OBJECTIVETo evaluate the application of different variable number tandem repeats (VNTR) locus in genotyping of Mycobacterium tuberculosis (M.tuberculosis) strains isolated from eight provinces in China, and to find the suitable locus-set of VNTR for epidemical strains in China.

METHODSAll 140 M.tuberculosis strains were randomly selected from 2800 M.tuberculosis strains isolated from eight provinces in China, 27 VNTR loci were used for typing all isolates. Discriminatory power (Hunter-Gaston Index, HGI) of every locus and different locus-set were analyzed by BioNumerics software. Meanwhile, Spoligotyping was used to identify Beijing family and non-Beijing family. Then the HGI of different locus-sets in two families was also evaluated.

RESULTSAll 140 isolates were clustered into Beijing kindred (112 strains, 80%) and non-Beijing kindred (28 strains, 20%) by Spoligotyping. The discriminatory power of Spoligotyping in 140 isolates was 0.4589. Every locus showed different polymorphism and HGI were from 0 to 0.809. The number of VNTR loci with HGI higher than 0.5 in all strains, Beijing family and non-Beijing family was 8, 7 and 14 respectively. 27 loci were combined into four groups which included 8, 12, 15 and 24 VNTR loci respectively. Four locus-sets showed different polymorphism, HGI of eight-locus, 12-locus, 15-locus, and 24-locus set in 140 strains was 0.9991, 0.9882, 0.9980 and 0.9986, and their discriminatory power were calculated in Beijing kindred (HGI: 0.9987, 0.9318, 0.9969 and 0.9975) and non-Beijing kindred (HGI: 1, 0.9894, 1 and 1).

CONCLUSIONDifferent VNTR locus and locus-set showed different discriminatory power in the selected M.tuberculosis strains isolated from China. Eight-locus set can be used in molecular epidemiological study of M.tuberculosis in China after standardization.

Bacterial Typing Techniques ; DNA, Bacterial ; genetics ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Tandem Repeat Sequences

PURPOSE: The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guerin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health. MATERIALS AND METHODS: A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates. RESULTS: All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level. CONCLUSION: The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.
Animals ; Cattle ; Classification/methods ; DNA Primers ; Genes, Bacterial ; Humans ; Multiplex Polymerase Chain Reaction/*methods ; Mycobacterium/classification/genetics/*isolation & purification ; Mycobacterium tuberculosis/classification/genetics/*isolation & purification ; Species Specificity

OBJECTIVETo identify the novel species 'Mycobacterium fukienense' sp. nov of Mycobacterium chelonae/abscessus complex from tuberculosis patients in Fujian Province, China.

METHODSFive of 27 clinical Mycobacterium isolates (Cls) were previously identified as M. chelonae/abscessus complex by sequencing the hsp65, rpoB, 16S-23S rRNA internal transcribed spacer region (its), recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobacterium. Clinical Mycobacterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed.

RESULTSThe 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 16S-23S rRNA internal transcribed spacer region (its), sodA, and recA genes as compared with the M. abscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences.

CONCLUSIONThe novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. chelonea/abscessus complex.

Bacterial Proteins ; genetics ; Base Sequence ; China ; epidemiology ; Cluster Analysis ; DNA, Bacterial ; genetics ; Humans ; Molecular Sequence Data ; Mycobacterium ; classification ; genetics ; isolation & purification ; Mycobacterium Infections, Nontuberculous ; epidemiology ; microbiology ; Mycobacterium chelonae ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Tuberculosis ; epidemiology ; microbiology

PURPOSE: Polymerase chain reaction (PCR) assay, introduced as a fast and sensitive diagnostic method, is useful in detecting Mycobacterium tuberculosis. The purpose of this study was to evaluate the usefulness of in-house PCR assay in the detection of Mycobacterium tuberculosis by comparing PCR results with conventional diagnostic techniques and Cobas Amplicor M. tuberculosis(TM) kit. MATERIALS and METHODS: We retrospectively assessed the diagnostic yield of in-house PCR method employed for the amplification IS6110 sequences in 2,973 specimens. We also compared in-house PCR with Cobas Amplicor M. tuberculosis(TM) kit in 120 specimens collected from June to July 2006. Routine acid-fast stain (AFS) and culture assay were also performed and analyzed. RESULTS: Of 2,973 cases, 2,832 cases (95.3%) showed consistent results between in house PCR, AFS and culture methods, whereas 141 (4.7%) displayed inconsistent results. The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 77.5%, 99.7%, 95.5%, and 98.0%, respectively for PCR; 49.2%, 100%, 100%, and 95.7%, respectively, for AFS method; and 80.7%, 100%, 100%, and 98.3%, respectively, for culture assay. Consistent results between PCR and Cobas Amplicor M. tuberculosis(TM) kit were shown in 109 cases (90.8%). The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 81.3%, 98.9%, 96.3%, and 93.5% respectively for PCR and 71.9%, 100%, 100%, and 90.7%, respectively, for Cobas Amplicor(TM) kit. CONCLUSION: In-house PCR and Cobas Amplicor(TM) kit show high sensitivity and specificity, and are reliable tests in the diagnosis of tuberculosis.
Humans ; Mycobacterium tuberculosis/*genetics/*isolation & purification ; Polymerase Chain Reaction/*methods ; Sensitivity and Specificity

