RESUMO
We aimed to identify an unique host transcriptional signature in peripheral blood mononuclear cells (PBMCs) in response to Mycobacterium leprae antigens to distinguish between patients with leprosy and non-leprosy controls for early diagnosis of the disease. Sixteen individuals were enrolled in the discovery cohort [eight patients with leprosy, comprising four multibacillary (MB) and four paucibacillary (PB); and eight non-leprosy controls, comprising four healthy house contacts (HHCs) and four endemic controls (ECs)]. The differences in the transcriptome response of PBMCs to M. leprae sonicate antigen were evaluated between leprosy patients and non-leprosy controls, and 12 differentially expressed genes (CCL2/MCP-1, IL-8, JAKM, ATP, ND1, SERP, FLJ10489, LINC00659, LOC34487, LOC101928143, MIR22, and NCF1C) were identified. The accuracy of the 12 differentially expressed genes was further validated for the diagnosis of leprosy using real-time quantitative PCR in 82 individuals (13 MB, 10 PB, 37 HHCs, and 22 ECs) in the validation cohort. We found that a 5 gene signature set IL-8, CCL2/MCP-1, SERP, LINC00659 and FLJ10489 had a suitable performance in discriminating leprosy from ECs. In addition, elevated expression of IL-8, CCL2/MCP-1, SERP and LINC00659 was associated with MB diagnosis compared with ECs, whereas increased expression of IL-8, CCL2/MCP-1, SERP and FLJ10489 was found to be useful biomarkers for PB diagnosis from ECs. Moreover, we found decreased expression of NCF1C among leprosy patients could distinguish leprosy from HHCs, whereas higher expression of CCL2 among MB than PB could distinguish different leprosy patients. In conclusion, among the 12 candidate host genes identified, a three gene signature IL-8, CCL2/MCP-1, and SERP showed the best performance in distinguishing leprosy patients from healthy controls. These findings may have implications for developing a rapid blood-based test for early diagnosis of leprosy.
Assuntos
Hanseníase , Mycobacterium leprae , Antígenos de Bactérias , Biomarcadores , Diagnóstico Precoce , Humanos , Hanseníase/diagnóstico , Leucócitos Mononucleares , Mycobacterium leprae/genética , TranscriptomaRESUMO
Leprosy is an infectious disease caused by Mycobacterium leprae (M. leprae), with about 210,000 new cases per year worldwide. Although numerous risk loci have been uncovered by genome-wide association studies, the effects of common genetic variants are relatively modest. To identify possible new genetic locus involved in susceptibility to leprosy, whole exome sequencing was performed for 28 subjects including 14 patients and 12 unaffected members from 8 leprosy-affected families as well as another case and an unrelated control, and then the follow-up SNP genotyping of the candidate variants was studied in case-control sample sets. A rare missense variant in mitochondrial ribosomal protein S5 (MRPS5), rs200730619 (c. 95108402T>C [p. Tyr137Cys]) was identified and validated in 369 cases and 270 controls of Chinese descent (Padjusted = 0.006, odds ratio [OR] = 2.74) as a contributing factor to leprosy risk. Moreover, the mRNA level of MRPS5 was downregulated in M. leprae sonicate-stimulated peripheral blood mononuclear cells. Our results indicated that MRPS5 may be involved in leprosy pathogenesis. Further studies are needed to determine if defective MRPS5 could lead to impairment of energy metabolism of host immune cells, which could further cause defect in clearing M. leprae and increase susceptibility to infection.
Assuntos
Povo Asiático/genética , Predisposição Genética para Doença , Hanseníase/genética , Proteínas Mitocondriais/genética , Polimorfismo de Nucleotídeo Único , Proteínas Ribossômicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , China/epidemiologia , Feminino , Regulação da Expressão Gênica , Humanos , Hanseníase/epidemiologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94-86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes. CONCLUSIONS/SIGNIFICANCE: Nested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.
