Genomic Reduction at TTC Repeats in the Bacterial Genome of Treated Cases of Hansen's Disease: A Possible Survival Mechanism of Mycobacterium leprae.

INTRODUCTION: Mycobacterium leprae has a small genome and a tendency of persisting as a very low-grade infection. The authors have shown earlier, that the changes in TTC repeats, in M. leprae genome may contribute to the restriction of the pathogenicity of the bacterium and its survival strategy in case of pure neural Hansen's disease. We suspect, that a similar genomic reduction if happens in treated cases of Hansen's disease, can be a determining factor for developing persisters and relapse. AIM: The present study aimed to find out if there was any evidence of genomic reduction in treated cases of Hansen's disease that showed microbiological nonresponse. METHODS: Skin biopsies were taken from treated cases of Hansen's disease at tertiary centers in Kolkata and at Raipur who had bacterial index (BI) unchanged or increased compared to their pretreatment BI. Analysis for the mutation in rpoB gene and folP1 gene were done to rule out rifampicin and dapsone resistance, respectively. The entire TTC repeat region of the bacteria was amplified by polymerase chain reaction and was subjected to sequencing. The obtained sequences were then analyzed by CLUSTALW. RESULTS: A total of 127 patients were included in the study of which in 52 the BI remained same and 75 had an increase in BI, even after 6 months of completion of multidrug therapy. Among the samples, 2 had positive rpoB gene mutation. No mutation was found in the folP1 gene. The TTC repeat of both the rpoB-resistant samples was found to have 17 copies, which matched their pretreatment copy number. In other 125 cases, 60 cases showed no change from their pretreatment TTC number. Of those 65 samples that showed evidence of genomic reduction, 11 samples showed one copy, 41 showed 2 copies, and 13 showed 3 copies deletion. We also observed a significant regional variation. CONCLUSION: We concluded that there was evidence of genomic reduction, which might lead to microbiological nonresponse in treated cases of Hansen's disease. This indicated a possibility of future persistence and relapse.

Mutation at codon 442 in the rpoB gene of Mycobacterium leprae does not confer resistance to rifampicin.

BACKGROUND: Rifampicin is the major drug in the treatment of leprosy. The rifampicin resistance of Mycobacterium leprae results from a mutation in the rpoB gene, encoding the ß subunit of RNA polymerase. As M. leprae is a non-cultivable organism observation of its growth using mouse food-pad (MFP) is the only Gold Standard assay used for confirmation of "in-vivo" drug resistance. OBJECTIVE: Any mutation at molecular level has to be verified by MFP assay for final confirmation of drug resistance in leprosy. MATERIAL AND METHODS: In the present study, M. leprae strains showing a mutation only at codon 442 Gln-His and along with mutation either at codon 424 Val-Gly or at 438 Gln-Val within the Rifampicin Resistance Determining Region (RRDR) confirmed by DNA sequencing and by high resolution melting (HRM) analysis were subjected for its growth in MFP. RESULT AND CONCLUSION: The M. leprae strain having the new mutation at codon 442 Gln-His was found to be sensitive to all the three drugs and strains having additional mutations at 424 Val-Gly and 438 Gln-Val were conferring resistance with Multi drug therapy (MDT) in MFP. These results indicate that MFP is the gold standard method for confirming the mutations detected by molecular techniques.

Report of rpoB mutation in clinically suspected cases of drug resistant leprosy: a study from Eastern India.

BACKGROUND: The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very efficient opportunity for the diagnosis of drug resistance by in vitro method. AIM: We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal. METHODS: A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplified by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI - BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences. RESULTS: A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp - 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specific for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT). LIMITATIONS: The major limitations of multiple-primer PCR amplification refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes. CONCLUSION: The study indicates the existence of rifampicin drug resistance in Eastern India.

Fite-Faraco staining in combination with multiplex polymerase chain reaction: a new approach to leprosy diagnosis.

BACKGROUND: Leprosy is not always an easy disease to diagnose, and patients can remain undiagnosed for longtime, not only at the peripheral clinics but also even at places with higher medical facilities, so, there is an urgent need for rapid and definitive modalities for leprosy diagnosis. This prospective study evaluates the ability of Fite-Faraco staining (FF staining) and multiplex polymerase chain reaction (PCR) over hematoxylin and eosin staining (H and E staining) and Ziehl-Neelsen staining (ZN staining). AIMS: The aim of this perspective study is to evaluate the effectiveness of FF staining in combination with multiplex PCR for the early and rapid diagnosis of leprosy than any other coexisting diagnosis tool. METHODS: Patients with new skin patches or nodules with or without evidence of nerve damage were selected for the study. Punch biopsy was collected according to standard procedures. Each biopsy sample was divided into two equal parts, one half was fixed in 4% (v/v) buffered neutral formalin and then accordingly embedded in paraffin. Sections were stained by three different methods: H and E staining for histopathological examination, ZN staining, and FF staining for detection of acid-fast bacilli (AFB). And the other part was subjected for DNA extraction and PCR was carried out by the obtained DNA sample. RESULTS: H and E staining, ZN staining, FF staining, and PCR yield 58.2%, 50.9%, 60%, and 67.7% successful diagnosis of leprosy. The true diagnostic performances for these techniques were as follows: H and E staining - sensitivity 70.6%, positive predictive value (PPV) 81.9%, negative predictive value (NPV) 53.6%. For ZN staining - sensitivity 59.9%, PPV 69%, NPV 45.7%. For FF staining - sensitivity 74.6%, PPV 85.9%, NPV 56.7%, and for PCR - sensitivity 87.8%, PPV 95.6%, NPV 71.2%. CONCLUSION: The combination of FF staining and PCR was shown to provide a rapid and definitive diagnosis in the majority of leprosy suspected cases with a higher positive likelihood ratio (+LR) of 7.76 and 2.716, respectively, than H and E staining of 2.244 and ZN staining of 1.378.

