Evaluation of a IS6110-Taqman real-time PCR assay to detect Mycobacterium tuberculosis in sputum samples of patients with pulmonary TB.

J Appl Microbiol. 2013 Apr;114(4):1103-8.

Lira LA, Santos FC, Carvalho MS, Montenegro RA, Lima JF, Schindler HC, Montenegro LM.

Departament of Imunology, Aggeu Magalhães Research Center – Fiocruz, Recife, Pernambuco, Brazil. lais.liraa@gmail.com

Abstract

AIM: Evaluate the IS6110-Taqman system performance in sputum samples from patients with pulmonary tuberculosis from health services in north-eastern Brazil as a diagnostic laboratory tool for pulmonary tuberculosis.

METHODS AND RESULTS: 165 sputum samples from respiratory symptomatic patients were evaluated in the IS6110-TaqMan assay: 66 patients with pulmonary tuberculosis and 99 without TB. When the IS6110-TaqMan assay was evaluated using culture and/or clinical response to the specific treatment as the gold standard, IS6110-TaqMan assay obtained a sensitivity of 87.9% and specificity of 98%. The performance of IS6110-TaqMan assay was also evaluated with the sputum smear microscopy, resulting in a sensitivity of 79.7% and specificity 94.8%.

CONCLUSIONS: The IS6110-TaqMan was rapid, sensitive and specific for the diagnosis of pulmonary TB.

SIGNIFICANCE AND IMPACT OF THE STUDY: IS6110-TaqMan assay is a promising auxiliary tool for the diagnosis of pulmonary TB when used in conjunction with routine laboratory tests, clinical and epidemiological criteria of the patient, thus increasing the sensitivity and specificity of diagnosis.

PMID: 23279625

 

Supplement

Conventional laboratory diagnosis of tuberculosis by smear and culture is still unsatisfactory as one of the main factors that contribute to the maintenance of high levels of the disease worldwide. Despite being curable and have specific treatment, TB still shows higher mortality rates. In face of such a situation it is necessary to develop studies on the use of new alternative diagnosis aiming faster and more effective results. Thus, this study evaluated a system for detection and quantification of Mycobacterium tuberculosis DNA by PCR in real time (qPCR) as an auxiliary tool in the laboratory diagnosis of patients with suspected pulmonary TB.

The biggest advantages of qPCR compared to conventional PCR are speed, greater accuracy, reproducibility and accuracy. Furthermore, analysis of the final product is made without the need for handling of the tube during processing, which reduces the risk of contamination by reducing false positives and false negatives. The results allow a better analysis because they are generated graphics that differentiate the positive and negative samples through cycles of DNA amplification (Figure 1). In addition, the reactions are compared to a standard curve of genomic DNA which may allow quantification of bacterial load of the patient. Therefore, we highlight the importance of the application of this technique in countries where TB has a high incidence, such as Brazil. It would enable results to be delivered with higher speed (1 to 2 days) and accuracy. Therefore, the possibility of an early and effective diagnosis will surely interfere with the transmission chain of the bacillus, and consequently, in disease control.

Laís Lira-1

Figure 1: Results demonstrates that amplification using the IS6110-Taqman real time-PCR with sputum samples from patients diagnosed with (positive samples) or without TB (negative samples). Positive samples: amplification up to cycle 30.

 

Contact:

Laís Ariane de Siqueira Lira

Biologist, Master in Public Health

Departament of Imunology, Aggeu Magalhães Research Center – Fiocruz, Recife, Pernambuco, Brazil.

E-mail: lais.liraa@gmail.com

Laís Lira-3

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann