Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens

Restrepo, Blanca I.; Gómez, Diana I.; Shipley, Gregory L.; McCormick, Joseph B.; Fisher‐Hoch, Susan P.

doi:10.1016/j.mimet.2006.03.016

Cited by 22 publications

(23 citation statements)

References 28 publications

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“…In addition, blood can contain PCR inhibiting factors. (29) Early diagnosis in patients under 15 years of age and the surveillance for infectious adults will result in greater detection of index cases (30) and have a positive impact on TB control programs.…”

Section: Discussionmentioning

confidence: 99%

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos

Lima

Montenegro

et al. 2009

J. bras. pneumol.

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Objective: To evaluate the performance of nested PCR (nPCR) in detecting the Mycobacterium tuberculosis complex in blood samples of patients suspected of having TB, in order to determine its potential for use as an auxiliary tool in the laboratory diagnosis of TB in children. Methods: Detection of the M. tuberculosis complex in blood samples using as a target the insertion sequence IS6110 of the genomic DNA of the bacillus. Blood samples of 120 patients were evaluated. All of the patients were under 15 years of age at the time of their treatment at public hospitals in the city of Recife, Brazil (between January of 2003 and August of 2005). Attending physicians at the hospitals diagnosed TB based on the criteria recommended by the American Thoracic Society. The nPCR amplified a 123-bp fragment with outer oligonucleotides (IS1/IS2) and, in the subsequent reaction, using inner oligonucleotides (IS3/IS4), generating an 81-bp amplicon. Results: Active or latent TB was found in 65 patients, TB was ruled out in 28 suspected cases, and 27 patients were TB-free (controls). The sensitivity of nPCR was 26.15% and was significantly higher for the extrapulmonary form of the disease (55.56%) than for the pulmonary form (18.18%). The specificity was 92.73%. Conclusions: Despite the difficulties in diagnosing TB in children and the low number of cases evaluated in the present study, nPCR in blood samples proved to be a rapid and specific technique, albeit one with low sensitivity. In order to establish its true usefulness in the diagnosis of paucibacillary forms, especially extrapulmonary TB, further studies need to be carried out with a larger sample of children and analyzing biological specimens other than blood.Keywords: Tuberculosis; Diagnosis; Blood; Polymerase chain reaction. ResumoObjetivo: Avaliar o desempenho da técnica nested PCR (nPCR) para detectar o complexo Mycobacterium tuberculosis em amostras de sangue de pacientes com suspeita de TB para sua possível utilização como uma ferramenta auxiliar no diagnóstico laboratorial da doença em crianças. Métodos: Detecção do complexo M. tuberculosis em amostras de sangue usando como alvo a sequência de inserção IS6110 do DNA genômico do bacilo. Foram avaliados 120 pacientes, menores de 15 anos de idade, de ambos os sexos, provenientes de hospitais públicos do Recife (PE), no período entre janeiro de 2003 e agosto de 2005. O diagnóstico de TB foi realizado pelo médico assistente do serviço de saúde de acordo com os critérios da Sociedade Torácica Americana. A nPCR amplificou um fragmento de 123 pb com oligonucleotídeos externos (IS1/IS2) e, na reação subsequente, com oligonucleotídeos internos (IS3/IS4), gerando um amplicon de 81 pb. Resultados: A TB ativa ou latente esteve presente em 65 pacientes, foi descartada em 28 suspeitos e 27 não tinham a doença (controles). A sensibilidade da nPCR foi de 26,15%, sendo significativamente maior na forma extrapulmonar (55,56%) em relação à pulmonar (18,18%), e a especificidade foi de 92,73%. Conclusões: Diante das dificuldades ...

