Clinical evaluation of an in-house novel primer sequence based PCR for conventional molecular diagnosis of pulmonary and extrapulmonary tuberculosis

Background:Tuberculosis is one of the chronic infectious bacterial disease that still remains as major cause of morbidity and mortality in the world. The limited role of diagnostic methods for correct and early detection of bacilli in different types of clinical samples remains uncertain increasing prevalence of TB. Aim & Objective: The purpose of present study is to develop an in-house primer to PCR targeting the fadE-20 gene for diagnostic applicability in detection of M.tuberculosis complex species from clinically suspected cases of pulmonary and extrapulmonary tuberculosis. Materials & Methods: This study was conducted in Genomix Carl Pvt. Ltd, Pulivendula, A.P during the period of 2 years 6 months from March 2017 to September 2019. A total of 233 different types of clinical samples were assayed in this study with optimized fadE gene primer and its accuracy was comparatively analyzed with conventional IS6110 sequence based PCR ,Acid fast Bacilli(AFB) smear microscopy and LJ-culture tests results. Results: In the present study The highest overall positivity rate of 85.4% was observed by using in-house fadE-20 PCR whereas overall positivity with IS6110 PCR was found to be 82.4%(192). The in-house fadE PCR test resulted in sensitivity of 100% & 96.1% for pulmonary and extrapulmonary culture positive specimens whereas IS6110 PCR resulted in sensitivity of 98.8% and 93.5%. respectively.Furthermore both the amplification tests gave consistently negative results from the negative control group therefore showing overall specificity of 100%. Conclusion: By comparative analysis of overall data from both amplification tests,It can be concluded from this study that the fadE-20 PCR appears to be a reliable tool for accurate identification of Mycobacterium tuberculosis complex in different types of pulmonary and extrapulmonary samples obtained from tuberculosis patients. Key wordsPulmonary and extra pulmonary tuberculosis, in-house primer, Polymerase chain reaction, Mycobacterium tuberculosis complex. www.ijcrt. org © 2020 IJCRT | Volume 8, Issue 2 February 2020 | ISSN: 2320-2882 IJCRT2002119 International Journal of Creative Research Thoughts (IJCRT) www.ijcrt. org 1157 Introduction Tuberculosis still continues as one of the world leading air borne chronic communicable mycobacterial infection causing severe health threat to humans despite the existence of laboratory methods for diagnosis of tuberculosis(TB) cases and effiicious chemotherapy treatments(ATT). TB globally leads to almost 9,0 million new infections each year and death toll of approximately 1.5 million per year (Kiran Chawla et al., 2015; Scherer et al. 2011). It is reported that India accounts for one quarter of the worlds TB buden sharing prevalence rate of 30% and mortality of almost 350,000 annually (S Kulkarni et al., 2012; Paras Singh et al., 2013). Eventhou,tuberculosis most commonly affects lungs(90%) but can also be reactivated by immune compromisation and disseminate outside lungs to other body sites leading to manifestation of Extrapumonary TB(EPTB 1520%)( S Kulkarni et al., 2012; Shavi Nagpal et al., 2016). In clinical practice by most of clinicians the corner stone for the confirmation of mycobacterial infections on patients with clinical signs, symptoms & radiographic image changes suggestive of tuberculosis are still made by conventional microbiological techniques like Acid fast Bacilli(AFB) Ziehl-Neelsen (ZN) staining smear microscopic examination and culture of clinical specimens on Lowenstein-Jensen(LJ) medium for identification of mycobacteria. But the limited role of conventional microbiological methods which are inadequate in sensitivity, specificity and lengthy for definitive identification of tuberculosis infection have made the disease undiagnosed. The emergence of molecular methods which provides higher degree of positive results with good specificity, reliability and fast in detection of Mycobacterium tuberculosis (MTB) from pulmonary and extrapulmonary samples have made suitable choice alternative to the traditional diagnostic methods. Among molecular methods,Polymearse chain reaction(PCR) targeting amplification of specific mycobacterial DNA sequence directly from variety of clinical specimens(both pulmonary and extrapulmonary) have attracted considerable importance for use in clinical practice in rapid diagnosis of mycobacterium tuberculosis infection with high sensitivity and specificity. Although primers targeting for IS6110 gene sequence present as multiple copies in genome which is mostly used in many laboratories that is considered to be specific but there are many literatures describing the presence of this insertion sequence in some bacteria species other than tubercle bacillus and its absence in some M.tuberculosis strains across the geography, reducing the sensitivity and specificity of PCR tests (S. Narayanan, 2013; T. Moatter et al., 1998). Hence the development of a robust genetic marker is importantly needed to establish a rapid and reliable molecular based PCR test for diagnosis of tuberculoss. With this background,in the current study we aimed to design and develop an In-house primer to PCR assay based on hypothetical novel fadE-20 gene sequence for rapid identification of M.tuberculosis complex in various clinical specimens obtained from suspected cases of pulmonary and extra-pulmonary tuberculosis.In addition,we also evaluated its potential diagnostic value by comparing with molecular IS6110 based PCR assay and conventional acid fast bacilli (AFB) smear microscopy and LJ medium culture methods results.fadE genes of mycobacterium tuberculosis complex encoding for acyl coenzyme A enzyme is reported (predicted) to be associated with host cell entry and suppressing immune defense mechanism to enable their increased survival causing host pathogenecity (M. Kaley et al., 2018; Rochelle van Wyk et al., 2019; F. Matthew et al., 2013). So with this baseline we focused at studying the clinical implications of fadE-20 gene that may lead to be a diagnostic marker for rapid diagnosis of tuberculosis. Materials and Methods: We performed this study in the Molecular Biology laboratory at GENOMIX-CARL, Research centre,Pulivendula, during the period of 2 years 6 months from March 2017 to September 2019. The samples undertaken for the current study were obtained from Genomix Molecular diagnostics Division,Hyderabad, that were received for the diagnosis of M.tuberculosis infection collected from different categories of TB cases attending the referred