Mitigating polymerase chain reaction/amplicon contamination in a high-risk high-burden mycobacterial reference laboratory in a resource-limited setting

Background: Nucleic acid amplification techniques have become important machineries in the diagnosis of several diseases in clinical laboratories. Polymerase chain reaction (PCR) contamination/amplicon contamination leading to false positivity remains a major concern in these laboratories. Prevention of these contaminations in establishing these molecular biology laboratories has been very crucial over the years. Although closed system PCRs have substantial reduction in the PCR contamination rates, the conventional probe-based hybridization methods continue to show occurrence of contamination for various reasons. The study involved checking the crucial parameters as well as the probable candidates of causing the contamination at a high-burden setting. Bringing out the most effective interventions in controlling the PCR contaminations for future endeavors stood as priority. The study explored the efficacies of different sets of interventions contributed to the process of reducing the contaminants. Methods: The detection of the contaminating PCR products or amplicons or contaminating organism is done by the genotype MTBDR plus V2 kits (Hains Life Sciences) based on DNA strip technology. Results: The pre- and post-cleaning as well as cleaning of the working surfaces was able to bring down the mean contamination percentage by 36.5%. The combined effect of cleaning the work surfaces, the automated pipetting devices, and the AC machines was able to bring down the mean contamination percentage to 53.5% reducing the contamination rate nearly to 94.6%. Conclusion: Regularly cleaning the work surfaces, the automated pipetting devices, the PCR machine, and the AC machines along with it filters and exposure of UV rays significantly lowers down the mean contamination percentage

Prevalence of rifampicin-resistant pediatric tuberculosis by cartridge-based nucleic acid amplification test at the intermediate reference laboratory under revised national tuberculosis control program India: A multidimensional approach

Background: Tuberculosis (TB) still remains the major public health threat in India. Early diagnosis, so as to initiate early treatment is a priority as any delay, may complicate the prognosis further leading to the failure of an effective control. India accounts for 6% incidence of pediatric TB cases in a population that has 40% as estimated latent TB cases. Implementation of cartridge-based nucleic acid amplification tests (CBNAATs) in diagnosis of pediatric TB has augmented the detection rates. The study involves a retrospective multidimensional analysis of the increased pediatric TB case detection by CBNAAT at the Intermediate Reference laboratory, State TB Demonstration and Training Centre in West Bengal, India. A total of 14,088 samples from pediatric patients coming from all the districts were tested from May 2014 to April 2018. This included pulmonary (sputum, bronchoalveolar lavage, gastric lavage, and gastric aspirate) and extrapulmonary (cerebrospinal fluid [CSF], pus, ascitic fluid, pericardial fluid, lymph node aspirate) samples. Although detection levels showed variation in the nature of the samples, the study explored percentage contribution of the types of samples and the proportion positive rates among them. Percentage-wise propensity with regard to age, clinical presentations, type of samples, and smear result were observed. The predominant geographical location in terms of incidences and periodic prevalence were studied. The resistant cases were retested with line probe assay by MTBDRplus and V2 Hains for concordance analysis. This was done based on the parameters in the erstwhile evaluations. Methods: Pulmonary and extrapulmonary pediatric samples were tested on CBNAAT by Xpert Mycobacterium tuberculosis rifampicin (RIF) (Cepheid) based on the manufacturer's instruction. All necessary aseptic measures were taken. The data were captured in the Xpert software automatically during the tests and exported to the Microsoft Excel sheets for further analysis. A defined study design against each and every objective was set up. Results: It was found that a point prevalence of 6%–7% of pediatric TB exists among the tested specimens every quarter. The periodic prevalence was found to be 5%, the incidence rates ranged from 4.5% to 5%. RIF resistance detection showing a seasonal variation ranged from 13% to 15% among the CBNAAT-positive cases. Gastric lavage showed a major detection in children below 6 months of age of whom collecting sputum samples were difficult. This prompted suboptimal detection levels due to the dearth of sample collection modalities at the peripheries. CSF accounted for 2.37% of positivity. Conclusions: The study concluded that more skilled collection centers for biological specimens are required to address the undetected pulmonary TB cases among the pediatric age group, especially below the 6 months of age. About 5% prevalence and around 4.9% incidence is an alarming situation in the TB control scenario of West Bengal. Focus on the universal drug-susceptibility testing is a prerequisite