Comparative evaluation of polymerase chain reaction and restriction enzyme analysis: Two amplified targets, hsp65 and rpoB, for identification of cultured mycobacteria

Cheunoy, Wattana; Prammananan, Therdsak; Chaiprasert, Angkana; Foongladda, Suporn

doi:10.1016/j.diagmicrobio.2004.09.006

Cited by 17 publications

(10 citation statements)

References 27 publications

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“…Mycobacterial DNAs were extracted by the boiling method [27]. Briefly, one loopful of M. tuberculosis colonies obtained from LJ medium was suspended in 200 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and boiled for 20 minutes.…”

Section: Methodsmentioning

confidence: 99%

Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

et al. 2010

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BackgroundSusceptibility testing of pyrazinamide (PZA) against Mycobacterium tuberculosis is difficult to perform because the acidity of culture medium that is required for drug activity also inhibits the growth of bacteria. In Thailand, very limited information has been generated on PZA resistance, particularly among multidrug-resistant tuberculosis (MDR-TB) isolated from Thailand. Only two studies on PZA susceptibility among Thai M. tuberculosis strains have been reported; one used a pyrazinamidase assay, and the other used the BACTEC 460 TB for PZA susceptibility testing. In this study, we determined the percentage of strains possessing pyrazinamide resistance among pan-susceptible M. tuberculosis and MDR-TB isolates by using the pyrazinamidase assay, BACTEC MGIT 960 PZA method and pncA sequencing, and assessed the correlation in the data generated using these methods. The type and frequency of mutations in pncA were also determined.ResultsOverall, 150 M. tuberculosis isolates, consisting of 50 susceptible and 100 MDR-TB isolates, were tested for PZA susceptibility by BACTEC MGIT 960 PZA, the pyrazinamidase assay and pncA sequencing. The study indicated PZA resistance in 6% and 49% of susceptible and MDR-TB isolates, respectively. In comparison to the BACTEC MGIT 960 PZA, the PZase assay showed 65.4% sensitivity and 100% specificity, whereas pncA sequencing showed 75% sensitivity and 89.8% specificity. Twenty-four mutation types were found in this study, with the most frequent mutation (16%) being His71Asp. Of these mutations, eight have not been previously described. The Ile31Ser and Ile31Thr mutations were found both in PZA susceptible and resistant isolates, suggesting that mutation of this codon might not play a role on PZA resistance.ConclusionsOur findings suggest that phenotypic susceptibility testing is still essential for the detection of PZA resistance, especially for MDR-TB isolates. Some mutations were not associated with resistance and could lead to misinterpretation of the genotypic methods. This information could be helpful for clinicians in managing tuberculosis patients and frequencies, and the types of pncA mutations should offer baseline information on PZA resistance.

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Section: Methodsmentioning

confidence: 99%

Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

et al. 2010

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“…For example, phylogenetic analysis of cloned and sequenced PCR-amplified Legionella 16S rRNA genes revealed a large diversity of uncultured Legionella spp., including L. bozemanii , L. worsleiensis , L. quateirensis, L. waltersii , and L. pneumophila in water from water treatment plants (Wullings and van der Kooij, 2006). Some studies used endonuclease digestion of PCR products for mycobacterial isolate identification (Cheunoy et al, 2005; Chimara et al, 2008). However, this technique requires establishment of restriction patterns for a collection of known mycobacterial species.…”

Section: Detection Methodsmentioning

confidence: 99%

Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A review

et al. 2017

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Opportunistic pathogens inhabiting premise (i.e., building) plumbing (OPPPs, e.g., L. pneumophila, M. avium complex, P. aeruginosa, Acanthamoeba, and N. fowleri) are a significant and growing source of disease. Because OPPPs establish and grow as part of the native drinking water microbiota, they do not correspond to fecal indicators, presenting a significant challenge to common and effective monitoring strategies. Further, different OPPPs present distinct requirements for sampling, preservation, and analysis, creating a significant impediment to their parallel detection. The aim of this critical review is to synthesize the state of the science of monitoring OPPPs and to identify a path forward for their simultaneous detection and quantification in a manner commensurate with the need for reliable data to inform risk assessment and mitigation. Water and biofilm sampling procedures, as well as factors influencing sample representativeness and detection sensitivity, are critically evaluated with respect to the five representative bacterial and amoebal OPPPs noted above. Available culturing and molecular approaches are discussed in terms of their advantages, limitations, and applicability. Knowledge gaps and research needs are identified.

