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

Abstract

Definitive and rapid diagnosis of extrapulmonary tuberculosis (EPTB) is challenging since conventional techniques have limitations due to the paucibacillary nature of the disease. To increase the sensitivity of detection of Mycobacterium tuberculosis (MTB) in EPTB specimens, we performed a nested PCR assay targeting several genes of MTB on EPTB specimens. A total of 100 clinical specimens from suspected cases of EPTB were processed. Standard staining for acid fast bacilli (AFB) was performed as the preliminary screening test. Extracted DNAs from specimens were subjected to Nested PCR technique for the detection of five different MTB target genes of IS6110, IS1081, hsp65kd, mbp64, and mtp40. On performing AFB staining, only 13% of specimens were positive, of which ascites fluid (33.3%), followed by pleural effusion (30.8%) showed the greatest AFB positivity rate. We demonstrated slight improvement in yields in lymph node which comprised the majority of specimens in this study, by employing PCR targeted to IS6110- and hsp65-genes in comparison to AFB staining. However, the yields in ascites fluid and pleural effusion were not substantially improved by PCR, but those from bone and wound were, as in nested PCR employing either gene, the same positivity rate were obtained for ascites fluid (33.3%), while for pleural effusion specimens only IS1081 based PCR showed identical positivity rate with AFB stain (30.8%). The results for bone and wound specimens, however, demonstrated an improved yield mainly by employing IS1081 gene. Here, we report higher detection rate of EPTB in clinical specimens using five different targeted MTB genes. This nested PCR approach facilitates the comparison and the selection of the most frequently detected genes. Of course this study demonstrated the priority of IS1081 followed by mtp40 and IS6110, among the five tested genes and indicates the effectiveness of any of the three genes in the design of an efficient nested-PCR test that facilitates an early diagnosis of paucibacillary EPTB cases, which are difficult to diagnose with the available standard.

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

“…Rosso et al 34 evaluated pleural fluid samples with the IS6110-TaqMan ® assay and obtained a better sensitivity (40%), probably because of the greater number of patients included in their study. Other research has also demonstrated a better performance of molecular tests in body fluids as compared with this study 13,[35][36][37] .…”

“…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).…”

Laboratory Diagnostic Tools for Syphilis: Current Status and Future Prospects

“…NAAT is a popular way to diagnose infectious diseases, especially for diseases caused by agents that are difficult to be cultured, such as Borrelia burgdorferi (the causative agent of Lyme disease) ( Shah et al., 2018 ) and Mycobacterium tuberculosis ( Khosravi et al., 2017 ). Because T. pallidum is so difficult to grow in culture, it is of great value to apply NAAT to the detection of T. pallidum ( Morshed et al., 2007 ).…”

Introduction and Application of the Interferon-γ Assay in the National Bovine Tuberculosis Control Program in South Korea

“…DNA was extracted from suspected bTB colonies for identification by polymerase chain reaction (PCR) using the DNeasy Blood and Tissue kit (Qiagen, Germany) according to the manufacturer's instructions. Primers were designed targeting the insertion elements (IS), IS1081 and IS6110, which specifically exist within the genome of Mycobacterium bovis (7,14,15). Multiplex PCR was performed for IS1081 and IS6110 in a reaction mixture (20 µl) ( Table 1) containing 10 pmol of each primer, 14 µl AccuPower hotstart PCR premix (Bioneer, South Korea), and 2 µl DNA template.…”