Evaluation of a nested-pcr for Mycobacterium tuberculosis detection in blood and urine samples

Abstract

The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8% and 52% in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29% and 26.9% in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease.

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

“…Molecular tests based on M. tuberculosis genomic fragment amplifi cation are rapid and sensitive techniques that can detect less than 10 bacilli per ml in a clinical sample 14,15 . Although conventional polymerase chain reaction (PCR) is useful for M. tuberculosis detection, the nested-PCR (nPCR) technique has greater sensitivity and specifi city for the test, indicating that it is a promising laboratory tool for diagnosing paucibacillary forms of TB, including the pleural form 6,14,16,17 .…”

“…Although conventional polymerase chain reaction (PCR) is useful for M. tuberculosis detection, the nested-PCR (nPCR) technique has greater sensitivity and specifi city for the test, indicating that it is a promising laboratory tool for diagnosing paucibacillary forms of TB, including the pleural form 6,14,16,17 .…”

“…This technique was performed using a modifi ed protocol, as fi rst utilized by Rossetti et al 14,21 . A water-containing negative control was included to assess any cross-contamination between the biological specimens.…”

“…This test was performed using a modifi ed protocol that was fi rst developed by Ritis et al 14,22 . For each amplifi cation, a positive control containing deoxyribonucleic acid (DNA) extracted from the M. tuberculosis H37Rv reference strain was included, along with a negative control (Milli-Q water) to exclude the possibility of cross-contamination.…”

“…The 12 samples that returned false-negative results in the test could likely be explained by the paucibacillary nature of the samples and because the pleural fl uid was an exudate arising from a hypersensitive reaction to the Mycobacterium 16 . Furthermore, as in other organic specimens, it is believed that pleural fl uid has inhibitory substances that can interfere with the PCR reaction, thus making it necessary to adopt procedures that can maximally and simultaneously remove those inhibitors 14,28 . Therefore, this necessity may allow for the determination between a low copy number and the absence of the target IS6110 region in clinical isolates 29 .…”