Comparison of the Sensitivities of Different PCR Assays for the Detection of Mycobacterium tuberculosis Complex Isolates from Five Regions of Cameroon

Abstract

The accurate diagnosis of tuberculosis caused by members of the Mycobacterium tuberculosis complex (MTBC) has remained a major challenge in clinical laboratories worldwide. Several studies have evaluated the use of highly specific in-house PCR assays targeting the IS6110, hupB, rpoB, oxyR, and IS1081 genes in the detection of MTBC species with reports on variable sensitivities depending on the geographical sourcing of isolates. In the present investigations, we evaluated the sensitivities of these PCR assays on 125 MTBC cultured isolates from five (West, Centre, Littoral, North West and South West) of the ten regions of Cameroon. Of this number, 124 (99.2%), 117 (93.6%), 123 (99.1%), 119 (95.2%) and 118 (94.4%) were positive by the IS6110, hupB, rpoB, oxyR, and IS1081-based PCR assays respectively. A total of 110 (88%) of the cultured isolates were also identified as MTBC by standard biochemical tests. Of this number, 109 (99.1%), 104 (94.5%), 109 (99.1%), 106 (96.4%) and 104 (94.5%) were positive in the IS6110, hupB, rpoB, oxyR, and IS1081-based PCR assays respectively. Concordant PCR results were obtained for 108 of the 125 samples. The 15 isolates that were negative biochemically scored sensitivities ranging from 100% (for the IS6110 assay) to 86.7% (for the hupB and oxyR assay). The combination of the IS6110 assay, which turned out to be the most sensitive, and each of the other assays gave 100% sensitivity. We conclude that the combined targeting of the IS6110 and rpoB genes is likely to yield the most sensitive PCR procedure for the diagnosis of MTBC infection in the five regions of Cameroon. F. Cho-Ngwa et al.

Diagnostic value of polymerase chain reaction targeting insertion sequence IS1081 for the diagnosis of pediatric tuberculosis

“…It was positive in 44% samples with maximum positivity in sputum (70%) which is comparable as reported by Rodrigues C et al, and Rishi et al 13,14 PCR IS1081 has shown 93.1% sensitivity (with 95% confidential interval) which is similar as reported by Fidelis et al, and Fatolahzadeh B. 15,16 This study showed that MGIT culture was positive in 44(44%) specimens, whereas IS1081 PCR showed that 74 (74%) specimens were positive for Mycobacterium tuberculosis. The difference was found to be statistically significant (p <0.05).…”

Evaluation of the performance of an in-house duplex PCR assay targeting the IS6110 and rpoB genes for tuberculosis diagnosis in Cameroon

“…It has been estimated that a rapid TB diagnostic test with at least 85% pooled sensitivity for smear-positive and smear-negative cases and 97% specificity could save approximately 400,000 lives annually [ 25 ]. This study determined the diagnostic accuracy of D-PCR using primers ( IS6110 and rpoB ) in direct sputum samples, hitherto established to be sensitive and specific [ 14 ].…”

“…Furthermore, four false-negative PCR results were recorded; two smear-positive/culture-positive, one culture-positive/smear-negative and one clinically diagnosed TB case. False negativity in nucleic acid amplification (NAA) tests for sputum has always been linked to the presence of inhibitors (including high amounts of genomic DNA), and a low number of bacilli [ 13 , 14 ]. However, because only presumptive identification of isolates (no speciation) was done before PCR, the possibility of detecting species other than Mycobacterium tuberculosis complex cannot be ruled out.…”

“…Although procedures abound on how to circumvent the problems of inhibition, most of the techniques are expensive or laborious and may either lead to loss of DNA pellets, the introduction of exogenous inhibitors and or damage to template [ 14 ]. The choice of sample dilution in this study was appealing because it is cheap, fast and easy to perform and in the end, suspected inhibition and /or low copy number was resolved in 85.7% (18/21) of samples subjected to this method.…”

“…A previous study to determine the sensitivity of 5 specific primers namely IS6110, IS1081, rpoB, oxyR and hupB on culture M. tuberculosis isolates in Cameroon, demonstrate a 100% sensitivity for IS6110 and rpoB genes when assayed in duplex PCR (D-PCR) [ 14 ]. As a follow up of these findings, this study was to evaluate the diagnostic accuracy of these two primers in sputum samples from clinical suspected patients using culture and Composite Reference Standard (CRS) as a gold standard to determine their suitability in routine diagnosis of pulmonary tuberculosis in Cameroon.…”