P1987 Rapid diagnosis of tuberculous pleuritis by real-time PCR

S572 P1987 Rapid diagnosis of tuberculous pleuritis by real-time PCR K. Baba, S. Pathak, L. Sviland, A.M. Dyrhol-Riise, N. Langeland, A.A. Hoosen, B. Asjø, T. Mustafa (Bergen, NO; Pretoria, ZA) Tuberculosis is a public health problem. The hallmark of tuberculosis control is prompt diagnosis and treatment. This is even more important in suspected tuberculosis pleuritis (TP) for which accurate diagnosis is very crucial since delay in chemotherapeutic intervention is associated with poor prognosis. Diagnosis of TP requires microscopy and culture both of which has limitation, microscopy is not sensitive and culture takes time. The nested PCR (N-PCR) is used to detect M. tuberculosis complex organisms and genus specific real time PCR is used for detection of a broader spectrum of mycobacteria since atypical mycobacteria is common in HIV-TB coinfected patients. The real-time PCR (RT-PCR) has the advantage of not being prone to contamination and quicker because it does not require post amplification detection. The aim of this study was to assess the diagnostic potential of nested PCR of the IS6110 gene to detect M. tuberculosis complex organisms and of RT-PCR for genus specific hsp65 detection in the diagnosis of TP. Thirty-six paraffin-embedded pleural tissue samples were taken from the archives of the department of Pathology Dr. George Mukhari Hospital/ Medunsa Campus University of Limpopo Pretoria South Africa. Twentyfive biopsies were from patients with clinical TP, and 11 biopsies from cases with other diseases were used as controls. Nested PCR was performed using the Mycobacterium tuberculosis complex specific IS6110 primer and real-time PCR was performed using the genus specific hsp65 primer (ABI 7500 RT-PCR system). Sixteen samples were positive by N-PCR and 15/16 was positive by RTPCR. Out of the 11 non-TB cases 2 were positive by both N-PCR and RT-PCR while additional 2 cases were positive by only RT-PCR. Using clinical diagnosis as gold standard the sensitivity of N-PCR and RT-PCR were 64% and 76% while specificity was 82% and 64%, respectively. The sensitivity of RT-PCR using N-PCR as gold standard was 94%. This study showed that RT-PCR is equally sensitive to N-PCR in diagnosing TP. Its lower specificity might be due to presence of atypical mycobacteria. Therefore RT-PCR is highly sensitive, not prone to contamination, and its quicker turn around time will lead to rapid diagnosis of TP. P1988 Diagnosis of mycobacterium infection by TB-rapid-antigentest in patients from Belarus S. Zaker, L. Titov, A. Bahrmand, E. Sagalchyk, L. Surkova (Tehran, IR; Minsk, BY) Background: Tuberculosis (TB) remains an important problem in the world, but TB diagnosis is often difficult. The routinely used procedures for TB diagnosis in clinical laboratory either show poor sensitivity (microscopy) or take several weeks (culture) before results are obtained. The aim of the study was to estimate sensitivity and specificity of developed for indication MTB in sputum kit “Rapid-Test”. Method: In this study we tested 288 sputum samples from patients living in different regions of Belarus with aim to detect Mycobacterium tuberculosis by developed Rapid-Test. Sputum samples from patients were concentrated and washed in the specific cassettes containing nitrocellulose membrane with absorbing pads above it. MBT were detected by addition anti-TB monospecific rabbit serum and goldprotein A solution. Positive samples were characterised by displaying a red-to brown colour in central area of cassettes in the contrast to the blue-purple colour in negative cases. This new rapid test was compared with the results of acid-fast staining microscopy, specific culture for TB bacilli and specific PCR amplification of TB DNA segments. Results: In this study 107 samples from healthy persons were used as control and 177 sputums from patients with tuberculosis confirmed by culture method. We found that developed Rapid-Test was more effective for M. tuberculosis detection compared with specimen microscopy. Efficiency of Rapid-test in compare with “gold standard” of culture

17th ECCMID / 25th ICC, Posters investigation was 88.5% for Belarussian samples. Compared with culture for Belarussian samples Rapid-Test was false positive in 22 cases, false negative in 19 cases, sensitivity was 81%, specificity 93%, positive predictive value 66.3% and negative predictive value 89.9%. Meanwhile Ziehl–Neelsen microscopy did not give false positive results, false negative results were registered in 23 cases, efficiency of microscopy was estimated as 88%, sensitivity 68.5%, specificity 94.8%, PPV 82% and NPV 89.7%. Conclusion: Therefore, we observed 25% more sensitivity and 20% specificity of rapid test compared with microscopy for Mycobacterium tuberculosis detection. P1989 Evaluation of a rapid automated assay for the isolation of Mycobacterium tuberculosis DNA directly from clinical samples, using magnetic particles J. Lys´en, H.K. Høidal Berthelsen, M. Espelund, S. Jeansson, A.T. Mengshoel, U.H. Refseth (Oslo, NO) Objectives: The objective of this study was to evaluate the performance of a fully automated DNA preparation system, the BUGS’n BEADS™ MYCOBACTERIUM (Genpoint, Norway), to isolate Mycobacterium tuberculosis complex DNA from clinical respiratory samples. Method: The BUGS’n BEADS™ principle was used to develop an automated customised application. Briefly, the automatic isolation begins with addition of specially coated magnetic particles to Nalc-NaOHtreated clinical samples. Following bacterial capture, the sample matrix is removed. A rapid lysis at RT releases DNA, which is then adsorbed on to the same magnetic particles. Beads are washed and the purified DNA is robotically transferred for NAAT analysis. Samples were analysed both by using an in-house developed real-time PCR targeting the M. tuberculosis complex, and the Strand Displacement Amplification (SDA) part of the ProbeTec™ ET Mycobacterium tuberculosis Complex (DTB) Direct Detection (Becton Dickinson). Results: Up to 48 samples can be simultaneously analysed using the automated BUGS’n BEADS™ system, with as little as 20 minutes of hands-on for each run, and requiring a total time to results of four hours. For smear positive samples, the results for the automated BUGS’n BEADS™ system in combination with real-time PCR were comparable to culture. The combination of SDA with the automated BUGS’n BEADS™ was comparable to the in-house Real-Time PCR. For the smear negative samples, we have observed so far that lowering the predefined BD cut-off from 3400 to 1000, enabled to improve sensitivity without worsening specificity. No inhibition was recorded for the SDA. Conclusions: We here present a fully automated system for preparation of M. tuberculosis complex DNA from clinical respiratory samples, compatible with in-house developed and commercial downstream detection systems. Used with respiratory samples, the system will give clinically relevant results within one working day, requiring minimal hands-on. The combination with SDA demonstrated successful removal of inhibition, while opening for further improvement of sensitivity. To further challenge the system, and evaluate lowering cut off, nonrespiratory samples will be included in a more extensive study. P1990 Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates M. Unzaga, R. Blanco, M. Fernandez, A. Morla, L. Garcia, C. Ezpeleta, J. Alava, R. Cisterna (Bilbao, ES) Objectives: The rapid determination of drug resistance in clinical isolates of Mycobacterium tuberculosis is essential for the initiation of effective chemotherapy and preventing further spread of drugresistant isolates. Drug susceptibility testing by conventional methods takes several weeks although some new liquid medium-based systems have been commercially introduced with a shortened incubation time. A commercially DNA strip assay (Genotype MTBDR assay (Hain Lifescience Nehren, Germany) based on a multiplex PCR in combination