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.

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.

Genetic, epidemiological and biological analysis of interleukin-10 promoter single-nucleotide polymorphisms suggests a definitive role for -819C/T in leprosy susceptibility.

Leprosy is a complex infectious disease influenced by genetic and environmental factors. The genetic contributing factors are considered heterogeneous and several genes have been consistently associated with susceptibility like PARK2, tumor necrosis factor (TNF), lymphotoxin-alpha (LTA) and vitamin-D receptor (VDR). Here, we combined a case-control study (374 patients and 380 controls), with meta-analysis (5 studies; 2702 individuals) and biological study to test the epidemiological and physiological relevance of the interleukin-10 (IL-10) genetic markers in leprosy. We observed that the -819T allele is associated with leprosy susceptibility either in the case-control or in the meta-analysis studies. Haplotypes combining promoter single-nucleotide polymorphisms also implicated a haplotype carrying the -819T allele in leprosy susceptibility (odds ratio (OR)=1.40; P=0.01). Finally, we tested IL-10 production in peripheral blood mononuclear cells stimulated with Mycobacterium leprae antigens and found that -819T carriers produced lower levels of IL-10 when compared with non-carriers. Taken together, these data suggest that low levels of IL-10 during the disease outcome can drive patients to a chronic and unprotective response that culminates with leprosy.

Utilidad de la biología molecular en el diagnóstico microbiológico de las infecciones por micobacterias / Utility of molecular biology in the microbiological diagnosis of mycobacterial infections

Las micobacterias constituyen un grupo de bacterias de gran interés en medicina, ya que, junto a especies telúricas y oportunistas, se hallan 2 especies (Mycobacterium tuberculosis y Mycobacterium leprae) de gran importancia en salud pública. A pesar de los esfuerzos realizados para su control, la tuberculosis (TB) sigue siendo en la actualidad uno de los problemas sanitarios de más trascendencia mundial. En los últimos años, la micobacteriología ha experimentado importantes avances tecnológicos. A pesar de ello, el diagnóstico temprano de la infección por micobacterias y, especialmente de la TB, sigue recayendo en el examen microscópico de las muestras teñidas de manera adecuada. En la actualidad, éste sigue siendo el procedimiento más simple, de mejor coste-efectividad y rapidez para proporcionar al clínico una orientación diagnóstica preliminar. El control efectivo de la TB se basa en la detección rápida de M. tuberculosis, seguido por la inmediata implementación del tratamiento antituberculoso adecuado.(AU)

La emergencia de cepas resistentes a los fármacos antituberculosos agudiza la necesidad de disponer de métodos rápidos de detección de M. tuberculosis y de resistencias. La disponibilidad de métodos de epidemiología molecular de fácil implementación y estandarización, que nos permitan identificar casos relacionados, es fundamental para identificar brotes epidémicos que ayuden a controlar la propagación de la TB. Aun reconociendo los evidentes progresos realizados en el diagnóstico molecular de las infecciones micobacterianas, las técnicas disponibles son todavía insuficientes. En esta revisión, describimos el estado actual de las principales técnicas moleculares para la detección directa de micobacterias en muestras clínicas, para su identificación, detección de resistencias a los principales fármacos antituberculosos y de epidemiología molecular. En cada caso, destacamos las ventajas y las limitaciones de ellas. En un próximo futuro la micobacteriología clínica evolucionará, con bastante probabilidad, hacia la universalización de las técnicas genéticas aplicadas al diagnóstico directo y la detección de resistencias. La epidemiología molecular de la TB se realizará, en sus diferentes aplicaciones, con técnicas más rápidas y automatizadas que las actuales(AU)

Species within the Mycobacterium genus are of major medical interest, since, together with environmental and opportunistic species, there are two species (Mycobacterium tuberculosis and Mycobacterium leprae) that remain an important public health challenge. Despite efforts to control tuberculosis (TB), this disease remains one of the most prominent health problems worldwide. In the last few years, mycobacteriology has experienced major technological advances. Nevertheless, the early diagnosis of mycobacterial infection and, especially of TB, is still based on microscopic examination of properly stained samples. At present, this procedure is still the simplest, fastest and most cost-effective method for preliminary diagnostic guidance. Effective control of TB is based on rapid detection of M. tuberculosis, followed by immediate implementation of the appropriate antituberculosis therapy. Because of the emergence of multidrug resistant strains, the development of rapid diagnostic methods, both for identification of M. tuberculosis and susceptibility testing, has become a pressing need. The availability of molecular epidemiology methods that are easy to implement and standardized and that would allow identification of related cases is of key importance to identify epidemic outbreaks and control the spread of TB. Despite the evident progress in the molecular diagnosis of mycobacterial infections, the available techniques are still inadequate(AU)

