Varalakshmi Vissa

Drug resistance surveillance and strain typing ofMycobacterium lepraeare necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR–high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) offolP1,rpoB, andgyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included threefolP1, tworpoB, and onegyrAmutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all thefolP1mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations.

Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.

Drug resistance surveillance identified six untreated leprosy patients in the Philippines withMycobacterium lepraefolP1mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked byM. lepraevariable-number tandem repeat (VNTR) strain types. In India,folP1mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target generpoB. These findings indicate that dapsone resistance is being transmitted.

Leprosy is a disease of the skin and peripheral nervous system caused by the obligate intracellular bacterium Mycobacterium leprae. The clinical presentations of leprosy are spectral, with the severity of disease determined by the balance between the cellular and humoral immune response of the host. The exact mechanisms that facilitate disease susceptibility, onset and progression to certain clinical phenotypes are presently unclear. Various studies have examined lipid metabolism in leprosy, but there has been limited work using whole metabolite profiles to distinguish the clinical forms of leprosy. In this study we adopted a metabolomics approach using high mass accuracy ultrahigh pressure liquid chromatography mass spectrometry (UPLC-MS) to investigate the circulatory biomarkers in newly diagnosed untreated leprosy patients. Sera from patients having bacterial indices (BI) below 1 or above 4 were selected, subjected to UPLC-MS, and then analyzed for biomarkers which distinguish the polar presentations of leprosy. We found significant increases in the abundance of certain polyunsaturated fatty acids (PUFAs) and phospholipids in the high-BI patients, when contrasted with the levels in the low-BI patients. In particular, the median values of arachidonic acid (2-fold increase), eicosapentaenoic acid (2.6-fold increase) and docosahexaenoic acid (1.6-fold increase) were found to be greater in the high-BI patients. Eicosapentaenoic acid and docosahexaenoic acid are known to exert anti-inflammatory properties, while arachidonic acid has been reported to have both pro- and anti-inflammatory activities. The observed increase in the levels of several lipids in high-BI patients may provide novel clues regarding the biological pathways involved in the immunomodulation of leprosy. Furthermore, these results may lead to the discovery of biomarkers that can be used to investigate susceptibility to infection, facilitate early diagnosis and monitor the progression of disease. Leprosy is an infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. M. leprae infects the skin and nerves, leading to disfigurement and nerve damage, with the severity of the disease varying widely. We believe there are multiple factors (genetic, bacterial, nutritional and environmental), which may explain the differences in clinical manifestations of the disease. We studied the metabolites in the serum of infected patients to search for specific molecules that may contribute to variations in the severity of disease seen in leprosy. We found that there were variations in levels of certain lipids in the patients with different bacterial loads. In particular, we found that three polyunsaturated fatty acids (PUFAs) involved in the inhibition of inflammation were more abundant in the serum of patients with higher bacterial loads. However, we do not know whether these PUFAs originated from the host or the bacteria. The variations in the metabolite profile that we observed provide a foundation for future research into the explanations of how leprosy causes disease.

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO¹, leprosy remains endemic in many countries with approximately 250,000 new cases each year.² The entire M. leprae genome has been mapped^3,4 and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites).⁵ Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci.^5,6,7 Variable number tandem repeat (VNTR)⁵ analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing^7,8,9, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA)¹⁰ has been used to study leprosy evolution and transmission in several countries including China^11,12, Malawi⁸, the Philippines^10,13, and Brazil¹⁴. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS).¹⁰ The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions.¹⁰ The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.

To explore the factors influencing the steady transmission of leprosy as indicated by new case detection rate in Qiubei county, Yunnan province, China despite the implementation of MDT for the last 25 years. Information related to case-finding was collected. ELISA and PCR were applied to detect anti-PGL-1 antibody in sera and Mycobacterium leprae in nasal secretions respectively, in leprosy patients, their household contacts and the general population. M. leprae by PCR was also detected from water in the highly endemic villages. VNTR typing was performed to explore the mode and chain of transmission of M. leprae. Prior to 2001, the proportion of new cases detected from the examination of household contacts of leprosy patients was low (number, compared to), while the proportion of patients whose identification was delayed by more than 2 years, was high (number, compared to). Qualities of these two indicators has been improved, along with the improvement of leprosy control program since 2001, but the detection rates has been steady at 4-5/100 000 during 1986 - 2010. The PGL-1 seropositivity rate was 20% - 30% in general population, with the peak rate (30%) detected in the teenage population in the endemic villages. In addition to the fact that M. leprae was detected in nasal secretion from patients, their contacts and from water, the M. leprae VNTR genotypes were found to be highly similar between skin biopsy and nasal secretion in untreated cases. Families with multi-cases were clustered and located in the Northern part of the County, and the genotypes of M. leprae were identical within those families. The percentage of clusters was considerably higher in Northern rather than Southern parts of the County. Results from this molecular study demonstrated evidence that transmission of leprosy within the families and in the endemic-villages was severe. M. leprae were detected in waters from the endemic villages and others areas which might have a relation to the continued transmission of leprosy.