Assuntos
Farmacorresistência Bacteriana , Técnicas de Genotipagem/métodos , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , China , Feminino , Genes Bacterianos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/isolamento & purificação , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Adulto JovemRESUMO
BACKGROUND: Detection and pathology analysis of Mycobacterium leprae using skin biopsy tissues are essential for leprosy diagnosis and monitoring response to treatment. Although formalin fixation of patient tissues may not be ideal for molecular studies, biopsy samples are the most accessible material from suspected cases. Therefore, clinical molecular laboratories must be able to utilize formalin-fixed, paraffin-embedded (FFPE) material. OBJECTIVE: To determine the best molecular method for diagnosing and monitoring leprosy in FFPE specimens, we developed a single-tube nested PCR (STNPCR) (131 bp) and SYBRGreen PCR (101 bp) assay using primers for the M. leprae-specific repetitive element (RLEP) gene and evaluated the results compared to those using previously established RLEP primers (372 bp). METHODS: FFPE biopsy samples obtained from 145 leprosy patients (during or after multidrug therapy (MDT)) and patients with 29 other confounding dermatoses were examined by the bacteria index (BI) and by simple PCR, STNPCR, and SYBRGreen PCR using primers amplifying a 372-bp, 131-bp or 101-bp fragment of RLEP, respectively. RESULTS: In leprosy patients receiving MDT, STNPCR showed a highest specificity of 100% and a positive predictive value (PPV) of 100%. For multibacillary (MB), paucibacillary (PB) and all leprosy patients, the highest sensitivities were 91.42%, 39.13%, and 67.92%, negative predictive values (NPVs) were 8.57%, 60.36%, and 32.07%, and the highest accuracies were 93.93%, 62.67%, and 74.81%, respectively, higher than the results of SYBRGreen PCR and simple PCR. For post-MDT leprosy patients, SYBRGreen PCR showed the highest sensitivity of 50.0%, highest specificity of 100%, a PPV of 100%, an NPV of 100% and the highest accuracy of 83.72% for MB patients, which were higher than those of STNPCR and simple PCR. STNPCR showed the highest sensitivity of 26.66% and 34.48%, highest specificity of 100% and 100%, a PPV of 100% and 100%, NPV of 72.50% and 60.21%, and highest accuracy of 75.00% and 67.24% for PB and leprosy patients, respectively, higher than those of SYBRGreen PCR and simple PCR. CONCLUSIONS: These findings suggest that STNPCR or SYBRGreen PCR (131-bp and 101-bp fragment amplification, respectively) for RLEP using FFPE specimens performs better as a diagnostic test and for monitoring response to MDT than does simple PCR based on 372-bp fragment amplification. Additionally, STNPCR showed increased sensitivity for PB diagnosis using FFPE specimens, which can be transferred remotely or retrieved from previous leprosy patients.
Assuntos
Formaldeído , Hanseníase/diagnóstico , Mycobacterium leprae/isolamento & purificação , Inclusão em Parafina/métodos , Reação em Cadeia da Polimerase/métodos , Biópsia/métodos , China , Primers do DNA , DNA Bacteriano/genética , Quimioterapia Combinada , Humanos , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Mycobacterium leprae/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sensibilidade e Especificidade , Pele/microbiologiaRESUMO
BACKGROUND: Leprosy, caused by Mycobacterium leprae, affects over 200,000 people annually worldwide and remains endemic in the ethnically diverse, mountainous and underdeveloped southwestern provinces of China. Delayed diagnosis of leprosy persists in China, thus, additional knowledge to support early diagnosis, especially early diagnosis of paucibacillary (PB) patients, based on the host immune responses induced by specific M. leprae antigens is needed. The current study aimed to investigate leprosy patients and controls in Southwest China by comparing supernatants after stimulation with specific M. leprae antigens in an overnight whole-blood assay (WBA) to determine whether host markers induced by specific M. leprae antigens improve the diagnosis or discrimination of PB patients with leprosy. METHODOLOGY/PRINCIPAL FINDINGS: Leprosy patients [13 multibacillary (MB) patients and 7 PB patients] and nonleprosy controls [21 healthy household contacts (HHCs), 20 endemic controls (ECs) and 19 tuberculosis (TB) patients] were enrolled in this study. The supernatant levels of ten host markers stimulated by specific M. leprae antigens were evaluated by overnight WBA and multiplex Luminex assays. The diagnostic value in PB patients and ECs and the discriminatory value between PB patients and HHCs or TB patients were evaluated by receiver operator characteristics (ROC) analysis. ML2044-stimulated CXCL8/IL-8 achieved the highest sensitivity of 100%, with a specificity of 73.68%, for PB diagnosis. Compared to single markers, a 3-marker combination model that included ML2044-induced CXCL8/IL-8, CCL4/MIP-1 beta, and IL-6 improved the diagnostic specificity to 94.7% for PB patients. ML2044-stimulated IL-4 and CXCL8/IL-8 achieved the highest sensitivity (85.71% and 100%) and the highest specificity (95.24% and 84.21%) for discriminating PB patients from HHCs and TB patients, respectively. CONCLUSIONS: Our findings suggest that the host markers induced by specific M. leprae antigens in an overnight WBA increase diagnostic and discriminatory value in PB patients with leprosy, with a particularly strong association with interleukin 8.
