Rapid diagnosis of tuberculous meningitis by a simplified nested amplification protocol

Liu, Peter Yuk-Fong; Shi, Zhi-Yuan; Lau, Yeu-Jun; Hu, Bor-Shen

doi:10.1212/wnl.44.6.1161

Cited by 62 publications

(67 citation statements)

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“…Recently, in the diagnosis of tuberculous meningitis (TBM), the detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid (CSF) samples using PCR has been widely used as a more rapid, sensitive, and specific diagnostic method than the conventional bacteriological detection methods, such as direct smear for acid-fast bacilli (AFB) and culture for M. tuberculosis (7,8,12,17,18). Use of the nested PCR assay has provided a remarkable increase in sensitivity and specificity of DNA amplification compared to the conventional single-step PCR assay (8,12,17,20).…”

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Novel Technique of Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA

Takahashi

Nakayama

2006

J Clin Microbiol

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The diagnosis of tuberculous meningitis (TBM) remains a complex issue because the most widely used conventional diagnostic tools, such as culture and PCR assay for cerebrospinal fluid (CSF) samples, are unable to rapidly detect Mycobacterium tuberculosis with sufficient sensitivity in the acute phase of TBM. Based on TaqMan PCR, we designed a novel technique consisting of an internally controlled quantitative nested real-time (QNRT) PCR assay that provided a marked improvement in detection sensitivity and quantification. We applied this novel technique to quantitatively detect M. tuberculosis DNA in CSF samples from patients with suspected TBM. For use as the internal control in the measurement of the M. tuberculosis DNA copy numbers in the QNRT-PCR assay, the original mutation (M) plasmid, which included an artificial random 22-nucleotide sequence within an inserted DNA fragment of the MPB64 gene of M. tuberculosis, was prepared. The QNRT-PCR assay showed high sensitivity and specificity that were approximately equivalent to those of the conventional nested PCR assay. Moreover, the QNRT-PCR assay made it possible to precisely and quantitatively detect the initial copy number of M. tuberculosis DNA in CSF samples. Therefore, compared to the conventional PCR assay, the QNRT-PCR assay can be considered a more useful and advanced technique for the rapid and accurate diagnosis of TBM. To establish the superiority of this novel technique in TBM diagnosis, it will be necessary to accumulate data from a larger number of patients with suspected TBM.

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confidence: 99%

Novel Technique of Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA

Takahashi

Nakayama

2006

J Clin Microbiol

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“…The sensitivity of these molecular methods has been reported to vary between 33% 15 and 90.5% 13 , and therefore the role of PCR in routine diagnosis remains unclear 4 , especially in resource-poor settings. Here we have examined the performance of two PCR-based methods, a commercially available kit (TB AMPLICOR, Roche Diagnostic Systems) and a nested PCR described by Liu and colleagues 13 , in the diagnosis of tuberculous meningitis in a tertiary care hospital in southeastern Brazil.…”

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confidence: 99%

“…The third CSF aliquot (200-500ml) was sent to the Laboratory of Molecular Parasitology, at the Faculty of Medicine of São José do Rio Preto, for DNA extraction and amplification by a nested PCR protocol that targets the species-specific MPB 64 protein gene from M. tuberculosis 13 . CSF samples were stored at -20°C until DNA extraction.…”

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confidence: 99%

“…Two rounds of PCR amplification were performed in a Genius thermal cycler (Techne). The first round used the pair of external primers (here named MPB1 and MPB2) originally described by Shankar and colleagues 20 , while the second round used the internal primers (here named MPB3 and MPB4) designed by Liu and colleagues 13 . All primers were supplied by Gibco, and their sequences were given in the original publications.…”

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Low sensitivity of polymerase chain reaction for diagnosis of tuberculous meningitis in southeastern Brazil

Brienze

et al. 2001

Rev. Soc. Bras. Med. Trop.

