- Email: [email protected]
P1986 Evaluation of real-time PCR assays, based in molecular beacon technology, for detection and quantification of Mycobacterium tuberculosis DNA
Tuberculosis and other mycobacterial infections: diagnostic microbiology Results: Our results were shown an equal sensitivity and specificity between this method and ELISA colorimetric assay of PCR products. Conclusions: THDA-ELISA DIG detection system can be used more cost-effectively than PCR-ELISA for molecular detection of Mycobacterium tuberculosis in developing countries. The other formats of this technology are under study in real-time format and mobile format. P1984 Utility of the Gen-Probe amplified MTD test for the tuberculosis diagnosis in formalin-fixed, paraffin-embedded histologic specimens M. Ros, D. Mariscal, P. Ferreros, N. Combalia, R. Orellana, J.A. V´azquez, D. Fontanals, M. Torra, M. Rey (Sabadell, ES) Objective: To value the relationship among the mycobacteria culture, clinical and histopathologic findings compatible with tuberculosis (TBC) and the results of the Gen-Probe amplified MTD test (MTDT) in formalin-fixed, paraffin-embedded histologic specimens. Methodology: The affected anatomic sites of 24 biopsies were: lymph nodes (9); skin (3); synovial (2); colon (2); lung (1); pleural (1); peritoneal (1), parotid gland (1); bone marrow (1); spleen (1); breast (1); vertebral (1). All had presence of granuloma with multinucleated giant cells and/or caseous necrosis. To detect AFB, all smears were stained by Ziehl–Neelsen (ZN). Only 9 (37.5%) of the specimens were cultured for mycobacteria. A MTDT was considered positive when the result was greater than 500.000 RLU. The clinical information of the 24 patients was reviewed and those that have received antituberculous treatment or/and grew mycobacteria in the culture were considered cases of TBC. Results: 23 of the biopsies had chronic granulomatous inflammation and 1 caseous necrosis. Only the specimen with necrosis presented ZN positive. In 7 specimens (29.1%) MTDT was positive, clinical information was suggestive of TBC and in 3 mycobacteria grew. One patient had MTDT negative, clinical information suggestive of TBC and a positive culture. In two cases, MTDT was negative, there was no culture but they were treated as TBC. Sensibility, specificity, PPV and NPV are 70%, 100%, 100% and 82.3% respectively. Conclusions: Considering that there are cases in which the corresponding tissues are not sent for TBC culture, the MTDT can be a useful technique for the diagnosis of TBC in formalin-fixed specimens when clinical or histopathologic suspicion exists. P1985 Direct identification of mycobacteria from BacTec MGIT 960 system tubes: comparative evaluation of INNO-LiPA Mycobacteria v2 and DNA AccuProbe assays E. Mokaddas, S. Ahmad (Dasma, Kuwait, KW) Objective: Species-specific identification of mycobacteria is of clinical relevance since treatment varies according to the species causing infection. This study evaluated the performance of two commercial probe assays; INNO-LiPA MYCOBACTERIA v2 (LiPA) and AccuProbe for species-specific identification of mycobacterial isolates in Kuwait directly from MGIT 960 system tubes. Methods: Seventy-three respiratory and extrapulmonary specimens flagged positive for growth by MGIT 960 system and smear-positive for mycobacteria were evaluated for identification. The AccuProbe assay was performed and interpreted according to the manufacturer’s instructions. The DNA extraction, PCR amplification and hybridisation steps were performed by following the instructions supplied with the LiPA kit. The results were validated by PCR amplification and DNA sequencing of 16S-23S internal transcribed spacer (ITS) region. Results: Each of the 73 tubes grew one mycobacterial culture. Both AccuProbe and LiPA identified 47 isolates as Mycobacterium tuberculosis complex (MTC) members and ITS region sequences of 8 randomly selected isolates were concordant. AccuProbe assay identified 26 isolates as non-tuberculous mycobacteria (NTM) with species-specific identification of 9 isolates. The ITS region sequences were concordant with the NTM status for 17 isolates and species-specific identification
