Reliable identification of mycobacterial species by PCR-restriction enzyme analysis (PRA)-hsp65 in a reference laboratory and elaboration of a sequence-based extended algorithm of PRA-hsp65patterns

Chimara, Érica; Ferrazoli, Lucilaine; Ueky, Suely Yoko Misuka; Martins, Maria Conceição; Durham, Alan Mitchell; Arbeit, Robert D.; Leão, Sylvia Cardoso

doi:10.1186/1471-2180-8-48

Cited by 105 publications

(97 citation statements)

References 41 publications

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“…In Brazil, the PRA-hsp65 technique proposed by Telenti et al 13 , and Devallois et al 14 has enabled the identifi cation of several species of NTM, the results of which correlated well with those from biochemical identification [13][14][15][16][17] . Beyond the molecular techniques, the sequencing of the specifi c genes rpoB and hsp65 has become the gold standard for identifying mycobacteria 18,19 .…”

Section: Methodsmentioning

confidence: 79%

“…The polymerase chain reaction and restriction enzyme analysis (PRA-hsp65) were performed using the techniques described in Telenti et al 13 The identifi cation was determined by comparing the sizes of the fragments with the algorithm described in the PRASITE site (http://app.chuv.ch/prasite/ index.html) 17 .…”

Section: Polymerase Chain Reaction and Restriction Enzyme Analysismentioning

confidence: 99%

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Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Lima

Duarte

Montenegro

et al. 2013

Rev. Soc. Bras. Med. Trop.

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Introduction:The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods: The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) Results: Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identifi cation of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the fi nal diagnosis from the attending physician. Conclusions: Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifi able using conventional biochemical and phenotypic techniques.

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Section: Methodsmentioning

confidence: 79%

Section: Polymerase Chain Reaction and Restriction Enzyme Analysismentioning

confidence: 99%

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Lima

Duarte

Montenegro

et al. 2013

Rev. Soc. Bras. Med. Trop.

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“…Every positive culture was subjected to Ziehl-Neelsen staining to confirm the presence of acid-fast bacilli (AFB) and cord formation, and to exclude contamination. Mycobacterial isolates were identified by conventional phenotypic methods and by PRA-hsp65 (Chimara et al, 2008). Unprocessed sputum aliquots for nucleic acid extraction were stored at 220 uC and sent to São Paulo IAL.…”

Section: Methodsmentioning

confidence: 99%

Rapid detection of Mycobacterium tuberculosis complex by real-time PCR in sputum samples and its use in the routine diagnosis in a reference laboratory

Pinhata

Cergole-Novella²,

Carmo³

et al. 2015

Journal of Medical Microbiology

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Tuberculosis (TB) is an infectious disease of global distribution, constituting a serious public health problem in Brazil. Sã o Paulo State, located in the south-east of Brazil, notified 16 580 new TB cases in 2013. The Instituto Adolfo Lutz is a public health reference laboratory for TB diagnosis for all the State. Considering that rapid and accurate diagnosis is essential for TB control, the aim of this study was to evaluate the use of an in-house real-time (RT)-PCR assay targeting the mpt64 gene in the routine diagnosis of TB, and to compare this technique with smear microscopy and culture. From August 2012 to October 2013, 715 sputum samples from 657 patients were included in the study. Smear microscopy, culture, phenotypic and PRAhsp65 identification of mycobacteria, and mpt64 RT-PCR were performed. With respect to confirmed TB cases (n562/657; 9.4 %), smear microscopy had a sensitivity of 82.3 %. Culture and RT-PCR showed the same sensitivity, i.e. 90.3 %. Specificity was 99.7, 99.4 and 98.6 % for smear microscopy, culture and RT-PCR, respectively. mpt64 RT-PCR showed high sensitivity and specificity for the detection of Mycobacterium tuberculosis complex in sputum samples. This technique can be deployed in laboratories that do not have a rapid test for TB available, enabling the performance of TB diagnosis in up to 5 h.

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“…2,3 Hsp65 restriction digestion with BstEII and HaeIII enzymes has been used extensively for NTM identification in multiple fields of study, while gyrB digestion with RsaI and TaqIhas been successfully used for M. tuberculosis (MTB) /M.africanum differentiation from M.bovis/ BCG. [4][5][6][7][8][9][10][11][12][13] In Sri Lanka, NTM were cultured in approximately 2-3% of all mycobacterial cultures done at the National Tuberculosis Reference Laboratory between 2005 and 2007 14,15 while a recent analysis of bronchoscopy cultures from patients in Kandy by Weeresekera et al showed that -13-14 % were positive for NTM isolates, including M.phocaicumand M. Smegmatis. 16 Data on the true (overall) NTM infection rate among patients with pulmonary and extra pulmonary disease in Sri Lanka is not available as cultures are not routinely performed in suspected pulmonary tuberculosis.…”

Section: Introductionmentioning

confidence: 99%

Efficacy and Cost of Molecular Identification of Clinical Mycobacterial Isolates in a Resource Limited Setting

Ratnatunga

Wickramasingha

Thevanesam

et al. 2017

SAARC J. Tuber. Lung Dis. HIV/AIDS

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introduction: Many molecular methods of identification of mycobacteria are now available. With many molecular consumables now being available at low prices, routine identification in clinical laboratories is now possible, even in low/ middle income settings that have basic molecular facilities. This study was conducted to optimize a low cost PCR-RFLP based identification that can be used in such a laboratory.

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Reliable identification of mycobacterial species by PCR-restriction enzyme analysis (PRA)-hsp65 in a reference laboratory and elaboration of a sequence-based extended algorithm of PRA-hsp65patterns

Cited by 105 publications

References 41 publications

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Rapid detection of Mycobacterium tuberculosis complex by real-time PCR in sputum samples and its use in the routine diagnosis in a reference laboratory

Efficacy and Cost of Molecular Identification of Clinical Mycobacterial Isolates in a Resource Limited Setting

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