title: Identification ofMycobacterium tuberculosisin 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 ofMycobacterium 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 ofIS6110, IS1081, hsp65kd, mbp64, andmtp40. 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 toIS6110- 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 onlyIS1081based PCR showed identical positivity rate with AFB stain (30.8%). The results for bone and wound specimens, however, demonstrated an improved yield mainly by employingIS1081gene. 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 ofIS1081followed bymtp40andIS6110, 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.
authors.affiliation: Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical SciencesAhvaz, Iran; Health Research Institute, Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical SciencesAhvaz, Iran.
authors.forename: Azar D
authors.initials: AD
authors.lastname: Khosravi
authors.affiliation: Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences Ahvaz, Iran.
authors.forename: Ameneh
authors.initials: A
authors.lastname: Alami
authors.affiliation: Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences Ahvaz, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Meghdadi
authors.affiliation: Aria Hospital Ahvaz, Iran.
authors.forename: Atta A
authors.initials: AA
authors.lastname: Hosseini
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D005798:Genes, Bacterial; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis*
keywords: Mycobacterium tuberculosis; clinical specimens; extrapulmonary tuberculosis; gene; nested PCR