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Section: Discussionmentioning

confidence: 99%

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos

Lima

Montenegro

et al. 2009

J. bras. pneumol.

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“…Additionally, an IS6110-targeted probe and respective flanking primers were retrieved from the study of Restrepo et al (2006) (Table 2).…”

Section: Design Of Taqman Probes and Respective Flanking Primersmentioning

confidence: 99%

Rapid identification of veterinary-relevant Mycobacterium tuberculosis complex species using 16S rDNA, IS6110 and Regions of Difference-targeted dual-labelled hydrolysis probes

Costa

Amaro

Ferreira

et al. 2014

Journal of Microbiological Methods

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“…Positive cultures were evaluated by smear microscopy (TB acid-fast stain; BD Diagnostics, Sparks, MD), and the mycobacterial species was confirmed by M. tuberculosis AccuProbe testing (Gen-Probe, Inc., San Diego, CA) (Brownsville or Texas Department of State Health Services) and/or highperformance liquid chromatography (HPLC) (Texas Department of State Health Services) (12). DNA was extracted from concentrated sputum, and M. tuberculosis was detected using an in-house TaqMan assay for IS6110, RD1, and IS1081, as described previously (10,13).…”

Section: Clinical Specimens (I) Specimens For Antigen Identificationmentioning

confidence: 99%

Validation of Mycobacterium tuberculosis Rv1681 Protein as a Diagnostic Marker of Active Pulmonary Tuberculosis

Pollock

Macovei²,

Kanunfre³

et al. 2013

J Clin Microbiol

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The development of an accurate antigen detection assay for the diagnosis of active tuberculosis (TB) would represent a major clinical advance. Here, we demonstrate that the Mycobacterium tuberculosis Rv1681 protein is a biomarker for active TB with potential diagnostic utility. We initially identified, by mass spectroscopy, peptides from the Rv1681 protein in urine specimens from 4 patients with untreated active TB. Rabbit IgG anti-recombinant Rv1681 detected Rv1681 protein in lysates and culture filtrates of M. tuberculosis and immunoprecipitated it from pooled urine specimens from two TB patients. An enzyme-linked immunosorbent assay formatted with these antibodies detected Rv1681 protein in unconcentrated urine specimens from 11/25 (44%) TB patients and 1/21 (4.8%) subjects in whom TB was initially clinically suspected but then ruled out by conventional methods. Rv1681 protein was not detected in urine specimens from 10 subjects with Escherichia coli-positive urine cultures, 26 subjects with confirmed non-TB tropical diseases (11 with schistosomiasis, 5 with Chagas' disease, and 10 with cutaneous leishmaniasis), and 14 healthy subjects. These results provide strong validation of Rv1681 protein as a promising biomarker for TB diagnosis.T uberculosis (TB) remains the second most common cause of death from an infectious disease in the world. According to a 2012 World Health Organization (WHO) report, there were an estimated 8.7 million new cases of TB globally in 2011 and approximately 1.4 million TB-related deaths (430,000 of which involved HIV-positive individuals) (1). Failure to control the spread of TB is due in large part to failure to adequately detect (and thus to efficiently treat) infectious cases (2); therefore, a major focus of the WHO's global plan to stop TB (3) is the development of new simple and cost-effective diagnostic methods to improve case detection. Historically, culture of Mycobacterium tuberculosis from a clinical specimen has been the gold standard for TB diagnosis; however, it is expensive, time-consuming, and not available worldwide. Thus, in resource-limited settings, diagnosis of active TB relies primarily on clinical evaluation and, where available, microscopic evaluation of sputum specimens. Sputum smears have widely varying sensitivities in different settings (20 to 80%) and are of limited utility in paucibacillary (e.g., pediatric and HIVassociated) TB (3-5). Due to the lack of accessible and effective diagnostic methods, many patients in resource-limited settings are treated empirically based on clinical information alone, exposing some patients to unneeded and potentially toxic drugs and missing others who have active TB.The ideal diagnostic method for tuberculosis would be a lowcost, point-of-care (POC), rapid, simple, and accurate test that could distinguish individuals with active TB from those with latent disease or no infection (including Mycobacterium bovis BCGvaccinated individuals) (2, 6). We previously demonstrated the identification by mass spectroscopy (MS) of four ...

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Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens

Cited by 22 publications

References 28 publications

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos

Rapid identification of veterinary-relevant Mycobacterium tuberculosis complex species using 16S rDNA, IS6110 and Regions of Difference-targeted dual-labelled hydrolysis probes

Validation of Mycobacterium tuberculosis Rv1681 Protein as a Diagnostic Marker of Active Pulmonary Tuberculosis

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