Dataset Information

Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

ABSTRACT: Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.

SUBMITTER: Leung ET

PROVIDER: S-EPMC3147870 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

Leung Eric T Y ET Zheng L L Wong Rity Y K RY Chan Edward W C EW Au T K TK Chan Raphael C Y RC Lui Grace G Lee Nelson N Ip Margaret M

Journal of clinical microbiology 20110518 7

Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hyperv ...[more]

PMID: 21593264

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Project description:The Beijing genotype is a lineage of Mycobacterium tuberculosis that is distributed worldwide and responsible for large epidemics, associated with multidrug-resistance. However, its distribution in Africa is less understood due to the lack of data. Our aim was to investigate the prevalence and possible transmission of Beijing strains in Mozambique by a multivariate analysis of genotypic, geographic and demographic data. A total of 543 M. tuberculosis isolates from Mozambique were spoligotyped. Of these, 33 were of the Beijing lineage. The genetic relationship between the Beijing isolates were studied by identification of genomic deletions within some Regions of Difference (RD), Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetivie Unit - variable number tandem repeat (MIRU-VNTR). Beijing strains from South Africa, representing different sublineages were included as reference strains. The association between Beijing genotype, Human Immunodeficiency Virus (HIV) serology and baseline demographic data was investigated. HIV positive serostatus was significantly (p=0.023) more common in patients with Beijing strains than in patients with non-Beijing strains in a multivariable analysis adjusted for age, sex and province (14 (10.9%) of the 129 HIV positive patients had Beijing strains while 6/141 (4.3%) of HIV negative patients had Beijing strains). The majority of Beijing strains were found in the Southern region of Mozambique, particularly in Maputo City (17%). Only one Beijing strain was drug resistant (multi-drug resistant). By combined use of RD and spoligotyping, three genetic sublineages could be tentatively identified where a distinct group of four isolates had deletion of RD150, a signature of the "sublineage 7" recently emerging in South Africa. The same group was very similar to South African "sublineage 7" by RFLP and MIRU-VNTR, suggesting that this sublineage could have been recently introduced in Mozambique from South Africa, in association with HIV infection.

Project description:BackgroundThe most prevalent strains of Mycobacterium tuberculosis (M.tb) in Beijing belong to the Beijing genotype family. The influence of Beijing genotype prevalence on the development of drug resistance, and the association of infection with Beijing genotype M.tb with population characteristics, in Beijing, however, are still unclear.MethodsIn this retrospective study, 1189 isolates were subjected to drug susceptibility testing (DST) and molecular epidemiological analysis, and differences in the percentage of drug resistance between Beijing and non-Beijing genotype strains were compared. The association between the occurrence of drug resistance and the prevalence of Beijing genotype M.tb was analyzed using statistical methods.ResultsThe Beijing genotype family was the dominant genotype (83.3%) among the 1189 M.tb isolates. Beijing genotype M.tb strains were more likely to spread among males [p = 0.018, OR (95% CI):1.127(1.004-1.264)] and people in the 45-64 age group [p = 0.016, OR (95% CI): 1.438 (1.027-2.015)]. On the contrary, non-Beijing genotype M.tb strains were more probably disseminated among the over 65 [p = 0.005, OR (95% CI):0.653 (0.474-0.9)] and non-resident population [p = 0.035, OR (95% CI):1.185(0.985-1.427)]. DST results showed that 849 (71.4%) strains were fully sensitive to first-line drugs, while 340 (28.6%) strains were resistant to at least one drug, and 9% (107/1189) were MDR-TB. The frequency of INH-resistance among Beijing genotype strains was significantly lower than that among non-Beijing genotype strains (p = 0.032). In addition, the Beijing genotype family readily formed clusters.ConclusionsOur findings indicate that male and middle-aged people were more probably be infected by Beijing genotype M.tb, older people and non-residents were more probably be infected by non-Beijing genotype M.tb. The high percentage of resistance to INH occurring in non-Beijing genotype strains suggested that non-Beijing genotype strains should be given much more interest in Beijing.

Project description:BackgroundDrug resistant (DR) and multi-drug resistant (MDR) tuberculosis (TB) is increasing worldwide. In some parts of the world 10% or more of new TB cases are MDR. The Beijing genotype is a distinct genetic lineage of Mycobacterium tuberculosis, which is distributed worldwide, and has caused large outbreaks of MDR-TB. It has been proposed that certain lineages of M. tuberculosis, such as the Beijing lineage, may have specific adaptive advantages. We have investigated the presence and transmission of DR Beijing strains in the Swedish population.Methodology/principal findingsAll DR M. tuberculosis complex isolates between 1994 and 2008 were studied. Isolates that were of Beijing genotype were investigated for specific resistance mutations and phylogenetic markers. Seventy (13%) of 536 DR strains were of Beijing genotype. The majority of the patients with Beijing strains were foreign born, and their country of origin reflects the countries where the Beijing genotype is most prevalent. Multidrug-resistance was significantly more common in Beijing strains than in non-Beijing strains. There was a correlation between the Beijing genotype and specific resistance mutations in the katG gene, the mabA-inhA-promotor and the rpoB gene. By a combined use of RD deletions, spoligotyping, IS1547, mutT gene polymorphism and Rv3135 gene analysis the Beijing strains could be divided into 11 genomic sublineages. Of the patients with Beijing strains 28 (41%) were found in altogether 10 clusters (2-5 per cluster), as defined by RFLP IS6110, while 52% of the patients with non-Beijing strains were in clusters. By 24 loci MIRU-VNTR 31 (45%) of the patients with Beijing strains were found in altogether 7 clusters (2-11 per cluster). Contact tracing established possible epidemiological linkage between only two patients with Beijing strains.Conclusions/significanceAlthough extensive outbreaks with non-Beijing TB strains have occurred in Sweden, Beijing strains have not taken hold, in spite of the proximity to high prevalence countries such as Russia and the Baltic countries. The Beijing sublineages so far introduced in Sweden may not be adapted to spread in the Scandinavian population.

Dataset Information

Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

Publications

Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

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