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1.
BMC Res Notes ; 4: 279, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21819571

RESUMO

BACKGROUND: Pleural tuberculosis (TB) diagnosis often requires invasive procedures such as pleural biopsy. The aim of this study was to evaluate the role of real-time polymerase chain reaction (PCR) for the IS6110 sequence of M. tuberculosis in pleural fluid specimens as a rapid and non-invasive test for pleural TB diagnosis. FINDINGS: For this cross-sectional study, 150 consecutive patients with pleural effusion diagnosed by chest radiography, who were referred for diagnostic thoracocentesis and pleural biopsy and met eligibility criteria, had a pleural fluid specimen submitted for real-time PCR testing. Overall, 98 patients had pleural TB and 52 had pleural effusion secondary to other disease. TB diagnosis was obtained using acid-fast bacilli (AFB) smear or culture for mycobacteria and/or histopathologic examination in 94 cases and by clinical findings in 4 cases. Sensitivity, specificity, positive and negative predictive values of PCR testing for pleural TB diagnosis were 42.8% (95% CI 38.4 - 44.8), 94.2% (95% CI 85.8 - 98.0), 93.3% (95% CI 83.6 - 97.7), and 48.5% (95% CI 44.2 - 50.4), respectively. The real-time PCR test improved TB detection from 30.6% to 42.9% when compared to AFB smear and culture methods performed on pleural fluid specimens, although the best sensitivity was achieved by combining the results of culture and histopathology of pleural tissue specimens. CONCLUSION: The real-time PCR test of pleural fluid specimens is a useful and non-invasive additional assay for fast diagnosis of pleural TB.

2.
J Virol Methods ; 177(1): 38-43, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21807028

RESUMO

Persistent infection with high-risk human papillomavirus (HR-HPV) has been associated with cervical cancer. Developing assays for the identification of these viral types is of great importance for monitoring patients and controlling strategies. The development of the MCHA (microplate colorimetric hybridization assay), a PCR-based method for identifying six of the most common HR-HPV types (HPV 16, 18, 31, 33, 39 and 45) is described. The MCHA combines the amplification with the GP5+/GP6+ consensus primers followed by PCR reverse hybridization with specific probes and detection through a colorimetric assay. The performance of the MCHA was evaluated using 108 DNA samples typed previously by the PapilloCheck(®). The agreement between both methods was 69.4% for HPV 16; 79.1% for HPV 45; 82.4% for HPV 18; 93.6% for HPV 31; 87.9% for HPV 33, and 17.6% for HPV 39. The assay had higher sensitivity than the Papillocheck(®), particularly for identifying HPV 16 and 18. The MCHA seemed to be sensitive and specific for the identification of the most prevalent HPV types in invasive cervical cancer, HPV 16, 18, 45, 33 and 31. It requires low-cost reagents and common laboratory apparatus.


Assuntos
Técnicas de Genotipagem/métodos , Hibridização de Ácido Nucleico/métodos , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Proteínas do Capsídeo/genética , Colorimetria , Feminino , Genótipo , Humanos , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/virologia , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/virologia
3.
Mem Inst Oswaldo Cruz ; 106(2): 194-9, 2011 03.
Artigo em Inglês | MEDLINE | ID: mdl-21537680

RESUMO

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2% (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85% and 98%, and 94% and 100%, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.


Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Colorimetria , DNA Bacteriano/análise , Humanos , Mycobacterium tuberculosis/genética , Sondas de Oligonucleotídeos/análise , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
4.
Mem. Inst. Oswaldo Cruz ; 106(2): 194-199, Mar. 2011. tab
Artigo em Inglês | LILACS | ID: lil-583945

RESUMO

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2 percent (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85 percent and 98 percent, and 94 percent and 100 percent, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.


Assuntos
Humanos , Mycobacterium tuberculosis , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Escarro , Tuberculose Pulmonar , Colorimetria , DNA Bacteriano , Mycobacterium tuberculosis , Sondas de Oligonucleotídeos , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
5.
Mem Inst Oswaldo Cruz ; 104(5): 710-4, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19820830

RESUMO

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC-->ACC (Ser-->Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100% of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , Farmacorresistência Bacteriana/genética , Isoniazida/farmacologia , Mutação/genética , Mycobacterium tuberculosis , Colorimetria/métodos , DNA Bacteriano/análise , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase
6.
Mem. Inst. Oswaldo Cruz ; 104(5): 710-714, Aug. 2009. ilus
Artigo em Inglês | LILACS | ID: lil-528078

RESUMO

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100 percent of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , Farmacorresistência Bacteriana/genética , Isoniazida/farmacologia , Mycobacterium tuberculosis , Mutação/genética , Colorimetria/métodos , DNA Bacteriano/análise , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase
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