Rapid Variable-Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens

Abstract

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.The causative pathogen of leprosy is Mycobacterium leprae. A continued incidence, defying global campaigns to eliminate leprosy even after years of rigorous case finding and the availability of multidrug therapy regimens (28,29,30,31), is attributed to subclinical human and environmental reservoirs of the pathogen (1,8,13). In recent years, molecular strain-typing methodologies have complemented conventional infectious disease epidemiology. With the publication in 2001 of the complete genome sequence of an isolate from Tamil Nadu, India, called the TN strain (4), selection of potential polymorphic genomic markers for strain typing was feasible. The first genetic markers that showed polymorphism were short tandem repeats (STRs) in the M. leprae genome. One was a 6-bp intragenic sequence in the rpoT gene, and the second, a trinucleotide (TTC) repeat element upstream of a pseudogene (17, 23). These sequences exhibit variable numbers of tandem repeats (VNTRs) when sequenced in different isolates. Based on these observations, we short-listed 44 loci (including the rpoT and TTC loci) by in silico analyses of the M. leprae genome and accomplished the screening of 11 STR loci, of which 9 were polymorphic when tested in a small panel of four human isolates derived from passage through armadillos (6). Five were minisatellites (6-to 50-bp repeat units), and four were microsatellites (1-to 5-bp repeat units). Since then, others have also shown that VNTR loci exist in M. lepra...

Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types

“…In 2001, the first reference M. leprae genome of the TN strain from a Tamil Nadu, India, leprosy patient was sequenced, offering new insight and opportunities for development of tools in investigating bacteriology, pathogenesis, and epidemiology. Mapping polymorphic loci, such as variable-number tandem repeats (VNTRs) (7,15) and single nucleotide polymorphisms (SNPs), has applications in strain typing for tracing transmission of leprosy. Four M. leprae lineages (SNP types 1, 2, 3, and 4) have been described on the basis of unique haplotypes derived from three SNPs that were identified by comparative genome sequencing following the availability of the TN strain genome sequence (28,29).…”

Comparação entre microssatélites e o gene Ml MntH como alvos para a identificação do Mycobacterium leprae por PCR na hanseníase

“…6 The diagnostic primers tested in this study were previously used for epidemiological mapping of the M. leprae bacillus in inter alia India, Indonesia and the Philippines. 4,7,8 However, these primers have not been routinely used given the difficulty of obtaining samples of genomic DNA from clinical material.…”

DNA Fingerprinting of <em>Mycobacterium leprae</em> Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

“…Multiple locus VNTR analysis (MLVA) 10 has been used to study leprosy evolution and transmission in several countries including China 11,12 , Malawi 8 , the Philippines 10,13 , and Brazil 14 . MLVA involves multiple steps.…”

“…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.…”

“…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.…”