Genomic characterization of Mycobacterium lepromatosis from ENL patients from India.

BACKGROUND: Leprosy is caused by Mycobacterium leprae and Mycobacterium lepromatosis. Both organisms cannot be cultured in vitro. M. lepromatosis was found to be associated mainly with diffuse lepromatous leprosy and with Lucio's phenomena initially. Later, M. lepromatosis was observed in borderline leprosy cases (BL), lepromatous leprosy cases (LL) and leprosy reactional cases (T1R and ENL). Although many cases are being reported with similar clinical features like Lucio phenomenon in India but M. lepromatosis was not isolated from these cases. The aim of this study was to screen MB patients and patients with type 2 reaction for the presence of M. lepromatosis. METHODOLOGY: We recruited a total of 75 multibacillary leprosy cases (45 MB cases without reaction and 30 type 2 reaction (ENL) cases) from TLM hospitals Purulia (West Bengal), Barabanki (Uttar Pradesh), Shahdara (Delhi) and PGIMER (Chandigarh), India. Punch biopsies of 5 mm were collected in 70% ethanol from all the study subjects. DNA was extracted followed by Hemi-nested PCR targeting 16S rRNA gene specific for M. lepromatosis. Further, PCR products were processed for Sanger sequencing for an absolute confirmation of M. lepromatosis. Whole genome sequencing was done to confirm the presence of M. lepromatosis. RESULT: We observed presence of M. lepromatosis in 4 necrotic ENL patients by heminested PCR. There was 100% 16S rRNA sequence similarity with M. lepromatosis FJ924 in one case, 98.96% in two cases and in one case it was 90.9% similarity by nucleotide BLAST (BLASTn) by using the NCBI website. On the basis of Sanger sequencing, we noted presence of M. lepromatosis in 3 necrotic ENL patients as one sample only gave 90.9% similarity by BLASTn. On the basis of de novo assembly and genome obtained, only one sample S4 with a 2.9 mb genome size was qualified for downstream analysis. Sixteen M. lepromatosis- specific proteins were identified in this case and the closest species was M. lepromatosis strain FJ924 based on whole genome level phylogeny. CONCLUSION: These results provide valuable insights into the prevalence of M. lepromatosis in ENL patients in different regions of India and contribute to our understanding of the genetic characteristics of this pathogen in the context of leprosy.

Survival of Mycobacterium leprae and association with Acanthamoeba from environmental samples in the inhabitant areas of active leprosy cases: A cross sectional study from endemic pockets of Purulia, West Bengal.

BACKGROUND: Mycobacterium leprae being an obligate intracellular parasite cannot be cultured in any artificial culture media but it has been shown to reside in wild armadillos in North America. Many studies suggested that M. leprae could be found in the environment and may have a role in continuing transmission of the disease. The exact role of the environment in the transmission dynamics is still speculative. The present study was undertaken to find out the presence of viable M. leprae around patients' environment like soil and water and association of free living pathogenic protozoa, Acanthamoeba which might play an important role in transmission of the disease. METHODS: Seven hundred soil and 400 water samples were collected from the surroundings of the houses of leprosy patients from endemic villages. Two hundred soil and 80 water samples were also collected from the surroundings of normal inhabitants from non-endemic villages as controls. These samples were screened for the presence of M. leprae and Acanthamoeba using DNA PCR. RNA was extracted from the PCR positive samples and Reverse Transcriptase - PCR targeting 16S rRNA gene region was performed for detection of viable M. leprae. RESULTS: We observed high PCR positivity in soil samples (218 out of 700; 31%) and water samples (73 out of 400; 18%). These samples when further screened for viability, it was observed that 106 soil samples (15% of total) and 34 water samples (8% of total) showed presence of 16S rRNA. We observed 18.3% of soil and 20.5% of water samples were PCR positive for Acanthamoeba. Soil samples from the control area, where no active leprosy case resided in the last 5 years, showed PCR positivity in 4 samples (2%) for M. leprae DNA in only soil samples with all water samples being negative. RT-PCR for all PCR positive soil samples was negative. Of the 106 soil samples positive for M. leprae RT-PCR, 30 samples were also positive for Acanthamoeba whereas out of 112 M. leprae RT-PCR negative but PCR positive samples only 10 samples were Acanthamoeba positive showing association of viability with presence of Acanthamoeba (p = .0021). Similarly, for water samples also, association of M. leprae viability with presence of Acanthamoeba was seen (p = .0009). CONCLUSION: This study suggests that the surrounding environment (soil and water) of leprosy patients contain viable M. leprae and the viability has association with Acanthamoeba which may provide a protective niche for M. leprae. This could play an important role in the focal transmission of the disease.

