Leprosy Drug Resistance Surveillance in Colombia: The Experience of a Sentinel Country.

An active search for Mycobacterium leprae drug resistance was carried out, 243 multibacillary patients from endemic regions of Colombia were included from 2004 to 2013 in a surveillance program. This program was a World Health Organization initiative for drug resistance surveillance in leprosy, where Colombia is a sentinel country. M. leprae DNA from slit skin smear and/or skin biopsy samples was amplified and sequenced to identify mutations in the drug resistance determining region (DRDR) in rpoB, folP1, gyrA, and gyrB, the genes responsible for rifampicin, dapsone and ofloxacin drug-resistance, respectively. Three isolates exhibited mutations in the DRDR rpoB gene (Asp441Tyr, Ser456Leu, Ser458Met), two in the DRDR folP1 gene (Thr53Ala, Pro55Leu), and one isolate exhibited mutations in both DRDR rpoB (Ser456Met) and DRDR folP1 (Pro55Leu), suggesting multidrug resistance. One isolate had a double mutation in folP1 (Thr53Ala and Thr88Pro). Also, we detected mutations outside of DRDR that required in vivo evaluation of their association or not with drug resistance: rpoB Arg505Trp, folP1 Asp91His, Arg94Trp, and Thr88Pro, and gyrA Ala107Leu. Seventy percent of M. leprae mutations were related to drug resistance and were isolated from relapsed patients; the likelihood of relapse was significantly associated with the presence of confirmed resistance mutations (OR range 20.1-88.7, p < 0.05). Five of these relapsed patients received dapsone monotherapy as a primary treatment. In summary, the current study calls attention to M. leprae resistance in Colombia, especially the significant association between confirmed resistance mutations and relapse in leprosy patients. A high frequency of DRDR mutations for rifampicin was seen in a region where dapsone monotherapy was used extensively.

Detection of stealthy small amphiphilic biomarkers.

Pathogen-specific biomarkers are secreted in the host during infection. Many important biomarkers are not proteins but rather small molecules that cannot be directly detected by conventional methods. However, these small molecule biomarkers, such as phenolic glycolipid-I (PGL-I) of Mycobacterium leprae and Mycobactin T (MbT) of Mycobacterium tuberculosis, are critical to the pathophysiology of infection, and may be important in the development of diagnostics, vaccines, and novel therapeutic strategies. Methods for the direct detection of these biomarkers may be of significance both for the diagnosis of infectious disease, and also for the laboratory study of such molecules. Herein, we present, for the first time, a transduction approach for the direct and rapid (30min) detection of small amphiphilic biomarkers in complex samples (e.g. serum) using a single affinity reagent. To our knowledge, this is the first demonstration of an assay for the direct detection of PGL-I, and the first single-reporter assay for the detection of MbT. The assay format exploits the amphiphilic chemistry of the small molecule biomarkers, and is universally applicable to all amphiphiles. The assay is only the first step towards developing a robust system for the detection of amphiphilic biomarkers that are critical to infectious disease pathophysiology.

SNP genotypes of Mycobacterium leprae isolates in Thailand and their combination with rpoT and TTC genotyping for analysis of leprosy distribution and transmission.

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.

Transmission of dapsone-resistant leprosy detected by molecular epidemiological approaches.

Drug resistance surveillance identified six untreated leprosy patients in the Philippines with Mycobacterium leprae folP1 mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked by M. leprae variable-number tandem repeat (VNTR) strain types. In India, folP1 mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target gene rpoB. These findings indicate that dapsone resistance is being transmitted.

A continuation: study and characterisation of Mycobacterium leprae short tandem repeat genotypes and transmission of leprosy in Cebu, Philippines.

OBJECTIVE: 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. METHODS: 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. RESULTS AND CONCLUSIONS: 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.

Molecular epidemiology of leprosy based on VNTR typing in Thailand.

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.

VNTR typing studies of Mycobacterium leprae in China: assessment of methods and stability of markers during treatment.

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.

Genetic diversity of mycobacterium leprae isolates from Brazilian leprosy patients.

INTRODUCTION: Leprosy is a chronic disease caused by infection with Mycobacterium leprae, an obligate intracellular parasite. A problem in studying the transmission of leprosy is the small amount of variation in bacterial genomic DNA. The discovery of variable number of tandem repeats (VNTRs) allowed the detection of strain variation in areas with a high prevalence of leprosy. Four genotypes of M. leprae based on three single-nucleotide polymorphism (SNPs) were also discovered to be useful for analysis of the global spread of leprosy. METHODS: In this present study, we examined the allelic diversity of M. leprae at 16 select VNTR and three SNP loci using 89 clinical isolates obtained from patients mainly from the neighbouring states of São Paulo and Rio de Janeiro Brazil. RESULTS AND CONCLUSION: By use of a PCR-RFLP-based procedure that allows the recognition of SNP types 3 and 4 without the need for the more expensive DNA sequencing steps, characterisation of the main M. leprae genotypes was easy. When applied on the study population, it was found that the SNP type 3 is most frequent in these two states of Brazil, and that VNTRs provided further discrimination of the isolates. Two Short Tandem Repeats (STRs) were monomorphic, with the remaining 14 STRs represented by two to 18 alleles. Epidemiological associations with township or state were not evident in this random collection and require further investigations. In phylogenetic trees, branches formed by all 16 STRs clearly separated SNP type 3 organisms from the other types while the allelic patterns of two minisatellite loci 27-5 and 12-5 were highly correlated with SNP type 3. This strain typing study provide the basis for comparison of M. leprae strain types within Brazil and with those from other countries, and informed selection of genomic markers and methods for future studies.