Assuntos
Antígenos de Bactérias/imunologia , Sangue/imunologia , Imunidade Inata , Imunoensaio/métodos , Hanseníase Paucibacilar/diagnóstico , Hanseníase Paucibacilar/imunologia , Mycobacterium leprae/imunologia , Adulto , China , Citocinas/análise , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Masculino , Curva ROC , Sensibilidade e Especificidade , Adulto JovemRESUMO
INTRODUCTION: Leprosy is a chronic infectious granulomas disease caused by Mycobacterium leprae that can manifest as a wide variety of immunological and clinical features. CASE SUMMARY: Here, we describe the case of a woman with clinical characteristics of borderline tuberculoid (BT) leprosy that manifested as 3 asymmetric skin lesions involving her hip and lower limbs. This unusual presentation was initially misdiagnosed as sarcoidosis because noncaseating granulomas are a histopathological feature of both diseases. Differentiation and the diagnosis of BT leprosy was achieved using real-time polymerase chain reaction (PCR) to amplify an M leprae specific DNA sequence and to detect serum antibodies specific to M leprae antigens. Accordingly, a 6-month course of multidrug therapy led to a marked improvement in the skin lesions. CONCLUSION: The use of auxiliary tests including real-time PCR to amplify an M leprae-specific DNA sequence, enzyme-linked immunosorbent assay, and dipstick detection of serum antibodies specific to M leprae antigens are good methods to obtain a correct diagnosis of BT leprosy.
Assuntos
Hanseníase Paucibacilar/diagnóstico , Dermatopatias Bacterianas/diagnóstico , Adulto , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Diagnóstico Diferencial , Feminino , Humanos , Mycobacterium leprae/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Sarcoidose/diagnósticoRESUMO
The purpose of this study was to evaluate peripheral neural impairment in leprosy patients by ultrasonography (US). The cross-sectional areas (CSAs) of the median (M), ulnar (U) and common fibular (CF) nerves were compared in 71 leprosy patients and 29 healthy controls, and the data were analyzed between the leprosy, multibacillary (MB)/paucibacillary (PB), reaction (R)/no reaction (NR), disability (D)/no disability (ND), and longer/shorter duration groups after treatment. We found that for the nerves located in upper limbs, the CSAs were significantly increased in the leprosy patients vs the controls; the PB group vs the MB group; the R group vs the NR group; the ND group vs the D group; and the longer duration group vs the shorter duration group at some positions of the M nerve and U nerve. In contrast, for the nerves located in lower limbs, the CSAs were significantly reduced in the leprosy patients vs the controls and in the longer duration group vs the shorter duration group at some positions of the CF nerve. This result indicated that nerve enlargement and neuratrophy coexist in leprosy patients.