Presence of viable Mycobacterium leprae in environmental specimens around houses of leprosy patients.

PURPOSE: Leprosy is a chronic systemic infectious disease caused by Mycobacterium leprae, one of the first organisms to be established as the cause for disease in humans. Because of high prevalence pockets of leprosy in the endemic regions, it is necessary to identify the possible sources of M. leprae in the environment and its mode of transmission. MATERIALS AND METHODS: Slit skin smears (SSSs) from lesions were collected in 70% ethanol from 50 leprosy cases staying in the leprosy resettlement village and hospital from a high endemic area. One hundred and sixty soil samples were collected from different areas around the leprosy hospital and from the resettlement village of cured leprosy patients where active cases also resided at the time of sample collection. M. leprae specific gene region (RLEP 129 bp) and 16S rRNA targets were used for polymerase chain reaction (PCR) based detection for the presence and viability of M. leprae. An rpoT region was also amplified to determine presence of numbers of 6 bp tandem repeats. RESULTS: All the SSS samples collected from patients showed three copies of rpoT region (6 bp tandem repeat, an ancient Indian type). Fifty-two soil samples showed presence of M. leprae DNA whereas M. leprae specific 16S rRNA gene was amplified in sixteen of these samples. PCR amplification and fragment length analysis showed 91 bp, i.e., three copies of the rpoT 6 bp tandem repeats from soil samples and similar three copies observed in patient samples. CONCLUSION: Presence of viable M. leprae in the soil having same rpoT genotype of M. leprae noted in patients suggests that it could be the same strain of M. leprae. M. leprae found in the soil could be the one that is excreted out by the patient. Significance of its viability in the environment and its pathogenicity with respect to transmission needs to be further explored. Findings of this study might provide possible insights for further exploration into understanding transmission patterns in leprosy and also will throw light on identifying potential for existence of extra human source or reservoirs of M. leprae, if any.

Genotyping of Mycobacterium leprae strains from a region of high endemic leprosy prevalence in India.

Leprosy is still a major health problem in India which has the highest number of cases. Multiple locus variable number of tandem repeat analysis (MLVA) and single nucleotide polymorphism (SNP) have been proposed as tools of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. Seventy slit skin scrapings were collected from Purulia (West Bengal), Miraj (Maharashtra), Shahdara (Delhi), and Naini (UP) hospitals of The Leprosy Mission (TLM). SNP subtyping and MLVA on 10 VNTR loci were applied for the strain typing of Mycobacterium leprae. Along with the strain typing conventional epidemiological investigation was also performed to trace the transmission chain. In addition, phylogenetic analysis was done on variable number of tandem repeat (VNTR) data sets using sequence type analysis and recombinational tests (START) software. START software performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. Diversity was observed in the cross-sectional survey of isolates obtained from 70 patients. Similarity in fingerprinting profiles observed in specimens of cases from the same family or neighborhood locations indicated a possible common source of infection. The data suggest that these VNTRs including subtyping of SNPs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci. The present study strongly indicates that multi-case families might constitute epidemic foci and the main source of M. leprae in villages, causing the predominant strain or cluster infection leading to the spread of leprosy in the community.

Single nucleotide polymorphism-based molecular typing of M. leprae from multicase families of leprosy patients and their surroundings to understand the transmission of leprosy.