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Two polymerase chain reaction (PCR) protocols showed low sensitivity (36% and 53% for TB AMPLICOR and MPB64 nested PCR, respectively), when compared with classic microbiological methods (73% and 54% for Ziehl-Neelsen staining and culture, respectively), in the diagnosis of tuberculous meningitis in 91 patients in southeastern Brazil. Only three PCR-positive, microbiologically negative patients were found. Analysis of sequential cerebrospinal fluid samples by nested PCR detected Mycobacterium tuberculosis DNA up to 29 days after the introduction of antituberculosis chemotherapy. Key-words: Diagnosis of tuberculous meningitis. PCR. Mycobacterium tuberculosis.Resumo Dois protocolos de reação em cadeia da polimerase (PCR) apresentaram baixa sensibilidade (36% e 53%, respectivamente), para TB AMPLICOR e PCR aninhado baseado no gene MPB64), quando comparados aos métodos microbiológicos clássicos (73% e 54% respectivamente para baciloscopia e cultura), no diagnóstico de meningite tuberculosa em 91 pacientes do sudeste do Brasil. Somente três pacientes apresentaram PCR positiva e microbiologia negativa. A análise de amostras seqüenciais de líquor com a PCR aninhada detectou DNA de Mycobacterium tuberculosis até 29 dias após a introdução de tratamento. Palavras-chaves: Diagnóstico da meningite tuberculosa. PCR. Mycobacterium tuberculosis.

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“…So far, results for a number of organisms have shown that PCR increases diagnostic sensitivity, specificity, and speed [13][14][15][16][17] .…”

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confidence: 99%

Polymerase chain reaction for the laboratory diagnosis of aseptic meningitis and encephalitis

Chesky

Scalco

Failace³

et al. 2000

Arq. Neuro-Psiquiatr.

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-A protocol for testing cerebrospinal fluid specimens using a range of PCR assays for the diagnosis of central nervous system infection was developed and used to test prospectively 383 specimens. PCR assays were used for the detection of adenovirus, Borrelia burgdorferi, enteroviruses, Epstein Barr virus, cytomegalovirus, herpes simplex virus, human herpes virus type 6, JC virus, Leptospira interrogans, Listeria monocytogenes, lymphocytic choriomeningitis virus, measles virus, mumps virus, Mycobacterium sp., Mycoplasma pneumoniae, Toxoplasma gondii and varicella zoster virus. Of the 383 specimens tested in this study, 46 (12.0%) were found to be positive. The microorganisms detected were CMV, enterovirus, Epstein Barr virus, herpes simplex virus, human herpes virus type 6, JC virus, L. monocytogenes, Mycobacterium genus, Toxoplasma gondii and varicella zoster virus. The introduction of the PCR protocol described has improved the diagnosis of a range of central nervous system infections in our laboratory. We believe however that further evaluation of these assays in immunocompromised patients is necessary to better determine the predictive value of positive PCR results in these patient groups.KEY WORDS: polymerase chain reaction (PCR), cerebrospinal fluid (CSF), central nervous system (CNS), lymphocytic meningitis, encephalitis. Reação em cadeia da polimerase no diagnóstico laboratorial das meningites e encefalites assépticasRESUMO -Foi desenvolvido um protocolo de PCR para analisar os principais patógenos que infectam o sistema nervoso central (SNC). Foram testadas 383 amostras de líquido-cefalorraquidiano (LCR), para detecção de: adenovirus, Borrelia burgdorferi, enterovirus, vírus Epstein Barr , citomegalovírus, vírus herpes simplex, herpes humano tipo 6, vírus JC, Leptospira interrogans, Listeria monocytogenes, vírus da coriomeningite linfocitária, vírus do sarampo, vírus da caxumba, Mycobacterium sp, Mycoplasma pneumoniae, Toxoplasma gondii e vírus da varicela zoster. Das 383 amostras de LCR testadas a PCR foi positiva em 46 (12,0%). Os microrganismos detectados foram: citomegalovírus, enterovirus, vírus Epstein Barr , herpes simplex, herpes humanao tipo 6, vírus JC, Listeria monocytogenes, Mycobacterium sp, Toxoplasma gondii e varicela zoster. A introdução da técnica de PCR através de um protocolo para análise do LCR otimizou o diagnóstico etiológico das infecções do SNC e tem se revelado um instrumento de grande potencial diagnóstico. Estudos futuros necessitam ser realizados para melhor avaliar os resultados da PCR em pacientes imunocomprometidos e assim determinar o valor preditivo positivo deste ensaio neste grupo de pacientes. PALAVRAS-CHAVE: reação em cadeia da polimerase (PCR), líquido cefalorraquidiano (LCR), sistema nervoso central (SNC), meningite asséptica, encefalite.

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Rapid diagnosis of tuberculous meningitis by a simplified nested amplification protocol

Cited by 62 publications

References 0 publications

Novel Technique of Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA

Novel Technique of Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA

Low sensitivity of polymerase chain reaction for diagnosis of tuberculous meningitis in southeastern Brazil

Polymerase chain reaction for the laboratory diagnosis of aseptic meningitis and encephalitis

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