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

Abstract

Background Tuberculosis (TB) remains a major public health concern in many low-income countries accounting for approximately two-thirds of deaths in people living with human immunodeficiency virus (HIV) infection. With prompt, accurate and appropriate treatment, almost all TB disease can be cured. The present study was to evaluate the diagnostic performance of an in-house duplex PCR (D-PCR) using IS1610 and rpoB specific primers in sputum samples from TB suspected patients. Methods A hospital-based cross-sectional study was conducted at the Limbe and Buea Regional Hospitals of the South West Region of Cameroon from June 2016 to April 2017. Sputum samples, decontaminated with hypertonic saline/sodium hydroxide solution were centrifuged and pellets processed for smear microscopy, culture and DNA extraction. Suspected inhibition was resolved by serial dilution of genomic DNA. Results were compared to culture as gold standard as well as a Composite Reference Standard (CRS). Results A total of 129 participants aged between 5 to 82 years were enrolled in to the study. The median age of the participants was 37 years (interquartile range, IQR: 27–50 years), with 54.3% being male. Forty-seven samples (36.4%) were positive by direct sputum microscopy, 49 (38%) by microscopy after concentration, 51 (39.5%) by culture and 62 (40.1%) by D-PCR. PCR inhibition was resolved in 85.7% (18/21) of the samples that had inhibition. The overall sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios and area under the curve AUC) of the D-PCR was 93.5, 94, 94, 94%, 15.6, 0.005 and 89.0% respectively using CRS as reference. The sensitivities of D-PCR observed among different sample categories were 95.7, 87.5 and 87.5% for smear-and culture-positives, smear-negative/culture-positive, and clinically diagnosed cases respectively. Conclusion IS1610 and rpoB duplex PCR using relatively cheap decontamination and DNA extraction methods in addition to simple serial dilutions to resolve PCR inhibition shows high sensitivity in the diagnosis of paucibacillary tuberculosis.

Diagnostic Performance of Multiplex PCR for Detection of Mycobacterium tuberculosis Complex in Presumptive Pulmonary Tuberculosis Patients and Its Utility in Smear Negative Specimens

“…It is possible that the bacillary load was too low to be detected by multiplex PCR, or there were some unrecognized PCR inhibitors in the samples. 13 These samples reiterate the role of culture as an important part of TB diagnosis.…”

“…These findings are supported by other recently published reports. For example, one report from Meriki et al 13 found the sensitivity and specificity of duplex PCR assay for all samples to be 93.5 and 94%, respectively; and for smear-negative culture positive samples, the sensitivity was 87.5%. Furthermore, a systematic review and meta-analysis to assess the overall accuracy of RT-PCR assay for TB diagnosis in different samples for individuals with active pulmonary and extrapulmonary infection 14 reported pooled sensitivity of 0.96 and pooled specificity of 0.92.…”

Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection

“…This higher positivity rate of IS 6110 -specific PCR over that of GeneXpert might be associated with a low concentration of target DNA in some samples owing to the uneven distribution of the microorganisms in clinical specimens and the presence of a moderately high quantity of human DNA, which is considered a common problem with the GeneXpert detection system ( Manke et al 2017 ; Meriki et al 2020 ). On the other hand, the IS 6110 , with a multiple-copy element within the tuberculosis genome, facilitates its detection by specific PCR in clinical samples even at a low concentration of target DNA ( Gonzalo-Asensio et al 2018 ).…”