Objective To evaluate the reliability and feasibility of two methods of multilocus variable number of tandem repeat analysis (MLVA) for strain typing of M. leprae, and to study whether short tandem repeat loci are stable and suitable for epidemiological study of leprosy. Methods Total DNA was extracted from skin biopsies of 20 new multibacillary (MB) patients from China diagnosed in 2006. To determine the copy numbers of short tandem repeats (STRs) for 13 loci, we amplified each locus individually by PCR, followed by sequence analysis of the amplicons. Separately, the same loci, plus four others were amplified by Multiplex PCRs (MP) using fluorescent primers and the copy number was identified by fragment length analysis (MP-FLA). MLVA was also performed at different times during treatment for a subset of the patients. Results and conclusions Genetic variability of M. leprae in China can be assessed in microsatellite loci. (GTA)9 and (TTC)21 loci are hypervariable, with array sizes of 25 repeat units or more. The expansion of the (GTA)9 locus is a characteristic of some M. leprae isolates in China. A high level of allele concordance was observed between PCR-sequencing and MP-FLA methods. However, MP-FLA method was cost- effective, rapid, high throughput and suitable for strain typing. Five of the 20 isolates of M. leprae were from patients residing in the same township in Qiubei County, Yunnan, and matched closely by MLVA. Three of these patients are family contacts of previously diagnosed patients, with intra-familial strain types being similar, suggesting infections from common sources and transmission chain(s). The VNTR patterns were highly similar in biopsy and slit skin smears (SSS) before treatment, and in the SSS collected at various time points during treatment. Taken together, VNTR strain typing is a useful tool for study of short range transmission in leprosy.

To study the stability and allelic diversity of tandem repeat loci in M. leprae in leprosy patients of Cebu, Philippines, and the suitability of multilocus variable number of tandem repeat (VNTR) analysis (MLVA) typing for detecting transmission. Seventy newly diagnosed leprosy patients consulting at the Leonard Wood Memorial, Cebu Skin Clinic Total DNA was extracted from slit skin smear (SSS) scrapings of each patient and used for amplification of 13 M. leprae VNTR loci by single locus or multiplex PCR. Number of repeats for each VNTR locus was obtained by DNA sequencing or fragment length analysis methods. Medical, social and geographic details were included in the molecular epidemiology database. Multiplex PCR (MP) and fragment length analysis (FLA) methods were found to be more efficient and accurate compared to single short tandem repeat (STR) amplification and DNA sequencing. Intra-patient MLVA patterns from four different samples were conserved in the minisatellites, while differences in one or more of the polymorphic and stutter prone microsatellites was observed, in four of five patients. The 13 loci could differentiate M. leprae strains in Cebu, however, MLVA patterns were stable enough during incubation and transmission between individuals within multi-case families. Thus M. leprae MLVA has potential for strain typing and transmission studies in Cebu.

Recently about 500 new cases of leprosy have been reported each year in Thailand. In addition to a steady rate of new case detection, Thailand is in Southeast Asia where leprosy is endemic in neighbouring countries; therefore, strain differentiation could be useful in tracing origins and routes of infection, and general leprosy surveillance. To identify suitable markers for differentiation of M. leprae strains in different global geographic regions and to determine the applicability of a systematic genotyping method for tracing leprosy transmission, variable nucleotide tandem repeats (VNTRs) of 14 loci were evaluated using DNA extracts from a total of 97 skin biopsies and slit skin smear samples. The alleles per locus ranged from 2–26 providing adequate strain differentiation. Microsatellite loci (GAA)21, (AT)17 are highly polymorphic followed by (GTA)9, (AC)8a, (AC)8b, and (AC)9. The minisatellites 6-7, 21-3 and 27-5 exhibited a limited number of alleles. The repeat of 23-3 showed no polymorphism. Overall, the strain types can be divided into two distinct Thai groups, according to the alleles at the (GGT)5 and 21-3 loci. However, there are no obvious geographical patterns of distribution of VNTR strain types. Closely matched VNTR profiles found in household members of two multi-case families suggested infection through a common source.