In this review, we describe the state of the art of the main molecular techniques for direct detection of mycobacteria in clinical samples, their identification, detection of resistance to the most important antituberculosis agents, and molecular epidemiology. In each case, we describe the advantages and limitations of current techniques. In the near future, clinical mycobacteriology will probably evolve to the universal use of genetic techniques for direct diagnosis and detection of resistance. The molecular epidemiology of TB will be performed, in its various applications, by faster and more automated techniques than those currently available(AU)

Utilidad de la biología molecular en el diagnóstico microbiológico de las infecciones por micobacterias / Utility of molecular biology in the microbiological diagnosis of mycobacterial infections

Las micobacterias constituyen un grupo de bacterias de gran interés en medicina, ya que, junto a especies telúricas y oportunistas, se hallan 2 especies (Mycobacterium tuberculosis y Mycobacterium leprae) de gran importancia en salud pública. A pesar de los esfuerzos realizados para su control, la tuberculosis (TB) sigue siendo en la actualidad uno de los problemas sanitarios de más trascendencia mundial. En los últimos años, la micobacteriología ha experimentado importantes avances tecnológicos. A pesar de ello, el diagnóstico temprano de la infección por micobacterias y, especialmente de la TB, sigue recayendo en el examen microscópico de las muestras teñidas de manera adecuada. En la actualidad, éste sigue siendo el procedimiento más simple, de mejor coste-efectividad y rapidez para proporcionar al clínico una orientación diagnóstica preliminar. El control efectivo de la TB se basa en la detección rápida de M. tuberculosis, seguido por la inmediata implementación del tratamiento antituberculoso adecuado. La emergencia de cepas resistentes a los fármacos antituberculosos agudiza la necesidad de disponer de métodos rápidos de detección de M. tuberculosis y de resistencias. La disponibilidad de métodos de epidemiología molecular de fácil implementación y estandarización, que nos permitan identificar casos relacionados, es fundamental para identificar brotes epidémicos que ayuden a controlar la propagación de la TB. Aun reconociendo los evidentes progresos realizados en el diagnóstico molecular de las infecciones micobacterianas, las técnicas disponibles son todavía insuficientes. En esta revisión, describimos el estado actual de las principales técnicas moleculares para la detección directa de micobacterias en muestras clínicas, para su identificación, detección de resistencias a los principales fármacos antituberculosos y de epidemiología molecular. En cada caso, destacamos las ventajas y las limitaciones de ellas. En un próximo futuro la micobacteriología clínica evolucionará, con bastante probabilidad, hacia la universalización de las técnicas genéticas aplicadas al diagnóstico directo y la detección de resistencias. La epidemiología molecular de la TB se realizará, en sus diferentes aplicaciones, con técnicas más rápidas y automatizadas que las actuales

Species within the Mycobacterium genus are of major medical interest, since, together with environmental and opportunistic species, there are two species (Mycobacterium tuberculosis and Mycobacterium leprae) that remain an important public health challenge. Despite efforts to control tuberculosis (TB), this disease remains one of the most prominent health problems worldwide. In the last few years, mycobacteriology has experienced major technological advances. Nevertheless, the early diagnosis of mycobacterial infection and, especially of TB, is still based on microscopic examination of properly stained samples. At present, this procedure is still the simplest, fastest and most cost-effective method for preliminary diagnostic guidance. Effective control of TB is based on rapid detection of M. tuberculosis, followed by immediate implementation of the appropriate antituberculosis therapy. Because of the emergence of multidrug resistant strains, the development of rapid diagnostic methods, both for identification of M. tuberculosis and susceptibility testing, has become a pressing need. The availability of molecular epidemiology methods that are easy to implement and standardized and that would allow identification of related cases is of key importance to identify epidemic outbreaks and control the spread of TB. Despite the evident progress in the molecular diagnosis of mycobacterial infections, the available techniques are still inadequate. In this review, we describe the state of the art of the main molecular techniques for direct detection of mycobacteria in clinical samples, their identification, detection of resistance to the most important antituberculosis agents, and molecular epidemiology. In each case, we describe the advantages and limitations of current techniques. In the near future, clinical mycobacteriology will probably evolve to the universal use of genetic techniques for direct diagnosis and detection of resistance. The molecular epidemiology of TB will be performed, in its various applications, by faster and more automated techniques than those currently available (AU)