The exact mode of transmission of leprosy is not clearly understood; however, many studies have demonstrated active transmission of leprosy around a source case. Families of five active leprosy cases and their household contacts were chosen from a high endemic area in Purulia. Fifty-two soil samples were also collected from different areas of their houses. DNA was extracted from slit-skin smears (SSS) and soil samples and the Mycobacterium leprae-specific RLEP (129 bp) region was amplified using PCR. Molecular typing of M. leprae was performed for all RLEP PCR-positive samples by single nucleotide polymorphism (SNP) typing and confirmation by DNA sequencing. SSS of these five patients and six out of the total 28 contacts were PCR positive for RLEP whereas 17 soil samples out of 52 showed the presence of M. leprae DNA. SNP typing of M. leprae from all RLEP PCR-positive subjects (patients and smear-positive contacts) and 10 soil samples showed the SNP type 1 genotype. M. leprae DNA from the five leprosy patients and the six contacts was further subtyped and the D subtype was noted in all patients and contacts, except for one contact where the C subtype was identified. Typing followed by subtyping of M. leprae clearly revealed that either the contacts were infected by the patients or both patients and contacts had the same source of infection. It also revealed that the type of M. leprae in the soil in the inhabited areas where patients resided was also of the same type as that found in patients.

Interleukin-17F single-nucleotide polymorphism (7488T>C) and its association with susceptibility to leprosy.

The objective of this study was to investigate the association, if any, between the interleukin-17F (7488T>C) (rs763780) polymorphism and susceptibility to leprosy and to elucidate the relationship between IL-17F genotypes and clinical profile of the disease. DNA was extracted from the peripheral venous blood of leprosy cases (n = 140), which were classified as per WHO classification into paucibacillary (PB) (n = 53) and multibacillary (MB) (n = 87) categories and healthy controls (n = 84) without any signs and symptoms of leprosy. The IL-17F (7488 T/C) polymorphism was genotyped using amplification refractory mutation system - polymerase chain reaction (Allele-specific amplification). In both PB and MB categories of leprosy cases, the homozygous TT genotype frequency was significantly higher than that of the healthy controls (78.70% vs. 29.76%, P < 0.05). The heterozygous TC genotype was higher in the controls than in the leprosy cases (57.14% vs. 17.68%, P < 0.05). TT genotype was more associated with the type 1 reactional states and tuberculoid/borderline tuberculoid groups in leprosy than the TC genotype. This study reveals that the IL-17F (7488T>C) single-nucleotide polymorphism is associated with susceptibility to leprosy and polymorphism confers decrease in risk of contracting leprosy in the north Indian cohort.

Cortisol and proinflammatory cytokine profiles in type 1 (reversal) reactions of leprosy.

PURPOSE: Cortisol levels in the circulation and at the sites of peripheral inflammation regulate type 1 (Reversal) reactions in leprosy akin to delayed type hypersensitivity reactions (DTH). In this study we determine the extent to which the differential mRNA expression of genes encoding cortisone-cortisol shuttle enzymes (11 ß hydroxysteriod dehydrogenase I & II (11 ß HSD I & II)), circulatory levels of proinflammatory cytokines (IL-6, IL-7, IP-10, IL-17F, IL-23, TNF-α, IL-1ß, PDGF BB and CRP) and cortisol are associated with development of type 1 reactions in leprosy. METHODS: Urine, blood and incisional skin biopsy samples from site of lesions were collected from 49 newly diagnosed untreated leprosy cases in T1R and 51 cases not in reaction (NR). mRNA expression levels of genes encoding 11 ß HSD I & II in skin biopsy samples were determined by realtime PCR. Cortisol levels from the lesional skin biopsies, serum and urine samples and serum proinflammatory cytokine levels were measured using ELISA. RESULTS: The mean expression ratios of 11 ß HSD I & II are significantly lower in leprosy cases with T1R when compared to the NR leprosy cases. Cortisol levels in lesional skin biopsies and in urine are significantly lower (p=0.001) in leprosy cases with T1R. Serum cytokine levels of IP-10, IL-17F, IL-IL-6 and TNF-α are significantly higher (p<0.05) in leprosy cases with T1R when compared the NR leprosy cases. CONCLUSION: Our study indicated an association of urinary and lesional skin cortisol levels with the manifestation of T1R in leprosy. IP-10, IL-17F, IL-6 and TNF-α can be potential prognostic serological markers and gene expression markers for early detection of type 1 reactions in leprosy.

Cohort study of the seasonal effect on nasal carriage and the presence of Mycobacterium leprae in an endemic area in the general population.