S571 of 7 of 9 isolates while two M. intracellulare strains were actually M. chimaera and M. avium complex sequevar MAC-C isolates. The LiPA identified 21 isolates as NTM with species (or complex)-specific identification of 19 isolates while no result was obtained for 5 isolates (no amplicons with LiPA primers). The ITS region sequencing confirmed the species (or complex)-specific identification by LiPA of all the 19 isolates while 2 isolates detected only as NTM by LiPA were identified as M. immunogenum and M. lentiflavum. The DNA sequencing also identified 4 isolates yielding no result in LiPA as M. kansasii and one isolate as M. chimaera. Conclusions: All the MTC isolates are correctly identified by both, the LiPA and AccuProbe assays. Although, AccuProbe assay identified only some (9 of 26) while LiPA identified most (19 of 26) NTM isolates to the species or complex level, the 4 M. kansasii isolates correctly identified by AccuProbe assay were not even detected by LiPA indicating variations in sequences targeted for LiPA amplification in M. kansasii strains in Kuwait. Supported by KURA grant MI02/04. P1986 Evaluation of real-time PCR assays, based in molecular beacon technology, for detection and quantification of Mycobacterium tuberculosis DNA D.P. Houhoula, A. Siatelis, N.J. Legakis, A. Tsakris, J. Papaparaskevas (Athens, GR) Objective: The evaluation of a conventional and two real-time PCR assays, in comparison to the standard culture for tuberculosis (Tb), and the patients’ clinical data. Materials and Methods: A total of 60 clinical samples submitted for Tb diagnosis, were tested using three PCR assays targeting IS6110. The samples were 30 (7 pulmonary and 23 extra-pulmonary) from 22 patients, for which Tb diagnosis was based either on positive culture, or clinical presentation, and 30 from 30 patients with other non-Tb diagnoses. The assays were a previously reported (Kox et al, JCM 1994) conventional PCR (C-PCR), a real-time PCR (RT1) using previously reported nested-PCR inner primers (Caws et al, JCM 2000) with a newly designed molecular beacon, and a novel real-time PCR (RT2). The Beacon Designer 5.0 software was used for all newly designed beacons (RT1, RT2) and primers (RT2). DNA quantification was performed using a standard curve of five positive concentrations (3.4 pg/mL, and 340, 34, 3.4, and 0.34 fg/mL) of DNA extracted from M. tuberculosis H37Rv strain. All samples were tested neat and diluted 1/10 for PCR inhibition. Real-time PCR runs were acceptable when the negative controls had undetectable cycle threshold (CT) values, and the first two positive DNA concentrations had CT values of 27 to 30 cycles. Results: Out of 30 samples from confirmed Tb cases, 28, 19 and 27 were positive using C-PCR, RT1, and RT2, respectively, corresponding to diagnostic sensitivities of 93%, 63% and 90%, respectively. Based on the DNA quantification performed, analytical sensitivities were 10 and 1 fg of DNA, for RT1 and RT2, respectively. When diluted specimens were examined, 5 inhibition cases were detected (1, 1 and 3 for C-PCR, RT1, and RT2, respectively). Combined performance of C-PCR+RT2 increased overall sensitivity to 100%. All 30 samples from non-Tb cases were negative with all assays (specificity 100%). RT2 quantification indicated that DNA extraction from tissue biopsies resulted in the lowest DNA yield compared to pleural/sputum samples (DNA load range 0.7– 659 fg, and 4.4–576 pg, respectively). However, a single pus specimen with DNA load of 2.7 ng was identified. Conclusions: RT2 and C-PCR were the most sensitive assays tested, and their combination resulted in 100% diagnostic sensitivity. Specificity was 100%. Real Time PCR, based on molecular beacon technology, seems to be a rapid and sensitive procedure for Tb diagnosis and DNA quantification in clinical samples.