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“…Based on the gene encoding the 65-kDa heat shock protein (hsp65), MAC can be identified and differentiated into species and types using PCR-restriction enzyme analysis (REA) (1,2,14). The present study demonstrates the usefulness of this method for rapidly differentiating MAC into M. avium types I, II, and III and M. intracellulare types I, II, III, and IV, providing useful epidemiological data.…”

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Distribution of hsp65 PCR-Restriction Enzyme Analysis Patterns among Mycobacterium avium Complex Isolates in Thailand

et al. 2006

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A total of 227 clinical Mycobacterium avium complex isolates from Thailand were differentiated into species and types by using PCR-restriction enzyme analysis of hsp65. The distribution of types showed the predominance of M. avium I (77%) in blood specimens, whereas M. intracellulare I was more commonly found in pulmonary specimens (44.2%). In addition, infections with M. avium were more likely to be found in younger adults (20 to 39 years old), while infections with M. intracellulare were more likely to be found in older adults (>60 years old). Our results provide the useful epidemiological information that some particular types have more invasive and virulent characters than others.The Mycobacterium avium complex (MAC) consists of two closely related species, M. avium and M. intracellulare, which are found widely in the environment, both in soil and water, and cause diseases in humans and animals (7). Both are capable of infecting diverse species, including birds, pigs, and humans, with consequences ranging from asymptomatic infection to clinically significant and even fatal disease. MAC is clinically important, as it is a frequent cause of disseminated disease and death in AIDS patients (5,8). Clinically as well as genetically significant differences between M. avium and M. intracellulare have been shown. Mycobacterium avium is the most common MAC species isolated from AIDS patients and is also a pathogenic bacterium isolated from animals, whereas M. intracellulare is more frequently isolated from immunocompetent patients, especially from individuals with pulmonary illnesses (4, 9).Based on the gene encoding the 65-kDa heat shock protein (hsp65), MAC can be identified and differentiated into species and types using PCR-restriction enzyme analysis (REA) (1, 2, 14). The present study demonstrates the usefulness of this method for rapidly differentiating MAC into M. avium types I, II, and III and M. intracellulare types I, II, III, and IV, providing useful epidemiological data.Two mycobacterial reference strains, M. avium ATCC 25291 and M. intracellulare ATCC 13950, and 227 clinical isolates of M. avium complex bacteria from different patients, identified by the biochemical method, were submitted for molecular identification by using hsp65 PCR-REA in the Molecular Mycology and Mycobacteriology Laboratory, Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, during 2003 and 2004. A total of 189 patients provided complete information on their sexes and ages; this group of patients consisted of 106 males and 83 females, with a mean age of 42.2 (range, 4 to 87) years. Clinical data about the human immunodeficiency virus (HIV) status of patients in this study were not completely available; only 67 (29.5%; 37 male and 30 female) of 227 patients confirmed that they were HIV ϩ patients. Genomic DNA was extracted by a boiling technique and used as a template for the hsp65 PCR-REA as previously described (1).Mycobacterium avium ATCC 25291 and M. intracellulare ATCC 13950 were identified a...

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Comparative evaluation of polymerase chain reaction and restriction enzyme analysis: Two amplified targets, hsp65 and rpoB, for identification of cultured mycobacteria

Cited by 17 publications

References 27 publications

Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A review

Distribution of hsp65 PCR-Restriction Enzyme Analysis Patterns among Mycobacterium avium Complex Isolates in Thailand

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