McFarland nephelometer as a simple method to estimate the sensitivity of the polymerase chain reaction using Mycobacterium tuberculosis as a research tool

Bollela, Valdes Roberto; Sato, Daisy Nakamura; Fonseca, Benedito Antônio Lopes da

doi:10.1590/s0100-879x1999000900003

Cited by 31 publications

(23 citation statements)

References 11 publications

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“…Moreover, M. tuberculosis detection by molecular methods may also play a role in laboratory safety and, therefore, in laboratory costs, since after the initial extraction procedure, only non-infectious materials are handled 15 . PCR is expected to be more specific and sensitive than the routine procedure for diagnosis, but it is also more costly 1 . Cost-effectiveness comparisons of PCR versus smear examination showed no advantages of the former for the diagnosis of tuberculosis 12 .…”

Section: Discussionmentioning

confidence: 99%

Nested-PCR using MPB64 fragment improves the diagnosis of pleural and meningeal tuberculosis

Martins

Paschoal

Nowakonski

et al. 2000

Rev. Soc. Bras. Med. Trop.

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Fluids in which Mycobacterium tuberculosis are seldom found, such as pleural and cerebrospinal liquids, are good candidates to be studied using PCR techniques. We detail our experience with a PCR assay applied to pleural and cerebrospinal fluids using the primer MPB64. Seventy three specimens were analyzed: 30 pleural fluids (PF), 26 pleural biopsies (PB) and 17 cerebrospinal fluids (CSF). The gold standard for the diagnosis of tuberculous meningitis was the positive culture for M. tuberculosis in CSF. Tuberculous pleural effusion was diagnosed when cultures of PF and/or PB were positive for M. tuberculosis, or the PB histology showed granulomas. Our results, compared to the gold standards employed, showed a sensitivity of 70%, specificity of 88%, positive predictive value of 82% and negative predictive value of 80%. The high specificity of the MPB64 fragment while still retaining a good sensitivity makes it very well suited for pleural and cerebrospinal tuberculosis diagnosis. Key-words: Tuberculosis. Diagnosis. PCR technique. Central nervous system. Pleura.Resumo O Mycobacterium tuberculosis é raramente encontrado em fluidos como o líquido pleural e o cérebroespinhal, tornando estas localizações de difícil diagnóstico. Apresentamos nossa experiência com uma técnica de PCR aplicada a líquido pleural e cerebroespinhal com o uso do primer MPB64. Sessenta e três espécimes foram analisados: 30 líquidos pleurais (PF), 26 biópsias pleurais (PB) e 17 líquidos cerebroespinhais (CSF). O gold standard para o diagnóstico de meningite tuberculosa foi a cultura positiva para M. tuberculosis no CSF. Tuberculose pleural era diagnosticada quando culturas do PF e/ou PB eram positivas para M. tuberculosis, ou a histologia da PB mostrava granulomas. Nossos resultados, comparados aos gold standards empregados, mostram sensitividade de 70%, especificidade de 88%, valor preditivo positivo de 82% e valor preditivo negativo de 80%. A elevada especificidade e boa sensibilidade do fragmento MPB64 o transformam em um bom parâmetro para o diagnóstico de tuberculose pleural e do líquido cérebroespinhal. Palavras-chaves: Tuberculose. Diagnóstico. Técnica de PCR. Sistema nervoso central. Pleura.

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Section: Discussionmentioning

confidence: 99%

Nested-PCR using MPB64 fragment improves the diagnosis of pleural and meningeal tuberculosis

Martins

Paschoal

Nowakonski

et al. 2000

Rev. Soc. Bras. Med. Trop.

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“…We verified the intrinsic sensitivity of strains in accordance with the protocol devised by Bollela et al (20) in order to establish the minimum concentration of bacilli that would be detectable by PCR with the various primers. Therefore, a suspension of Mtb H37Rv was prepared and adjusted to a 1.0 McFarland standard turbidity level (equivalent to 3 x 10 8 bacilli/mL).…”

Section: Methodsmentioning

confidence: 99%

Análise de diferentes primers utilizados na PCR visando ao diagnóstico da tuberculose no Estado do Amazonas

Ogusku¹,

Salem²

2004

J. bras. pneumol.

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Background: Various primers are being tested for the detection of Mycobacterium tuberculosis DNA. The accuracy of the polymerase chain reaction (PCR) depends on the target sequence used and whether the test will be performed in culture or in clinical specimens. Objectives:To identify DNA sequences, specifically those commonly reported as targets for diagnosis of tuberculosis (TB), in clinical samples of M. tuberculosis strains.Method: Eighty-one clinical samples from suspected TB patients were initially processed and submitted to bacilloscopy (smear) and culture, and PCR was performed with specific primers for the following targets: IS6110, 65 kDa, 38 kDa and MPB64.Results: Smear and culture results were negative in 24 samples, as was the PCR. The 19 samples testing smear positive, as well as the isolated strains, were 100% positive on PCR, with the exception of the 89.4% result from PCR with MPB64 primers. In the 38 smear negative and culture positive samples, PCR results were inconsistent. The primers specific for amplifying the 123 bp IS6110 fragment gave the highest positivity (92.1%), diagnostic agreement (0.9143), co-positivity (94.7%) and co-negativity (100%). Conclusion:The IS6110, 38 kDa, MPB64 and 65 kDa sequences were found in the genome of all M. tuberculosis strains isolated in patients from the state of Amazonas. The protocol for processing the clinical samples prior to PCR analysis and the specific primers used to amplify the 123bp IS6110 fragment showed a greater efficiency in diagnosing pulmonary (paucibacillary) tuberculosis in comparison to published data.

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“…Standard M. tuberculosis suspensions were prepared by growing the M. tuberculosis H37Rv laboratory strain (ATCC 27294) as per routine protocol to the turbidity of a 0.5 McFarland standard; the equivalent concentration of x 8 CFU/ml (Bollela et al, 1999, Global Laboratory Initiative, 2014, Murray and Baron, 2007. The optical turbidity was measured using the PhoenixSpec Nephelometer (BD Diagnostics, Franklin lakes, NJ, USA) against a standard curve.…”

Section: Preparation Of M Tuberculosis Suspensions For Laboratory Evmentioning

confidence: 99%

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis

Omar

Peters

Ismail

et al. 2015

Journal of Microbiological Methods

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Background: Modern molecular-based approaches for the detection of M. tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore® -Molecular Transport Medium (PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems. Conclusions: Collecting and transporting sputum from TB suspects in PS-MTM offers safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.

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McFarland nephelometer as a simple method to estimate the sensitivity of the polymerase chain reaction using Mycobacterium tuberculosis as a research tool

Cited by 31 publications

References 11 publications

Nested-PCR using MPB64 fragment improves the diagnosis of pleural and meningeal tuberculosis

Nested-PCR using MPB64 fragment improves the diagnosis of pleural and meningeal tuberculosis

Análise de diferentes primers utilizados na PCR visando ao diagnóstico da tuberculose no Estado do Amazonas

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis

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