S571 of 7 of 9 isolates while two M. intracellulare strains were actually M. chimaera and M. avium complex sequevar MAC-C isolates. The LiPA identified 21 isolates as NTM with species (or complex)-specific identification of 19 isolates while no result was obtained for 5 isolates (no amplicons with LiPA primers). The ITS region sequencing confirmed the species (or complex)-specific identification by LiPA of all the 19 isolates while 2 isolates detected only as NTM by LiPA were identified as M. immunogenum and M. lentiflavum. The DNA sequencing also identified 4 isolates yielding no result in LiPA as M. kansasii and one isolate as M. chimaera. Conclusions: All the MTC isolates are correctly identified by both, the LiPA and AccuProbe assays. Although, AccuProbe assay identified only some (9 of 26) while LiPA identified most (19 of 26) NTM isolates to the species or complex level, the 4 M. kansasii isolates correctly identified by AccuProbe assay were not even detected by LiPA indicating variations in sequences targeted for LiPA amplification in M. kansasii strains in Kuwait. Supported by KURA grant MI02/04. P1986 Evaluation of real-time PCR assays, based in molecular beacon technology, for detection and quantification of Mycobacterium tuberculosis DNA D.P. Houhoula, A. Siatelis, N.J. Legakis, A. Tsakris, J. Papaparaskevas (Athens, GR) Objective: The evaluation of a conventional and two real-time PCR assays, in comparison to the standard culture for tuberculosis (Tb), and the patients’ clinical data. Materials and Methods: A total of 60 clinical samples submitted for Tb diagnosis, were tested using three PCR assays targeting IS6110. The samples were 30 (7 pulmonary and 23 extra-pulmonary) from 22 patients, for which Tb diagnosis was based either on positive culture, or clinical presentation, and 30 from 30 patients with other non-Tb diagnoses. The assays were a previously reported (Kox et al, JCM 1994) conventional PCR (C-PCR), a real-time PCR (RT1) using previously reported nested-PCR inner primers (Caws et al, JCM 2000) with a newly designed molecular beacon, and a novel real-time PCR (RT2). The Beacon Designer 5.0 software was used for all newly designed beacons (RT1, RT2) and primers (RT2). DNA quantification was performed using a standard curve of five positive concentrations (3.4 pg/mL, and 340, 34, 3.4, and 0.34 fg/mL) of DNA extracted from M. tuberculosis H37Rv strain. All samples were tested neat and diluted 1/10 for PCR inhibition. Real-time PCR runs were acceptable when the negative controls had undetectable cycle threshold (CT) values, and the first two positive DNA concentrations had CT values of 27 to 30 cycles. Results: Out of 30 samples from confirmed Tb cases, 28, 19 and 27 were positive using C-PCR, RT1, and RT2, respectively, corresponding to diagnostic sensitivities of 93%, 63% and 90%, respectively. Based on the DNA quantification performed, analytical sensitivities were 10 and 1 fg of DNA, for RT1 and RT2, respectively. When diluted specimens were examined, 5 inhibition cases were detected (1, 1 and 3 for C-PCR, RT1, and RT2, respectively). Combined performance of C-PCR+RT2 increased overall sensitivity to 100%. All 30 samples from non-Tb cases were negative with all assays (specificity 100%). RT2 quantification indicated that DNA extraction from tissue biopsies resulted in the lowest DNA yield compared to pleural/sputum samples (DNA load range 0.7– 659 fg, and 4.4–576 pg, respectively). However, a single pus specimen with DNA load of 2.7 ng was identified. Conclusions: RT2 and C-PCR were the most sensitive assays tested, and their combination resulted in 100% diagnostic sensitivity. Specificity was 100%. Real Time PCR, based on molecular beacon technology, seems to be a rapid and sensitive procedure for Tb diagnosis and DNA quantification in clinical samples.