Improved detection of Mycobacterium tuberculosis using two independent PCR targets in a tertiary care centre in South India

Abstract

Introduction: Tuberculosis (TB) causes significant morbidity and mortality worldwide as one of the leading infectious diseases. In India, more than 1.8 million new cases occur every year. Rapid and accurate diagnosis of TB would improve patient care and limit its transmission. This study aimed to evaluate a dual target polymerase chain reaction (PCR) diagnostic assay to detect Mycobacterium tuberculosis from pulmonary and extra-pulmonary samples at a tertiary care centre in South India. Methodology: Samples were collected from patients with a low index of suspicion of TB. Acid-fast smears were performed by Auramine O fluorescent microscopy and PCR was performed by using two site-specific primer pairs targeting IS6110 by nested PCR and TRC 4 by conventional PCR. Amplified products for IS6110 and/or TRC 4 were indicative of M. tuberculosis. Results: Among 114 (19 pulmonary and 95 extra-pulmonary) samples tested by PCR assay, 12 (11%) were positive for both IS6110 and TRC 4 , of which 11 (10%) were non-respiratory and one was (1%) respiratory in origin. PCR for TRC 4 alone was positive for eight (7%) nonrespiratory and two (2%) respiratory samples, while IS6110 alone tested positive for six (5%) non-respiratory samples and one (1%) respiratory sample. Of a total of 29 PCR positive samples, 17 (15 %) were acid-fast smear positive. Conclusion: Although the target site of IS6110 is specific for M. tuberculosis, some strains from South India may lack this region. Therefore, the use of an additional target site (TRC 4 ) is required for improved detection of M. tuberculosis.

Use of Endo-Ovarian Tissue Biopsy and Pelvic Aspirated Fluid for the Diagnosis of Female Genital Tuberculosis by Conventional versus Molecular Methods

“…Further, the conventional methods showed 99% to 100% specificity with a low sensitivity, ranging from 21.78% to 42.08% while H & E staining showed a sensitivity of 51.48%. However, multi-gene PCR method was found to have a much higher sensitivity of 70.29% for MTB64 gene, 86.63% for 19 kDa antigen gene and TRC4 element and 88.12% for 32 kDa protein gene [47], [58]. The specificity of multi-gene PCR was 100%.…”

“…However, one should use varied combination of parameters (or genes) to overcome the limitations such as the false positivity by way of contaminations, false negativity, dead bacilli and asymptomatic TB at different sites. Thus, the increase in PCR sensitivity and decrease in false negative results were achieved using dual targets for the detection of M. tuberculosis [58]. Even technical considerations, such as the use of suitable controls, standard strains, suitable conditions and the retesting of doubtful positive samples considerably influence the sensitivity and specificity.…”

“…32 kDa protein gene showed higher sensitivity and specificity than any other gene due to the presence of infectious organisms representing Mycobacterium genus. Even 19 kDa antigen gene and TRC4 element showed better sensitivity (86.63%) and specificity (100%) indicating the infection due to MTB complex [47], [58]. Thus, these genes could be emerging future diagnostic biomarkers in the detection of FGTB [42], [43].…”

Utility of multiplex real-time PCR in the diagnosis of extrapulmonary tuberculosis

“…Among the two smear positives, one tissue sample (lymph node biopsy) grew M. avium and the other pus sample grew M. abscessus. The remaining four smear negative samples, which grew NTM, were pus samples and were identified as M. fortuitum (1) and M. abscessus (3). All these six samples were PCR negative for both targets.…”

Microcalcificaciones cerebrales en un recién nacido con tuberculosis congénita

“…En la mayoría de los estudios se ha utilizado como blanco de la PCR la secuencia de inserción IS6110, presente en múltiples copias en el genoma de la mayoría de las cepas de M. tuberculosis (9). Para el tratamiento de los recién nacidos con tuberculosis se utilizan 10 a 15 mg/kg por día de isoniacida, 10 a 20 mg/kg por día de rifampicina, 15 a 30 mg/kg por día de pirazinamida y 20 a 30 mg/kg por día de estreptomicina o 15 a 25 mg/kg por día de etambutol durante dos meses, seguidos de isoniacida y rifampicina durante 4 a 10 meses, dependiendo de la gravedad del cuadro clínico.…”