Mycobacterium lentiflavum has recently been described as an emerging human pathogen without regard to the immune status of the host. We herein report on M. lentiflavum isolated from a respiratory specimen of a patient. Although the organism described in this case seems to be a colonizer of a lung destroyed by tuberculosis, the current methods for species identification of nontuberculous mycobacteria have to be re-evaluated so as not to underestimate these organisms.
Aged ; Humans ; Male ; Mycobacterium/genetics/*isolation & purification ; Tomography, X-Ray Computed ; Tuberculosis, Pulmonary/diagnosis/*microbiology/radiography

Bacterial Typing Techniques ; China ; Humans ; Interspersed Repetitive Sequences ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Ribotyping

OBJECTIVETo prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).

METHODSThe CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA.

RESULTSCFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability.

CONCLUSIONThe reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.

Bacterial Proteins ; genetics ; standards ; Fluoroimmunoassay ; methods ; Gene Amplification ; Mycobacterium tuberculosis ; isolation & purification ; Reference Standards

Cryoelectron Microscopy ; Mycobacterium smegmatis ; genetics ; ultrastructure ; RNA, Ribosomal ; ultrastructure ; Ribosomal Proteins ; genetics ; isolation & purification ; Ribosomes ; genetics ; ultrastructure

OBJECTIVETo access the application of spacer oligotyping (Spoligotyping) and Multiple Locus VNTR(MLVA) in epidemiological studies of Mycobacterium tuberculosis.

METHODS224 clinical isolates of M. tuberculosis were collected and typed by Spoligotyping and MLVA respectively, to compare the results of both methods and to access their application in epidemiological studies of M. tuberculosis.

RESULTSData from Spoligotyping showed that 224 strains presented 55 kinds of genotypes. Of these, 39 were represented by a unique isolate, with the remaining 185 isolates being grouped in 16 clusters whereas the result of MLVA showed that 224 strains presenting 160 kinds of genotypes. Of these, 132 were represented by a unique isolate, with the remaining 92 isolates being grouped in 28 groups. Data from the combination of Spoligotyping and VNTR showed that 224 strains presenting 179 kinds of genotypes. Of these, 159 were represented by a unique isolate, with the remaining 65 isolates being grouped in 20 groups. There was significant difference noticed among M. tuberculosis between Hunan and Anhui in the proportion of Beijing family (P < 0.001). The proportion of Beijing family in Anhui was higher than that in Hunan.

CONCLUSIONResults from this direct comparison studies demonstrated that MLVA analysis was more effective than Spoligotyping in discriminating individual M. tuberculosis isolates. However, Spoligotyping had an advantage over MLVA in identifying Beijing family strains and M. bovis. Taking Spoligotyping as a first-line typing technique and VNTR as second-line typing technique, the arrangement would improve the effectiveness of epidemiological investigation and pathological inspection of tuberculosis. The strains in different regions seemed to have had different characteristics.

China ; epidemiology ; Genotype ; Humans ; Mycobacterium bovis ; genetics ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Oligonucleotide Array Sequence Analysis ; Tandem Repeat Sequences ; Tuberculosis ; epidemiology

Forty mycobacterial strains comprising clinical Indian isolates of Mycobacterium tuberculosis (28 field isolates +1H37 Rv) and Mycobacterium bovis (10 field isolates +1 AN5) were subjected to restriction fragment length polymorphism analysis (RFLP) using IS6110 and IS1081 probes. Most of these strains originated from dairy cattle herd and human patients from Indian Veterinary research Institute (IVRI) campus isolated from the period of 1986 to 2000. Our study showed presence of 8 copies of IS6110 in most of the M.tuberculosis (96.6%) strains irrespective of their origin with the exception of one M.tuberculosis strain with presence of an extra copy (3.4%). All M.bovis strains showed a single copy of IS6110 on the characteristic 1.9kb restriction fragment. RFLP analysis with IS1081 invariably showed the presence of 5 copies in all isolates of M.bovis and M.tuberculosis at the same chromosomal location. Similarity of IS6110 RFLP fingerprints of M.tuberculosis strains from animals and human suggested the possibility of dissemination of single M.tuberculosis strain among animals as well as human. It was not possible to discriminate within the isolates of either M.tuberculosis or M.bovis, when IS1081 was used as target sequence. The IS6110 RFLP is a valuable tool for disclosing transmission chain of M. tuberculosis and M. bovis among humans as well as animals
Animals ; Bacterial Typing Techniques ; Cattle ; DNA Fingerprinting/*veterinary ; DNA, Bacterial/*genetics ; Deer ; Humans ; India/epidemiology ; Mycobacterium bovis/classification/*genetics/isolation&purification ; Mycobacterium tuberculosis/classification/*genetics/isolation & purification ; Polymerase Chain Reaction/veterinary ; Polymorphism, Restriction Fragment Length ; Zoonoses/epidemiology