Assuntos
Hanseníase/diagnóstico por imagem , Doenças do Sistema Nervoso Periférico/diagnóstico por imagem , Nervos Espinhais/diagnóstico por imagem , Idoso , Feminino , Humanos , Hanseníase/complicações , Masculino , Nervo Mediano/diagnóstico por imagem , Pessoa de Meia-Idade , Doenças do Sistema Nervoso Periférico/complicações , Nervo Fibular/diagnóstico por imagem , Nervo Ulnar/diagnóstico por imagemRESUMO
Leprosy has long been thought to have a strong genetic component, and so far, only positional cloning and genomewide association studies have been used to study the genetic susceptibility to leprosy,while whole exome sequencing (WES) approach has not yet been applied. In this study, we used WES approach on four leprosy patients and four healthy control relatives from two leprosy families. We found three new susceptible loci of leprosy, one in GAL3ST4 and two in CHGB. We went on to validate the findings of WES using 151 leprosy cases and 226 healthy controls by Sanger sequencing. Stratified by gender, GAL3ST4 was found to be the susceptible gene only for the female population, and CHGB48 and CHGB23 were susceptibile to leprosy for the male population, respectively). Moreover, the gene expression levels of the three susceptible loci were measured by real-time PCR after the stimulation by M. leprae antigens in the PBMC (peripheral blood mononuclear cells) of 69 healthy people. The results showed that the female subjects with high frequent genotype in GAL3ST4 had a fivefold elevated expression. We suggest the polymorphisms in GAL3ST4 in different population are associated with increased risk of leprosy.
Assuntos
Cromogranina B/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Hanseníase/genética , Sulfotransferases/genética , Alelos , Estudos de Casos e Controles , Biologia Computacional/métodos , Bases de Dados Factuais , Feminino , Expressão Gênica , Loci Gênicos , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Razão de Chances , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Fatores Sexuais , Sequenciamento do ExomaRESUMO
Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China.
Assuntos
Genótipo , Hanseníase/microbiologia , Mycobacterium leprae/genética , Polimorfismo de Nucleotídeo Único , RNA Ribossômico 16S/genética , Adulto , China , Feminino , Humanos , Masculino , Mutação de Sentido Incorreto , Mycobacterium leprae/isolamento & purificação , Pele/microbiologiaRESUMO
Leprosy is the disabling outcome of chronic infection with Mycobacterium leprae. The disease often evades early detection, particularly now that fewer clinicians are able to confidently diagnose the disease following the integration of leprosy control measures within general health services in many countries. Although leprosy is officially eliminated in China, endemic regions remain in some difficult-to-reach, underdeveloped areas in Southwest China. In order to better understand the extent of M. leprae infection and identify new leprosy cases in a timely manner, simple tools that can detect infection and the early disease are required. In this report we evaluated the performance of antigen-specific ELISA, the NDO-LID rapid diagnostic test, and antigen-specific whole blood assays (WBA) as potential diagnostic tools. Our data support the use of antibody detection tests and WBA to facilitate the diagnosis of multibacillary and paucibacillary leprosy, respectively. These tools could be invaluable for increased, but simplified, monitoring of individuals in order to provide referrals for clinical exam and early leprosy diagnosis.
Assuntos
Antígenos de Bactérias/sangue , Hanseníase/sangue , Mycobacterium leprae/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , China , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hanseníase/genética , Hanseníase/patologia , Pessoa de Meia-Idade , Mycobacterium leprae/genética , Mycobacterium leprae/patogenicidadeRESUMO
We evaluated the sensitivity and specificity of a nested-polymerase chain reaction (PCR) method for detection of Mycobacterium leprae DNA from whole blood. Whole-blood specimens were subjected to nested-PCR amplification of M. leprae repeat DNA sequences in 49 multibacillary (MB) and 30 paucibacillary (PB) leprosy patients, 96 household contacts (HHCs), 18 tuberculosis (TB) patients, and 35 normal healthy individuals. M. leprae DNA was detected in 95.92% (47/49) of MB, 70% (21/30) of PB, and 6.25% (6/96) of HHC, but it was not detected in 18 TB or 35 normal controls. The sensitivities of the anti-bovine serum albumin (ND-O-BSA) immunoglobulin M (IgM) and antifusion protein of ML0405-ML2331 IgG for MB were 97.96% and 89.8%, and these values for PB were 70% and 53.33%. However, the ND-O-BSA enzyme-linked immunosorbent assay (ELISA) had lower specificity, with relatively high false-positive results for TB patients (16.67%) and normal healthy controls (10%). Based on these promising findings, we propose the use of nested PCR of whole-blood samples along with ELISA test for early detection of leprosy cases.