Leprosy continues to be a significant health problem in certain pockets in developing countries. Better understanding of the transmission and source of the infection would help to decipher the transmission link, leading to control of the spread of the disease. The nose is considered to be a portal of entry, suggesting an aerial route for transmission through droplet infection. The evidence suggests that many individuals from endemic countries carry Mycobacterium leprae in their nasal cavities without having obvious symptoms of leprosy. The objective of the present study was to assess the presence of M. leprae on the nasal mucosa in the general population from a leprosy-endemic pocket. M. leprae detection was carried out using PCR targeting RLEP. Four hundred subjects from an area highly endemic for leprosy were included in the study and followed up during three different seasons--winter, summer, and monsoon--for evidence of nasal exposure to M. leprae. PCR positivity for M. leprae was observed in 29%, 21% and 31% of the samples collected in winter, summer and the monsoon season, respectively. Twenty-six individuals from the cohort showed amplification for M. leprae for all seasons. Our results are consistent with reports in the literature showing widespread exposure to M. leprae in the endemic community. The results also suggest possible association of the environmental conditions (climate) with the transmission pattern and levels of exposure to M. leprae. However, the present study indicated that the population from highly endemic pockets will have exposure to M. leprae irrespective of season.

Single nucleotide polymorphisms typing of Mycobacterium leprae reveals focal transmission of leprosy in high endemic regions of India.

Earlier studies indicate that genotyping of Mycobaterium leprae based on single-nucleotide polymorphisms (SNPs) is useful for analysis of the global spread of leprosy. In the present study, we investigated the diversity of M. leprae at eight SNP loci using 180 clinical isolates obtained from patients with leprosy residing mainly in Delhi and Purulia (West Bengal) regions. It was observed that the frequency of SNP type 1 and subtype D was most predominant in the Indian population. Further, the SNP type 2 subtype E was noted only from East Delhi region and SNP type 2 subtype G was noted only from the nearby areas of Hoogly district of West Bengal. These results indicate the occurrence of focal transmission of M. leprae infection and demonstrate that analysis by SNP typing has great potential to help researchers in understanding the transmission of M. leprae infection in the community.

Increased serum circulatory levels of interleukin 17F in type 1 reactions of leprosy.

PURPOSE: Leprosy is a chronic infectious disease caused by Mycobacterium leprae affecting mainly skin and peripheral nerves. Acute inflammatory episodes in the borderline immunological spectrum of the disease cause severe nerve and tissue damage leading to deformities. Finding of any serological marker for leprosy reactions will help in prediction of reactions and in early treatment intervention. The objective of this study was to measure the serum circulatory levels of Interleukin 17F (IL 17F) and to correlate the levels with type 1 and type 2 reactional states and with clinico-histopathological spectrum of leprosy. We studied IL 17F to delineate its role and its clinical implications in leprosy reactions. METHODS: Patients were classified based on the Ridley DS and Jopling WH Classification and blood samples (5 ml each) were collected from 80 active untreated leprosy cases in Type 1 reaction (T1R), 21 cases in Type 2 (Erythema Nodosum Leprosum ENL) reaction (T2R), 80 cases without reaction (NR), and 94 non-leprosy cases (NL). Serum was separated and measured for IL 17F levels using ELISA (Commercial Kits, R&D Systems Inc., USA). RESULTS: IL 17F levels were significantly higher in the T1R group when compared to the NR group (p < 0.001). The borderline lepromatous group showed the highest levels of IL 17F among the other groups in the disease spectrum. Bacteriological index (BI) showed negative correlation with the IL 17F levels. CONCLUSION: The results specify that serum circulatory levels of IL 17F are elevated during T1Rs in the borderline spectrum of the disease and thus may play a role in the regulation of inflammatory responses associated with reactions in leprosy.

Serum samples from patients with mycobacterial infections cross-react with HIV structural proteins Gp41, p55 and p18.

