macrophages and human Schwann cells while human monocyte-derived macrophages needed to be pre-activated with VitD to show the same impact. Parkin also impacted on the constitutive production of MCP-1. The regulatory activity of parkin on cytokine production was found to be independent of the canonical TLR-NFκB signalling pathway. We also tested association of IL6 and CCL2 gene expression levels in whole blood assays with PARK2 polymorphisms. For both cytokines, we found significant associations with those PARK2 variants that were established leprosy susceptibility factors. Hence, our results show that genetic PARK2 variants that are correlated with leprosy susceptibility are also correlated with production of these cytokines following stimulation with M. leprae sonicate.

A genomewide association study in Chinese patients with leprosy detected association signals in 16 single-nucleotide polymorphisms (SNPs) belonging to 6 loci, of which 4 are related to the NOD2 signaling pathway and are Crohn’s disease susceptibility loci. Here, we studied these 16 SNPs as potential leprosy susceptibility factors in 474 Vietnamese leprosy simplex families. We replicated SNPs at HLA-DRDQ, RIPK2, CCDC122-LACC1, and NOD2 as leprosy susceptibility factors in Vietnam. These results validated the striking overlap in the genetic control of Crohn’s disease and leprosy. Leprosy is a chronic infectious disease of the skin and peripheral nerves and continues to contribute to the public health burden in several countries [1]. The causal agent, Mycobacterium leprae, shows high host specificity and displays low genomic diversity across strains. Only a small proportion of exposed individuals become infected and develop clinically overt disease [2]. The clinical presentation of leprosy can be broadly classified into 2 groups: paucibacillary leprosy (PB), characterized by a small number of anesthetized skin lesions with a lack of detectable M. leprae bacilli, and multibacillary leprosy (MB), characterized by numerous skin lesions with an abundance of M. leprae bacilli. Susceptibility to leprosy per se and the development of the clinical subtypes depend on human genetic factors [2]. Forward human genetics has led to the positional identification of leprosy risk variants in PARK2, LTA, and HLA-C [3–5]. A genome-wide association study (GWAS) conducted in Chinese patients with leprosy identified associations in 6 genes: CCDC122, LACC1(formerly C13orf31), NOD2, TNFSF15, HLADR ,a ndRIPK2 [6]. Three of these genes (TNFSF15, NOD2, RIPK2) are part of the NOD2 signaling pathway, and 4 have been associated with Crohn’s disease (TNFSF15, NOD2, HL ADR, LACC1 )[ 7]. In addition, a trend toward association was observed for the PARK2-interacting LRRK2 gene, which is associated with both Parkinson’s disease and Crohn’s disease [6]. To assess whether these findings may be extended beyond the Chinese population, we performed an association study among 474 Vietnamese simplex families (2 parents and 1 leprosy affected child) with the 16 leprosy-associated single-nucleotide polymorphisms (SNPs) identified in the Chinese GWAS.

Experimental evidence suggested the existence of unidentified leprosy susceptibility loci in the human leukocyte antigen (HLA) complex. To identify such genetic risk factors, a high-density association scan of a 1.9mega-base (Mb) region in the HLA complex was performed. Among 682 single-nucleotide polymorphisms (SNPs), 59 were associated with leprosy (P ,.01) in 198 Vietnamese single-case leprosy families. Genotyping of these SNPs in an independent sample of 292 Vietnamese single-case leprosy families replicated the association of 12 SNPs (P ,.01). Multivariate analysis of these 12 SNPs showed that the association information could be captured by 2 intergenic HLA class I region SNPs (P 5 9.4 3 10 29 )—rs2394885 and rs2922997 (marginal multivariate P 5 2.1 3 10 27 and P 5 .0016, respectively). SNP rs2394885 tagged the HLA-C*15:05 allele in the Vietnamese population. The identical associations were validated in a third sample of 364 patients with leprosy and 371 control subjects from North India. These results implicated class I alleles in leprosy pathogenesis.

Background. Leprosy is a chronic infectious disease caused by Mycobacterium leprae. The Mitsuda reaction is a delayed granulomatous skin reaction elicited by intradermal injection of heat-killed M. leprae. Interestingly, results of the Mitsuda test are positive in the majority of individuals, even in areas not endemic for M. leprae. Like leprosy, the Mitsuda reaction is thought to be genetically controlled, but its mode of inheritance is unknown, although the role of the NRAMP1 gene has previously been reported. Methods. We conducted a segregation analysis of quantitative Mitsuda reactivity in 168 Vietnamese nuclear families ascertained through patients with leprosy. Results. We found strong evidence ( ) for a major gene controlling the Mitsuda reaction independently 9 P ! 10 of leprosy clinical status. Subsequent linkage analysis showed that this major gene was distinct from NRAMP1. Under the major-gene model, ∼12% of individuals are homozygous for the recessive predisposing allele and are predicted to display high levels of Mitsuda reactivity (mean, ∼10 mm, versus 5 mm in other individuals). Conclusion. We provide evidence that the Mitsuda reaction is controlled by a major gene. Our study paves the way for the identification of this gene and should provide novel insight into the mechanisms involved in granuloma formation, especially in M. leprae infection. Leprosy is a chronic infectious disease, caused by Mycobacterium leprae, that mainly affects skin and peripheral nerves [1]. Despite a dramatic decrease in prevalence during the past 15 years, partly due to the efficacy of multidrug therapy [2], the worldwide leprosy incidence is still approaching 500,000 novel cases/year. Whereas most infected individuals do not develop clinical disease, even after sustained exposure to M. leprae, others present a broad spectrum of clinical symptoms. Leprosy ranges from the tuberculoid form, characterized by mature, well-delineated, and well-differentiated