Diagnosing leprosy: revisiting the role of the slit-skin smear with critical analysis of the applicability of polymerase chain reaction in diagnosis.

BACKGROUND: Diagnosing leprosy is challenging, especially in early-stage cases, and the need for a sensitive diagnostic tool is urgent. Polymerase chain reaction (PCR) holds promise as a simple and sensitive diagnostic tool, but its usefulness in the Indian context requires further evaluation. Slit-skin smear (SSS) remains the conventional method of leprosy detection. Hence, this study was undertaken to evaluate and compare the diagnostic efficacy of PCR versus that of SSS. METHODS: Punch biopsy of skin and SSS were obtained from the active margins of lesions. Cases were clinically grouped according to whether they were multibacillary (MB) or paucibacillary (PB) and classified into tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL), lepromatous (LL), histoid, and indeterminate groups after clinicopathological correlation. DNA was extracted from biopsy specimens, and multiplex PCR was carried out incorporating primers intended for the amplification of a specific 372-bp fragment of a repetitive sequence of Mycobacterium leprae DNA. RESULTS: Among 164 patients, PCR was positive in 82.3%. The sensitivity of PCR was significantly greater (P < 0.0001) than that of SSS in both the MB (85.9% vs. 59.8%) and PB (75.4% vs. 1.8%) subgroups; the difference in sensitivity in the PB subgroup is remarkable. Positivity by PCR and SSS was found in 100% of LL and histoid leprosy, but PCR had significantly greater (P < 0.0001) positivity in BT leprosy and was of definite increased value in indeterminate and TT leprosy. CONCLUSIONS: Polymerase chain reaction had higher sensitivity compared with SSS, especially in diagnostically challenging and PB cases. Thus, the use of this costly but sensitive tool should be restricted to this subgroup, because SSS is sufficiently sensitive in the diagnosis of LL and histoid leprosy.

Multiplex PCR technique could be an alternative approach for early detection of leprosy among close contacts--a pilot study from India.

BACKGROUND: Implementation of Multi drug Therapy (MDT) regimen has resulted in the decline of the total number of leprosy cases in the world. Though the prevalence rate has been declining, the incidence rate remains more or less constant and high in South East Asian countries particularly in India, Nepal, Bangladesh, Pakistan and Srilanka. Leprosy, particularly that of multibacillary type spreads silently before it is clinically detected. An early detection and treatment would help to prevent transmission in the community. Multiplex PCR (M-PCR) technique appears to be promising towards early detection among contacts of leprosy cases. METHODS: A total of 234 paucibacillary (PB) and 205 multibacillary (MB) leprosy cases were studied in a community of an endemic area of Bankura district of West Bengal (Eastern India). They were assessed by smear examination for acid-fast bacilli (AFB) and M-PCR technique. These patients were treated with Multidrug Therapy (MDT) as prescribed by WHO following detection. A total of 110 MB and 72 PB contacts were studied by performing M-PCR in their nasal swab samples. RESULTS: 83.4% of MB patients were observed to be positive by smear examination for AFB and 89.2% by M-PCR. While 22.2% of PB patients were found to be positive by smear examination for AFB, 80.3% of these patients were positive by M-PCR. Among leprosy contacts (using M-PCR), 10.9% were found to be positive among MB contacts and 1.3% among PB contacts. Interestingly, two contacts of M-PCR positive MB cases developed leprosy during the period of two years follow up. CONCLUSION: The M-PCR technique appears to be an efficient tool for early detection of leprosy cases in community based contact tracing amongst close associates of PB and MB cases. Early contact tracing using a molecular biology tool can be of great help in curbing the incidence of leprosy further.

Development and application of a new efficient and sensitive multiplex polymerase chain reaction (PCR) in diagnosis of leprosy.

India contributes about 80% of the global leprosy case load including case of fresh infection and reinfection. Due to lack of gold standard, diagnosis is done mainly based on routine clinical signs and symptoms, smear and histopathological evidences. There is a lot of lacunae in early confirmatory diagnosis in terms of sensitivity and specificity, especially in paucibacillary tuberculoid type. Moreover, the classification of different classes of leprosy is very important for selection of proper therapeutic schedule. Hence this study was undertaken to develop a multiplex polymerase chain reaction for the diagnosis and strain differentiation of M leprae. A multiplex polymerase chain reaction was developed using the primers R1 and R2 (a) amplifying 372bp DNA target from a repetitive sequence of M leprae and this repetitive sequence (372bp) that was used as a target DNA for amplification was reported to be specific for M leprae was not present in 20 mycobacterium species other than M leprae and primers TTCA and TTCB (b) amplifying (201bp) DNA target of variable sizes from the regions flanking TTC repeats of M leprae genome. This multiplex polymerase chain reacton developed in our laboratory revealed that the number of repeats at each locus might be variable among M leprae but they are found mostly in multibacillary (as the bacterial load is higher in multibacillary) type.