BACKGROUND: Infection with Mycobacterium leprae is associated with a high frequency of false positive results in a variety of serological assays. Our studies have found cross-reactivity to HIV structural proteins in serum samples from leprosy patients, irrespective of the type of disease, treatment duration, age and gender and from a few patients with active TB disease. METHODS: Western blot (WB) analysis revealed that sera from HIV negative leprosy patients across the spectrum showed high reactivity with p18, Gp41 and p55 and lower reactivity with other HIV proteins. The reactivity appeared to be specific; western blot-positive samples were negative in ELISA and in several rapid tests for HIV. Cross-reactivity was not found in sera from patients with leishmaniasis or from normal healthy individuals. RESULTS: None of the WB reactive leprosy patients seroconverted to HIV positivity within 6 months to 1 year after Western blot testing. BLAST analysis revealed that envelope antigens of HIV (Gp41, Gp120 and Gp160) contained amino acid sequences similar to M. leprae ML0470, putative integral membrane protein, Rv0740, mmpL9 (M. tuberculosis). Core (gag) antigens (p18) had similarities to ML0406, but polymerase antigens (p52) had similarities to PE_PGRS (M. tuberculosis, H37Rv). Nucleotide sequence analysis, on the other hand, did not reveal any significant homology between M. leprae or M. tuberculosis and HIV. CONCLUSIONS: The occurrence of these high false-positive rates in M. leprae-infected individuals suggests a possible complication of serodiagnosis of HIV in regions where mycobacterial infections are endemic. There is a need for caution in reporting HIV infection among leprosy patients. Our observations emphasise the value of the various rapid assay kits for HIV, where this false positivity is not observed.

Increased level of urinary nitric oxide metabolites in leprosy patients during type 2 reactions and decreased after antireactional therapy.

OBJECTIVES: To assess the urinary nitric oxide metabolites in lepromatous patients in ENL (type 2 reactions) and to compare these metabolites after subsidence of reactions following antireactional therapy. Further to compare the levels in a group of lepromatous leprosy patients without reactions. DESIGN: The initial urine samples were collected from lepromatous leprosy patients when they came with ENL before commencing antireactional therapy and repeat samples were taken after resolution of ENL. Morning urine samples were collected from LL patients without reactions. Nitrites and nitrates in urine were measured using commercially available kit. Mean levels of nitric oxide metabolites of LL patients with ENL and without ENL were compared by student's 't' test. The level during ENL and after resolution was compared by paired 't' test. RESULTS: The nitric oxide metabolites were analyzed in 14 LL patients with ENL and after resolution of ENL and in 5 LL patients without reaction. The level of urinary nitric oxide metabolite is higher in LL patients in ENL reaction compared to LL patients without reaction (P < 0.04). These levels were reduced significantly with resolution of reaction following antireactional therapy (P < 0.004). CONCLUSION: The findings of this study suggested that the NO/NOM excretion is increased in leprosy patients during ENL episodes. With antireactional therapy (steroids) and clinical improvement the levels are reduced.

Evaluación de Técnicas Serodiagnósticas para la Lepra. Estudio en una población endémica del sur de la India / tab

Para evaluar la utilidad de los antígenos disacárido natural (PGL1) y 35 kDa en determinaciones serológicas de lepra sobre todo para la detección de grupos de alto riesgo para contraer la enfermedad, se llevó a cabo este estudio en una población endémica del sur de la India. De 3.346 casos y sus convivientes y vecinos, se obtuvieron muestras de suero de 2.994 y 2.875 individuos y se cribaron para detectar antígenos frente al PGL1 y 35 kDa respectivamente. Mientras que la positividad total para contactos y casos de lepra era del 3,3% para anticuerpos PGL1, la positividad para el anticuerpo 35 kDa era del 6,3%. La seropositividad para la población contacto era del 2,7% y 5,4% para anticuerpos PGL1 Y 35 kDa, respectivamente. Los pacientes lepromatosos y bordeline lepromatosos presentaron positividades del 35,1% para anticuerpos PGL1 y 45,7% para el 35 kDa. El seguimiento de los contactos reveló que la mayoría de los analizados permanece (>90%9 seronegativos para ambos anticuerpos y la mayoría de nuevos casos se presentaron precisamente en el grupo seronegativo. El ensayo demuestra claramente que las técnicas serológicas específicas no son lo suficientemente sensibles para su aplicación, tanto para el diagnóstico como para identificar individuos en riesgo de contraer lepra en esta población endémica del sur de la India (AU)

In order to evaluate the usefulness of natural disaccharide (PGL1) and 35 kDa antigens based serology in diagnosis of leprosy and in detecting high risk groups for leprosy, this study was conducted in an endemic population in South India. 90%) remained seronegative for both the antibodies and most of the new cases emerged form the seronegative group. The study clearly indicates that specific serological assays are not sensitive enough for application, both for diagnosis and for identifying any individuals at risk for leprosy in the south Indian endemic population (AU)

Seroprevalence of HIV infection among leprosy patients in Agra, India: trends and perspective.

