First-time detection and identification of the Mycobacterium tuberculosis Complex members in extrapulmonary tuberculosis clinical samples in south Tunisia by a single tube tetraplex real-time PCR assay

Abstract

IntroductionTunisia has one of the highest burdens of extrapulmonary tuberculosis (EPTB) among tuberculosis (TB) cases but the contribution of MTBC-mediated human EPTB is unknown. EPTB diagnosis is challenging due to the paucibacillary nature of clinical samples. Therefore, a need of a simplified molecular method for sensitive and specific TB detection and differentiation of MTBC members caused EPTB remains a priority to an early diagnosis, optimize successful anti-TB treatment and minimize transmission. We evaluated the performance of a single tube tetraplex Taq Man real time PCR for EPTB detection and differentiation between MTBC members directly on extrapulmonary samples.Materials and methodsExtrapulmonary samples obtained from clinically suspected EPTB patients from 2013 to April 2015 were tested by Ziehl Neelsen Staining, mycobacterial culture and qPCR assay for RD1, RD9, RD12 and ext-RD9 targets (MTBC-RD qPCR). The performance of qPCR was compared to a reference standard based on MTBC culture and/or at least two criteria of a composite reference standard (CRS) including clinical, radiological, histopathological and therapeutic findings.ResultsEPTB was identified in 157/170 (92.4%) of included patients of whom 99 (63%) were confirmed by culture and 58 (36.9%) by CRS criteria. The sensitivity and specificity of qPCR, in comparison to the reference standard were 100% (157/157) and 92.3% (12/13), respectively. The sensitivity of qPCR was statistically significant as compared to culture and smear microscopy (P< 0.001). QPCR results showed M. bovis identification in 77.1% of extrapulmonary samples in occurrence to lymphadenitis infection. M. tuberculosis and M.bovis BCG were detected in 21.6% and 1.3% of cases, respectively.ConclusionsMTBC–RD qPCR proved to be a rapid and sensitive assay for simultaneously TB detection and MTBC members identification on extrapulmonary samples within 1.5 days after sample receipt. Its high sensitivity could make this method a useful tool in diagnosing TB in addition to routine conventional methods and TB clinical parameters.

Rapid detection of mycobacterium tuberculosis complex by real-time polymerase chain reaction (PCR) in pulmonary and extra-pulmonary samples in Casablanca, Morocco

“…On the other hand, DNA can be detected from non-cultivable or dead MTC bacteria for the cases treated [ 25 ]. Therefore, our study confirms that 1) the use of culture or the PCR alone cannot be a criterion to determine the negativity of the results and 2) a comparison with the clinical data is strongly necessary when making decisions during diagnosis [ 26 ]. Real-time PCR showed a high sensitivity (95.2%) and a good specificity (95.4%) on pulmonary samples, notice that the same rates were reported by other authors [ 14 , 27 ].…”

A first insight into genetic diversity of Mycobacterium bovis isolated from extrapulmonary tuberculosis patients in South Tunisia assessed by spoligotyping and MIRU VNTR

“…In South Tunisia, almost 77% of clinically and bacteriologically diagnosed cases of EPTB are attributed to M . bovis [7]. To our knowledge, this is the first study that investigates molecular characterization of M .…”

“…In South Tunisia, Siala et al have detected M . bovis by qPCR in almost 77% of extrapulmonary samples taken from patients with EPTB [7]. In addition, LTB was estimated to be 50% of all EPTB cases of which cervical localization was the most frequent (70% to 90%) [5, 8].…”

“…bovis is still spreading in Tunisia causing bTB with a frequency of 64.4% in 2014–2015, whereas Lamine-khmiri et al , reported a frequency of 35% in 2013 [9, 10]. Consequently, the risk of contracting EPTB is still high since the control measures for herd, livestock and unpasteurized dairy products as well as milk are consistently declining [1, 7]. Nevertheless, no study evaluated the risk of transmission of zoonotic TB due to M .…”

Detection of a streptomycin-resistant Mycobacterium bovis strain through antitubercular drug susceptibility testing of Tunisian Mycobacterium tuberculosis complex isolates from cattle

“…A previous study demonstrated that raw milk consumers are at high risk of being infected with M. bovis [ 30 ]. Numerous clinical cases of human extrapulmonary tuberculosis due to M. bovis have been recently documented in Tunisia [ 31 , 32 ] and the consumption of unpasteurized dairy products has been indicated as the most likely source of transmission [ 33 ]. Implementation of effective and comprehensive strategies to control bTB and to prevent zTB are therefore of primary importance in the country.…”