VNTR typing of Mycobacterium leprae in South Indian leprosy patients.

OBJECTIVES: To study the suitability, stability and diversity of short tandem repeat (STR) genomic markers to elicit strain variation in the Mycobacterium leprae isolates within leprosy patients from Andhra Pradesh and Tamil Nadu states in South India. MATERIALS AND METHODS: Slit skin smear (SSS) samples were collected from lesions and various body sites of newly diagnosed leprosy patients. The SSSs from each patient were pooled, except in the case of five patients. Total DNA was extracted from SSS samples. M. leprae STRs were amplified from the DNA either by multiplex PCR (MP) or single PCR methods. The number of repeats for each STR locus (the STR allele) was obtained either by fragment length analysis (FLA) or by DNA sequencing of the PCR amplicons. RESULTS AND CONCLUSION: Multiplex PCR minimised the use of DNA and reagents, and together with FLA, was time and cost effective for STR strain typing. After examination of the isolates of South Indian origin at 13 STR loci, it was determined that the alleles for (AC)8b, (GGT)5, 6-3a (rpoT), 21-3, 27-5, and 23-3 were conserved in two study populations. In a family from Andhra Pradesh, the M. leprae STR patterns in two patients were identical in 16 of 18 loci which indicate a common source of infection. Fourteen of 15 STR loci showed no intra-patient variation in the five patients tested in Tamil Nadu. Altogether, these studies indicate the suitability of STR strain typing for assessing short-range transmission chains.

Identification and comparison of Mycobacterium leprae genotypes in two geographical regions of Colombia.

OBJECTIVE: To evaluate and establish genomic strain typing markers suitable for the identification of transmission patterns of leprosy in different regions of Colombia. DESIGN: Patients from Agua de Dios, Barranquilla and Cartagena cities and neighbouring towns were enrolled during 2006-2007. Slit skin smears or biopsies were obtained from newly detected untreated patients, and those undergoing multidrug therapy. DNA was extracted from the clinical samples and tested using 15 different short tandem repeat and three SNP polymorphic markers. RESULTS AND CONCLUSION: Differences or similarities between strain types from the northeast (n = 20) and central regions of Colombia (n = 18) were noted. The alleles at two loci, 27-5 and 12-5 were different in the M. leprae in the two regions. The other microsatellite loci may be useful for further intra-population differentiation. There was strong association of 27-5 and 12-5 alleles with the SNP types. The 4-5 combination of alleles was associated with SNP type 3, while the 5-4 combination was mostly associated with SNP type 1, 2 or 4. The SNP type 4 m. leprae isolates were seen in patients in the northeast, but not in the central part.

Population-based molecular epidemiology of leprosy in Cebu, Philippines.

To address the persisting problem of leprosy in Cebu, Philippines, we compiled a database of more than 200 patients who attend an established referral skin clinic. We described the patient characteristics in conventional demographic parameters and also applied multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) and single nucleotide polymorphism (SNP) typing for Mycobacterium leprae in biopsied skin lesion samples. These combined approaches revealed that transmission is ongoing, with the affected including the young Cebuano population under 40 years of age in both crowded cities and rural areas of the island. The emergence of multicase families (MCF) is indicative of infection unconstrained by standard care measures. For the SNPs, we designed a low-cost PCR-restriction fragment length polymorphism typing method. MLVA in M. leprae was highly discriminatory in this population yet could retain broad groups, as defined by the more stable SNPs, implying temporal marker stability suitable for interpreting population structures and evolution. The majority of isolates belong to an Asian lineage (SNP type 1), and the rest belong to a putative postcolonial lineage (SNP type 3). Specific alleles at two VNTR loci, (GGT)5 and 21-3, were highly associated with SNP type 3 in this population. MLVA identified M. leprae genotype associations for patients with known epidemiological links such as in MCFs and in some villages. These methods provide a molecular database and a rational framework for targeted approaches to search and confirm leprosy transmission in various scenarios.

Rapid variable-number tandem-repeat genotyping for Mycobacterium leprae clinical specimens.

Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n = 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n = 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.