Detection and Identification of Mycobacterium tuberculosis in Joint Biopsy Specimens by rpoB PCR Cloning and Sequencing

Abstract

Osteoarticular tuberculosis (OAT) is an extrapulmonary tuberculosis and accounts for 1 to 3% of all tuberculosis cases. We used an rpoB PCR-plasmid TA cloning-sequencing method to detect and identify tubercle bacilli in surgical specimens from patients suspected of having OAT. By comparing the similarities of the rpoB sequences determined with those in GenBank, Mycobacterium tuberculosis was detected in 23 of 43 samples. Three of the 23 positive samples had mutations at codon 531, which are commonly observed in rifampin-resistant M. tuberculosis strains. Our results suggest that the rpoB PCR-TA cloning-sequencing method developed, which detects M. tuberculosis and which simultaneously determines its rifampin susceptibility, can also be used efficiently for the diagnosis of OAT.The World Health Organization reported that nearly a third of the world's population suffers from tuberculosis (TB). Each year 8 million individuals have active disease, and 2 million deaths occur annually (28). With this resurgence, cases of extrapulmonary TB have also shown an increase. Approximately, 10 to 11% of extrapulmonary TB cases involve joints and bones (1 to 3% of all reported TB cases).Thus, the estimated global prevalence of latent joint and bone TB is approximately 19 million to 38 million cases (17). Moreover, since osteoarticular tuberculosis (OAT) can cause functional disability, it should be accurately diagnosed and treated early.As the identification of mycobacterial species from clinical samples usually requires culture (26), the diagnosis of OAT depends upon microbiologic testing (i.e., smear or culture) and the histologic examination of tissue samples. Although culture is the "gold standard," it may take 6 to 8 weeks before a positive culture is detected (22), unless the radiometric BACTEC 460 method or the nonradiometric BACTEC 960/ Mycobacteria Growth Indicator Tube method (4) is used.In recent years, several nucleic acid-based techniques have been developed for the rapid detection of Mycobacterium tuberculosis in clinical samples. By testing sputa and bronchoalveolar lavage specimens, mycobacteria can be detected and identified by PCR or PCR-linked methods (1, 18). However, unlike pulmonary TB samples, such as sputa and bronchoalveolar lavage specimens, from which tubercle bacilli are concentrated for culture and further testing, joint biopsy samples usually contain only a small number of bacteria. This causes difficulties with culture and staining for acid-fast bacilli (8,17,19) that necessitate the use of molecular biology-based methods.In the present study, rpoB PCR-plasmid TA cloning-sequencing for Mycobacterium species was applied directly to clinical specimens from patients suspected of having OAT without culture. rpoB encodes the ␤ subunit of RNA polymerase (3), and recently, partial rpoB DNA sequences containing the Rif r region, which is related to rifampin resistance, have been used to identify Mycobacterium species (5, 10, 11) and non-Mycobacterium species (12,13,14,15,16).M. tuberculosis is readily dif...

Drug-Resistant Tubercular Uveitis

“…MDR-TB was diagnosed by MTBDRplus assay in 32.14% of 30 PCR-positive TBM cases (4), and rifampin resistance was detected by rpoB gene sequencing in 13% of OATB cases (5). Mutations for rifampin resistance at codons 531, 516, and 526, as seen in our patients, have been documented from other extrapulmonary sites (4,5).…”

Tuberculose ostéoarticulaire extravertébrale

“…Dans quatre cas d'arthrites tuberculeuses, une nested PCR a été positive dans tous les cas [43]. Dans une étude sud-coréenne [44], recherchant par PCR la partie rpoB de l'ADN, 53 % des 43 prélèvements chirurgicaux d'origine articulaire étaient positifs. Cette technique reste mal validée dans les TOA et de nombreux problèmes techniques existent, restreignant son utilisation en pratique.…”

High-Level Rifampin Resistance Correlates with Multiple Mutations in the rpoB Gene of Pulmonary Tuberculosis Isolates from the Afghanistan Border of Iran

“…Correlations between high mutation rates, the geographic distribution of mutations, antibiotic resistance, and virulence in bacteria have been reported in several studies (9,20,33,37). Knowledge of geographic variations is important for monitoring rifampin (rifampicin) resistance within a defined population of patients infected with Mycobacterium tuberculosis, as the prevalence of the mutations studied so far varies for M. tuberculosis strains isolated from different countries (24,26,29,33,36). In 2004, the prevalence of tuberculosis in Iran was reported to be 17 per 100,000, and at the southern border of Iran (Zabol province) where tuberculosis is endemic, the prevalence was 141 per 100,000 (20).…”