Evaluation of 85 A‐C intergenic region PCR primers for detection of Mycobacterium leprae DNA in urine samples

detection of Mycobacterium leprae DNA in urine samples Brazil has the second largest number of recorded cases of leprosy among countries in the world. Leprosy may be classified into three clinical forms: tuberculoid (TT), lepromatous (LL), and dimorphous or borderline (BB) leprosy. Borderline leprosy may be further classified into tuberculoid (BT), borderline (BB), and lepromatous (BL) leprosy. Some clinical forms evolve from indeterminate leprosy (LI). The diagnosis is based on the clinical evaluation of the skin lesions associated with neurological manifestations, microscopic detection of acid-fast bacillus (AFB) in slit-skin smears, and histopathological examination. AFB detection is the most frequently used laboratory test; however, a negative result cannot rule out TT or LI leprosy. Polymerase chain reaction (PCR) has been used for the detection of Mycobacterium leprae in slit-skin smear, blood, nasal cavity, skin tissues, and urine. The main PCR targets are genes that encode protein antigens of 36 kDa, 18 kDa, 85 kDa, 65 kDa and 16S rRNA gene of M. leprae. Antigen 85 complex consists of three components named A, B, and C encoded by fbpA, fbpB, and fbpC, respectively. The 85A-C intergenic region as target for TaqMan real-time and conventional PCR became a useful tool for the detection of M. leprae in skin biopsy in which bacilli were undetectable by AFB staining. This study evaluated the performance of PCR 85A-C to detect M. leprae DNA in urine samples, owing to its easy application and feasibility for leprosy diagnosis. Seventy-three leprosy patients (36 under anti-leprosy therapy) and 50 healthy individuals were selected by clinical and laboratory findings. Two first-emission urine samples from each individual were subjected to DNA extraction and PCR 85A-C. Specificity was analyzed using DNA from Mycobacterium tuberculosis H37Rv, Mycobacterium bovis AN5, and M. leprae DNA obtained from the skin lesion of an infected armadillo. Sensibility was carried out with M. leprae DNA in 90 lg ⁄ ml, 0.90 lg ⁄ ml, 0.30 lg ⁄ ml, and 0.15 lg ⁄ ml. Data were analyzed by McNemar test using Statistica 6.0 (StatSoft, Inc. Tulsa, OK, USA), with P = 0.05 as significant value. PCR 85A-C amplified a specific fragment of 250 bp from M. leprae DNA with a detection limit up to 0.90 lg ⁄ ml. Table 1 and Fig. 1 show PCR 85A-C results in urine of the patients and healthy control group. PCR was positive in 4.1% (3 ⁄ 73) of leprosy patients (two TT leprosy and one under anti-leprosy therapy LL leprosy). The occurrence of positive PCR in urine of nontreated leprosy patients (5.4%) was significantly higher (P = 0.000) than in patients undergoing anti-leprosy treatment (2.8%). Although no statistical difference was observed in PCR positivity between treated and nontreated TT leprosy patients (P = 0.6831), it was observed in LL leprosy patients (P = 0.000). PCR 85A-C sensibility in detecting M. leprae in urine was lower (4.1%) than that detected by Martinez et al. (2006) in skin biopsy (82.6%). In PCR targeting pra gene in urine sample, Parkash et al. (2004) revealed better PCR positivity (37.5%, 6 ⁄ 16) when compared with that found in current work using 85A-C-PCR. Probably, this difference is related to the primer target in M. leprae genome. Among nontreated leprosy patients, PCR 85A-C was positive only in individuals classified as TT leprosy. This positivity may be related to the immune response-mediating cell, which is predominant in this clinical setting resulting in free DNA being excreted by kidneys. Otherwise higher PCR positivity was observed in LL (P = 0.000) than in TT (P = 0.683) leprosy patients undergoing treatment. Jamil et al. (1993) reported that in the initial phase of anti-leprosy therapy, bacilli are killed and eliminated from tissues, increasing their excretion in urine. The above issue highlights that in LL leprosy patients positive PCR may be the consequence from treatment and not caused by cell-mediated immune response that is not predominant in LL leprosy.