Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

Abstract

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39–31.27% for rpoB-PCR, 36.44–60.83% for IS6110- PCR, 75.29–92.93% for nested-rpoB PCR, and 87.98–99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15–100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results.

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples

“…Another work, which evaluated pleural fragments by qPCR using the same molecular target and extraction method, yielded similar results to this research (a sensitivity of 52.8% and specificity of 93.3%) 34 . In agreement with our study, Meghdadi et al 38 , using PCR targeting IS6110, demonstrated a sensitivity and specificity of 48.6% and 100%, respectively.…”

“…To evaluate the positivity of biological samples, the threshold value of amplification (C t ) was established for positive and negative cases. The resulting C t was 25 (25)(26)(27)(28)(29)(30)(31)(32)(33) and 34 (34)(35)(36)(37)(38) for the positive and negative tests, respectively (data not shown).…”

Identification of Mycobacterium tuberculosis in Clinical Specimens of Patients Suspected of Having Extrapulmonary Tuberculosis by Application of Nested PCR on Five Different Genes

“…Variation in sensitivity is one of the obstacles that prevent the full standardization of PCR technique in the laboratories of clinical analysis centers. To overcome this problem, PCR can be improved by a series of modifications to be able to detect the low number of MTB bacilli (Meghdadi et al, 2015). Alternatively, combination of two PCR techniques in the form of nested-PCR will increases its sensitivity and specificity in comparison to the conventional single PCR (da Cruz et al, 2011).…”

Comparison of conventional and molecular methods in diagnosis of extrapulmonary (cutaneous) tuberculosis in a tertiary care hospital in Delhi

“…They were diagnosed as M. tuberculosis infection. Out of study by Meghdadi et al [38] showed 20% of PCR positivity. Hijidin et al [31] had reported PCR positivity of 5.1% which is very lower as compared to this study.…”