The Mitsuda test, which measures the specific immune response against intradermally injected lepromin, has a high prognostic value for susceptibility or resistance to the lepromatous form of leprosy. A sib-pair linkage analysis between the Mitsuda response and the NRAMP1 gene was done among 20 nuclear families with leprosy (totaling 118 sibs) from Ho Chi Minh City, Vietnam. All family subjects were genotyped for several intragenic and flanking NRAMP1 markers, leading to the definition of a fully informative NRAMP1 haplotype. Significant linkage was observed between NRAMP1 and Mitsuda reaction when considered either as a quantitative (P

To determine the extent of HIV infection among female commercial sex workers (CSW), to identify risk factors, and to provide baseline data for developing and targeting prevention measures.A total of 968 female CSW were enrolled in a cross-sectional study from August 1995 to October 1996. Information was obtained from confidential face-to-face interview, physical examination, and laboratory testing.A total of 65.5% of female CSW reported inconsistent condom use. Overall seroprevalence was 5.2%. The highest seroprevalence (9.5%) was detected in An Giang province, a border area adjacent to Cambodia. Out of seven HIV isolates in An Giang province, six were characterized as Thai subtype E and one as subtype B. Multiple logistic regression analysis showed an independent significant association between HIV seroprevalence and the following: ageor = 30 years [odds ratio (OR), 5.1; 95% confidence interval (CI), 1.7-15.2]; high frequency of sex (20 times per week; OR, 13.5; 95% CI, 3.6-50.2); inconsistent condom use (OR, 2.8; 95% CI, 1.01-8.0; sign of genital ulcers (OR, 18.1; 95% CI, 1.8-182); venereal warts (OR, 9.0; 95% CI, 2.5-33.0); brothels as sex venue (OR, 7.0; 95% CI, 2.0-24.3); and working at the border area (OR, 5.1; 95% CI, 2.4-11.0). Brothels as work-sites were significantly related to inconsistent condom use and the socioeconomic background of clients. Only 0.5% of CSW reported injecting drug use.Female CSW at brothels who reported inconsistent condom use and ulcerous sexually transmitted disease, particularly in the border area with Cambodia, had greater risk of HIV infection. Brothels were more frequently used as sex venues in the border area and were more likely to be visited by occasional clients who were difficult to access. Drug use among female CSW in this region was rare. The development of prevention measures should be based on these results.

Leprosy reversal reactions type 1 (T1R) are acute immune episodes that affect a subset of leprosy patients and remain a major cause of nerve damage. Little is known about the relative importance of innate versus environmental factors in the pathogenesis of T1R. In a retrospective design, we evaluated innate differences in response to Mycobacterium leprae between healthy individuals and former leprosy patients affected or free of T1R by analyzing the transcriptome response of whole blood to M. leprae sonicate. Validation of results was conducted in a subsequent prospective study. We observed the differential expression of 581 genes upon exposure of whole blood to M. leprae sonicate in the retrospective study. We defined a 44 T1R gene set signature of differentially regulated genes. The majority of the T1R set genes were represented by three functional groups: i) pro-inflammatory regulators; ii) arachidonic acid metabolism mediators; and iii) regulators of anti-inflammation. The validity of the T1R gene set signature was replicated in the prospective arm of the study. The T1R genetic signature encompasses genes encoding pro- and anti-inflammatory mediators of innate immunity. This suggests an innate defect in the regulation of the inflammatory response to M. leprae antigens. The identified T1R gene set represents a critical first step towards a genetic profile of leprosy patients who are at increased risk of T1R and concomitant nerve damage., Author Summary Leprosy type 1 reversal reactions (T1R) are an important cause of nerve damage in leprosy patients and accurate prediction of patients at increased risk of T1R is a major challenge of current leprosy control. The incidence of T1R differs widely from 6% to 67% of leprosy patients in different leprosy endemic settings. Whether or not this reflects the impact of unknown environmental triggers or differences in the genetic background across ethnicities is not known. We performed a comparative transcriptome analysis between leprosy patients affected and free of T1R in response to M. leprae antigens. As the discovery sample we enrolled cured leprosy patients who had been diagnosed with T1R at the time of leprosy diagnosis and leprosy patients who had never undergone T1R (retrospective arm). Whole genome transcriptome analysis after stimulation of blood with M. leprae antigen resulted in the definition of a T1R signature gene set. We validated the T1R gene set in RNA samples obtained from T1R-free patients at the time of leprosy diagnosis and followed for 3 years for development of T1R (prospective arm). These results confirm the role of innate factors in T1R and are a first step towards a predictive genetic T1R signature.