Comparative study of different diagnostic modalities of m. tuberculosis and evaluation of rifampicin resistant strains using line probe assay versus conventional proportional method

ABSTRACT

Tuberculosis remains a major health problem around the world being responsible for 3 million deaths each year. The spread of TB is expected to worsen as infection with multi-drug resistance M. tuberculosis isolates [MDR-M] increases in many countries. Rifampicin is an important component of effective multi-drug therapies of TB. Thus, Rif-resistance means that short course therapy is no longer an option and those second-line drug susceptibilities are required in order to make an informed choice for alternative therapy. Furthermore, resistance to Rif serves as a useful surrogate marker for the detection of MDR isolates. The aim of this study is to shed some light on the line probe assay [LiPA] in comparison to the conventional proportion method for detecting Rif susceptibility genotype and phenotype of M. tuberculosis clinical isolates. This study was conducted on 154 patients with age ranged from 18 to 72 years who were divided into 2 groups. The first group included 130 cases [80 males and 50 females] suspected of having pulmonary TB, and the second group included 24 cases [18 males and 6 females] suspected of having extrapulmonary TB. All cases were subjected to tuberculin skin test. The clinical specimens were collected according to the site of presenting symptoms. At least 3 successive samples of sputum, urine and stool were collected for each case. Contaminated specimens [98 sputum, 13 bronchial lavage, 7 urine and one stool samples] were subjected to a digestion-decontamination procedure. Aseptically collected specimens [13 peritoneal aspirate, 19 pleural aspirate, one fallopian tube and 2 lymph node samples]. All used for smear staining with Kinyoun's stain and culture. Mycobacterial isolates on L-J slants were biochemically typed by niacin accumulation, nitrate reduction and heat stable catalase test. The susceptibility of M. tuberculosis to Rif was performed using both the proportion method on MB7H10 agar and LiPA using LiPA Rif TB strips. The distribution of tuberculous patients according to clinical specimens among all cases showed that 98 sputum samples revealed 26 positive culturers. Two sputum samples were positive by stained smear after processing but negative by either direct smear or culture. The overall positivity of sputum samples were 28 specimens. One positive culture/negative Kinyoun's stain was recovered from fallopian tube specimen. Tuberculin skin test revealed positive induration diameter in 28 [96.6%] out of 29 tuberculous patients, whereas the tuberculin skin test was positive in 78 [62.4%] out of 125 negative smear/culture cases. Direct Kinyoun's stain showed positive smear in 15 out of 154 specimens, which were also positive by L-J culture. Also 12 positive cultures were recovered from the 139 negative direct smears. The sensitivity and specificity of direct Kinyoun's stain versus L-J culture were 55.6% and 100% respectively, and the negative and positive predictive values were 91.4% and 100% respectively. Stained smears after processing revealed positive smear in 22 out of 119 patients, of which 20 were positive by L-J culture. However 6 positive cultures were recovered from 97 negative smears. Its sensitivity and specificity versus L-J culture were 76.9% and 97.8% respectively, and the positive and negative predictive values were 90.9% and 93.8% respectively. There was a statistically significant difference in comparing direct Kinyoun's smear and Kinyoun's smear after processing of the 115 contaminated specimens. The results of mycobacterial biochemical reactions revealed 24 M. tuberculosis and 3 mycobacteria other than tuberculosis [MOTT] isolates. As regard Rif-susceptibility testing of the 24 M. tuberculosis isolates, 8[33.3%] isolates were Rif-resistant and 16 [66.7%] were Rif sensitive by the proportion method, whereas 6[25%] isolates were Rif resistant and 18 [75%] were Rif-sensitive by LiPA. The type of rpoB gene mutations in the 6 Rif-resistant M. tuberculosis isolates by LiPA showed that the most frequently detected mutations were H526Y [57.1%] and S531L [28.6%] and H526D 14.3%. One strain showed double mutations atH526Y and S531L. It is concluded that LiPA is a simple technique which provides clear results requiring only a basic knowledge of molecular technique