Evaluation of in-house mpt64 real-time PCR for rapid detection of Mycobacterium tuberculosis in pulmonary and extra-pulmonary specimens

Sethi, Sunil; Yadav, Rakesh; Mewara, Abhishek; Dhatwalia, Sunil Kumar; Sharma, Mukut; Gupta, Dheeraj

doi:10.1016/j.bjid.2012.08.010

Cited by 11 publications

(14 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The mpt64 (also called mpb64 or Rv1980c) gene sequence present in RD1 region, encoding an antigenic protein (205aa, 22Á4 kDa) is highly conserved in organisms of MTBC (Yamaguchi et al 1989;Kumar et al 2011). mpt64 is a well-known and well-characterized marker for detection of MTBC and its sensitivity and specificity has already been established in PCR and real-time PCR assays (Mitarai et al 1995;Bhanu et al 2005;Therese et al 2005;Sethi et al 2012). Hence, this study was aimed at developing a LAMP assay based on the mpt64 gene sequence (Table 1) which was evaluated with sputum samples of suspected pulmonary TB patients attending a tertiary care centre.…”

Section: Introductionsupporting

confidence: 93%

See 1 more Smart Citation

A loop-mediated isothermal amplification assay for the diagnosis of pulmonary tuberculosis

Sharma

Tewari

Dhatwalia

et al. 2019

Lett Appl Microbiol

View full text Add to dashboard Cite

Quantitated Mycobacterium tuberculosis (M.tb) H37Rv DNA was used to analyse the sensitivity and the specificity was assessed using DNA isolated from the reference strain H37Rv, 12 nontuberculous mycobacterium (NTM) species and five nonmycobacterium species. Furthermore, performance of the assay was evaluated on the sputum samples and compared with smear microscopy, culture and PCR. mpt64 (also called mpb64 or Rv1980c) loop‐mediated isothermal amplification (LAMP) successfully detected 1 pg DNA within 40 min and successfully rejected NTMs and other bacterial species tested. It specifically detected all the 119 confirmed TB cases and 100 of the 104 control cases. The resulting sensitivity and specificity of LAMP assay was found to be 100% (95% CI: 96·79–100%) and 96·15% (95% CI; 90·44–98·94%) respectively. Significance and Impact of the Study Loop‐mediated isothermal amplification (LAMP) is a technique for isothermal DNA amplification suitable for cost‐limited settings as it prevents the use of sophisticated instruments. Using mpt64 antigenic protein gene, we developed a LAMP assay especially for organisms of the M. tuberculosis complex. mpt64 LAMP assay showed 100% sensitivity and detected all the bacteriologically and clinically positive TB cases not detected by smear, culture or PCR methods.

show abstract

Section: Introductionsupporting

confidence: 93%

“…; Sethi et al . ). Hence, this study was aimed at developing a LAMP assay based on the mpt64 gene sequence (Table ) which was evaluated with sputum samples of suspected pulmonary TB patients attending a tertiary care centre.…”

Section: Introductionmentioning

confidence: 97%

A loop-mediated isothermal amplification assay for the diagnosis of pulmonary tuberculosis

Sharma

Tewari

Dhatwalia

et al. 2019

Lett Appl Microbiol

View full text Add to dashboard Cite

show abstract

“…1 ). Twenty-one [ 10 , 24 , 25 , 27 , 29 , 31 , 32 , 39 , 44 , 45 , 47 , 49 , 51 , 52 , 54 , 57 , 60 , 61 , 64 , 66 , 67 ] reported detection of pulmonary TB (PTB), fifteen [ 23 , 30 , 33 , 35 , 37 , 38 , 40 – 42 , 50 , 53 , 55 , 56 , 59 , 62 ] reported detection of extra-pulmonary TB (EPTB) and ten [ 26 , 28 , 34 , 36 , 43 , 46 , 48 , 58 , 63 , 65 ] reported on both types of pathological sample. Table 1 summarises the main characteristics of the included studies.…”

Section: Resultsmentioning

confidence: 99%

“…Smear microscopy is widely used in the diagnosis of TB but has drawbacks owing to low and variable sensitivity values (0–40%) and cannot readily differentiate between MTB and non-tuberculous mycobacteria (NTM) [ 70 – 72 ]. Culture identification for MTB also has variable sensitivities (0–80%) in different TB specimens [ 63 , 73 – 75 ] with turn-a-round time of 2–10 weeks requiring the use of skilful technicians [ 76 ]. Diagnosis of TB from tissue samples is usually made by histopathological examination that depends on the presence of granulomatous inflammation and caseous necrosis [ 70 , 77 ].…”