This study compares the results of HIV seroprevalence, which was carried out in two phases, i.e., 1989 to 1993 and 1999 to 2004. Although the number of leprosy patients screened for HIV infection in the second phase is less (2125) as compared to those screened during the first phase (4025), a rise in HIV infection from 0.12% to 0.37% is certainly disturbing since this area appears to be endemic for both the infections. During the study period, the Out Patient department attendance of a few types of leprosy patients like borderline and borderline lepromatous have risen, whereas others like borderline tuberculoid and polar tuberculoid have declined in the second phase as compared to that of the first phase. The trend over a decade suggests that HIV infection is low among the leprosy patients when compared with other risk groups. Follow-up of these patients at an interval of six months, revealed that none of them downgraded into a severe form of leprosy nor developed ARC or AIDS. In this study, it appears that neither infection precipitated the other. The occurrence of downgradation as well as reversal reactions and neuritis (both chronic and acute) was not observed among the leprosy patients. None of them developed erythema nodosum leprosum reactions. Similarly, the HIV-positive leprosy cases did not develop either AIDS related complex (ARC) or full blown case of AIDS.

Utility of serodiagnostic tests for leprosy: a study in an endemic population in South India.

In order to evaluate the usefulness of natural disaccharide (PGL1) and 35 kDa antigens based serology in diagnosis of leprosy and in detecting high risk groups for leprosy, this study was conducted in an endemic population in South India. Out of 3346 cases and their households and neighbouring household contacts, serum samples from 2994 and 2875 individuals were screened for antibodies against PGL1 and 35kDa antigens respectively. While the overall positivity for contacts and leprosy cases was 3.3% for PGL1 antibody, the positivity for 35 kDa antibody was 6.3%. The positivity for contact population was 2.7% and 5.4% for PGL1 and 35 kDa antibodies, respectively. Lepromatous and borderline lepromatous patients showed positivity of 35.1% for PGL1 antibody and 45.7% for 35 kDa antibody. Follow-up of contacts showed that the majority (>90%) remained seronegative for both the antibodies and most of the new cases emerged from the seronegative group. The study clearly indicates that specific serological assays are not sensitive enough for application, both for diagnosis and for identifying any individual at risk for leprosy in the south Indian endemic population.

Ultrastructural study of Schwann cells and endothelial cells in the pathogenesis of leprous neuropathy.

Peripheral nerve biopsies from 4 borderline tuberculoid (BT) and 4 lepromatous (LL) patients who were on multidrug therapy were investigated by light and electron microscopic studies. The variation of diameters and distribution of myelinated and unmyelinated fibers between BT and LL patients were not significant. This study has shown significant changes in peripheral nerves and endoneural blood vessels. It was revealed that besides Schwann cells (SC), the endothelial cells (EC) of endoneural blood vessels frequently harbor M. leprae. In BT, peripheral nerves in addition to the degenerative changes of SC and presence of perineural and perivascular cuffing by mononuclear cells, the endoneurial blood vessels showed thickening of basement membrane with hypertrophy of EC leading to narrowing or complete occlusion of lumen. On the other hand, peripheral nerves of LL patients were infiltrated with large number of M. leprae shown to be present in the electron transparent zone (ETZ) of the SC. The EC of endoneurial blood vessels were found to be loaded with M. leprae, and this bacillary loaded EC was found to release M. leprae into the lumen through its ruptured membrane.

A major susceptibility locus for leprosy in India maps to chromosome 10p13.

Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is prevalent in India, where about half of the world's estimated 800,000 cases occur. A role for the genetics of the host in variable susceptibility to leprosy has been indicated by familial clustering, twin studies, complex segregation analyses and human leukocyte antigen (HLA) association studies. We report here a genetic linkage scan of the genomes of 224 families from South India, containing 245 independent affected sibpairs with leprosy, mainly of the paucibacillary type. In a two-stage genome screen using 396 microsatellite markers, we found significant linkage (maximum lod score (MLS) = 4.09, P < 2x10-5) on chromosome 10p13 for a series of neighboring microsatellite markers, providing evidence for a major locus for this prevalent infectious disease. Thus, despite the polygenic nature of infectious disease susceptibility, some major, non-HLA-linked loci exist that may be mapped through obtainable numbers of affected sibling pairs.