Assuntos
DNA Bacteriano/sangue , Diagnóstico Precoce , Hanseníase/diagnóstico , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Animais , Anticorpos Antibacterianos/sangue , Bovinos , China , DNA Bacteriano/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina M , Hanseníase/microbiologia , Mycobacterium leprae/isolamento & purificação , Sensibilidade e Especificidade , Soroalbumina Bovina/imunologiaRESUMO
OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Assuntos
Hanseníase/transmissão , China/epidemiologia , Humanos , Hanseníase/epidemiologia , Epidemiologia Molecular , Prevalência , Microbiologia da Água , Poluentes da ÁguaRESUMO
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.
Assuntos
Hanseníase Multibacilar/microbiologia , Repetições Minissatélites , Mycobacterium leprae/genética , China/epidemiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Variação Genética , Genótipo , Humanos , Hanseníase Multibacilar/epidemiologia , Epidemiologia Molecular/métodos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
OBJECTIVE: Multiple locus variable number-tandem repeat (VNTR) analysis (MLVA) had been proposed as a means of strain typing for tracking of source and studying the transmission chain of pathogens. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. METHODS: MLVA on 7 VNTR loci was applied to the strain typing on prevalent Mycobacterium leprae isolates collected from Qiubei county, Yunnan province during 2002-2006 in the study on the relationship between geographic distribution and genotypes of M. leprae. The strain typing, combined with conventional epidemiological investigation was performed to trace the transmission chain. RESULTS: (1) Phylogenetic analyses through application of PAUP 4.0, The M. leprae were grouped into A, B, C, D and E strains according to the allelic range 9, 11-13, 15-26 and > 26 on the GTA9 locus. The strains with 9 copies on GTA9 locus, was named A. (2) Genotypes of strains from the five multi-case families located at North and North-West parts were similar and belonged to A strains. VNTR patterns of intra-family were identical or similar but not identical inter-family. (3) Not only A cluster appeared higher proportion in total isolates but also distributes cluster, indicating ongoing transmission from recent findings. CONCLUSION: VNTR strain typing was suitable to trace the short chain of transmission in both small area and intra-families. Multi-case families might constitute epidemic foci and source of M. leprae in villages, causing the predominant strain or cluster which tends to be those identified in multi-case families and resulted in the spreading of leprosy. A long-term study was underway to reveal whether A strain was predominant strain and to observe the evolution of M. leprae in this spatially and temporally defined endemic population.
Assuntos
Genótipo , Hanseníase/microbiologia , Mycobacterium leprae/genética , Feminino , Humanos , Masculino , Repetições Minissatélites/genética , Epidemiologia Molecular , Mycobacterium leprae/classificação , Filogenia , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: To understand the genotypic mapping of Mycobacterium leprae identified in China and to compare with those from other countries to select suitable alleles for epidemiological investigation in the transmission chain of leprosy. METHODS: Various number of tandem repeat(VNTR) in genomic DNA of Mycobacterium leprae was used in the present genotyping study. 33 skin biopsies from Wenshan prefecture,Yunnan province and 17 from other parts of China were studied. DNA extracted from skin biopsies of leprosy patients was subjected to PCR followed by agarose gel analysis and DNA sequencing to determine the number of repeats. RESULTS: Loci GGT-5,12-5,21-3 and 23-3 were as highly homogenous as 100%; The homogeneity of loci AC-8, 18-8, 27-5 and rpoT were 97%, 94%, 97% and 85% respectively. Loci GTA-9, AC-9 and 6-7 showed significant allelic diversity in isolates and the diversity of GTA-9 in Mycobacterium leprae isolated from China was also different from those identified other countries. We had subjected loci GTA-9 and the ten loci to phylogenetic tree analysis respectively. CONCLUSION: The present study revealed that the genotype of Mycobacterium leprae identified from China was close to the strains from the Philippines and India although a few loci were somehow differentiate. Locus 12-5 manifested as only 3 copies in China whereas 4-5 copies predominating in other countries. 12-5 locus might serve as a useful marker to diffrentiate Chinese strains from those in other countries. However, further study on the diversity of GTA-9 was needed in China. The molecular typing of Mycobacterium leprae from different geographic areas might be useful in studying the transmission of leprosy.