Section: Discussionmentioning

confidence: 99%

Effectiveness of real-time polymerase chain reaction assay for the detection of Mycobacterium tuberculosis in pathological samples: a systematic review and meta-analysis

et al. 2017

View full text Add to dashboard Cite

BackgroundRapid and accurate diagnosis of tuberculosis (TB) is key to manage the disease and to control and prevent its transmission. Many established diagnostic methods suffer from low sensitivity or delay of timely results and are inadequate for rapid detection of Mycobacterium tuberculosis (MTB) in pulmonary and extra-pulmonary clinical samples. This study examined whether a real-time polymerase chain reaction (RT-PCR) assay, with a turn-a-round time of 2 h, would prove effective for routine detection of MTB by clinical microbiology laboratories.MethodsA systematic literature search was performed for publications in any language on the detection of MTB in pathological samples by RT-PCR assay. The following sources were used MEDLINE via PubMed, EMBASE, BIOSIS Citation Index, Web of Science, SCOPUS, ISI Web of Knowledge and Cochrane Infectious Diseases Group Specialised Register, grey literature, World Health Organization and Centres for Disease Control and Prevention websites. Forty-six studies met set inclusion criteria. Generated pooled summary estimates (95% CIs) were calculated for overall accuracy and bivariate meta-regression model was used for meta-analysis.ResultsSummary estimates for pulmonary TB (31 studies) were as follows: sensitivity 0.82 (95% CI 0.81–0.83), specificity 0.99 (95% CI 0.99–0.99), positive likelihood ratio 43.00 (28.23–64.81), negative likelihood ratio 0.16 (0.12–0.20), diagnostic odds ratio 324.26 (95% CI 189.08–556.09) and area under curve 0.99. Summary estimates for extra-pulmonary TB (25 studies) were as follows: sensitivity 0.70 (95% CI 0.67–0.72), specificity 0.99 (95% CI 0.99–0.99), positive likelihood ratio 29.82 (17.86–49.78), negative likelihood ratio 0.33 (0.26–0.42), diagnostic odds ratio 125.20 (95% CI 65.75–238.36) and area under curve 0.96.ConclusionsRT-PCR assay demonstrated a high degree of sensitivity for pulmonary TB and good sensitivity for extra-pulmonary TB. It indicated a high degree of specificity for ruling in TB infection from sampling regimes. This was acceptable, but may better as a rule out add-on diagnostic test. RT-PCR assays demonstrate both a high degree of sensitivity in pulmonary samples and rapidity of detection of TB which is an important factor in achieving effective global control and for patient management in terms of initiating early and appropriate anti-tubercular therapy.Systematic review registrationPROSPERO CRD42015027534.Electronic supplementary materialThe online version of this article (10.1186/s13643-017-0608-2) contains supplementary material, which is available to authorized users.

show abstract

“…In a study by Sethi et al 35 , M. tuberculosis infections in extra-pulmonary specimens (22) were detected with a higher analytical sensitivity by an mpt64 reverse transcription-PCR assay when compared with the IS6110 PCR reaction (these sensitivities were 50% vs. 18%, respectively). In our study, we observed a greater sensitivity in our nPCR system approach, even though the IS6110 target was used.…”

mentioning

confidence: 97%

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

Montenegro

Silva

Lima

et al. 2013

Rev. Soc. Bras. Med. Trop.

View full text Add to dashboard Cite

Introduction: This study evaluated the performance of an in-house nested-PCR system for the detection of the Mycobacterium tuberculosis complex in pleural fl uid, blood and urine samples from pleural effusion tuberculosis patients by health services physicians in Pernambuco, Brazil. Methods: A prospective double-blind study with 37 hospitalized patients of both sexes, aged over 15, was used to investigate the diagnosis of pleural effusion. The criteria used to defi ne the cases included the demonstration of bacillus in biological samples by smear or culture or by a granulomatous fi nding in the histopathological examination, associated with an evident response to specifi c treatments to each clinical situation. Pleural fl uid, blood and urine samples were collected and subjected to routine tests and the nested PCR technique to assess for M. tuberculosis amplifi cation. Results: In total, 37 pleural effusion patients took part in the study, of whom 19 (51.3%) had tubercular etiologies and 18 (48.7%) had etiologies from other causes. When the pleural fl uid, blood and/or urine sample in-house nested-PCR sensitivities were evaluated simultaneously, the results were positive regardless of the biological specimen (the sensitivity was 84.2%); however, when the blood and/or urine samples were analyzed together, the sensitivity was 72.2%. When the pleural fl uid samples were evaluated alone, the sensitivity was only 33.3%. Conclusions: The performance of the diagnostic pleural tuberculosis nested-PCR was directly related to the diversity of the samples collected from the same patient. Additionally, this study may identify a need to prioritize non-invasive blood and urine collection for this diagnosis.

show abstract

Evaluation of in-house mpt64 real-time PCR for rapid detection of Mycobacterium tuberculosis in pulmonary and extra-pulmonary specimens

Cited by 11 publications

References 5 publications

A loop-mediated isothermal amplification assay for the diagnosis of pulmonary tuberculosis

A loop-mediated isothermal amplification assay for the diagnosis of pulmonary tuberculosis

Effectiveness of real-time polymerase chain reaction assay for the detection of Mycobacterium tuberculosis in pathological samples: a systematic review and meta-analysis

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

Contact Info

Product

Resources

About