BMC Public Health 2007, 7:356 http://www.biomedcentral.com/1471-2458/7/356 intermediate (26%–75%); and high (>75%) pretest prob- abilities of active PTB. Classifications were made by four respiratory specialists (2 with 10 years of experience and 2 with 20 years of experience in diagnosing TB). This was an estimate of disease probability based on clinical history, physical examination, other laboratory data available besides microbiological tests, and chest radiographs eval- uation performed using a validated form [19]. Radiographic analysis Chest radiographs were classified as typical, compatible, atypical and normal. Typical were those considered as having any parenchymal infiltrate or cavity localized in the upper zone (defined as the area above the posterior third rib); compatible were those presenting a miliary pat- tern, pleural effusion or thoracic adenopathy, and atypical those showing any other abnormality [19]. Case definition PTB cases were defined as those with a positive culture for MTB in the respiratory specimen or those with clinical and radiological improvement after six months of solely anti- TB treatment, as judged by three different chest physicians in a blinded review, not involved in this study [20]. Non- PTB was considered in patients whose acid-fast smear and culture for MTB were negative and who had no chest radi- ographic changes after six months of follow-up. PCR results were not available for routine care or for the panel of experts. Gold standard criteria for SNPTB final diagnosis included all PTB cases, confirmed or not by culture. Routine laboratory process All sputum specimens were processed at the Public Refer- ence Laboratory. All sputum specimens were tested by the Ziehl- Neelsen method, cultured in Lowentein Jensen and identified according to Kubica's method [21]. The presence of the amplified fragment derived from IS6110 insertion element sequence in PCRs positives was checked by electrophoresis with 2% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light [16]. The positive and negative controls were included in electrophoresis analysis. The PCR colorimetric dot-blot assay was performed as previously published [16]. Briefly, the biotinylated PCR products were transferred to a nylon membrane and hybridization was performed with a specific probe. The detection of hybridization was performed using a conju- gated streptavidin-alkaline phosphatase probe. The posi- tive reaction was obtained by adding BCIP and NBT (5- bromo-4-chloro-3-indoyl phosphate and nitro blue tetra- zolium). The positive and negative controls were included for each set of PCR. To detect specimen inhibitors in neg- ative results, a tube of PCR mix for each specimen was spiked with purified DNA target. All PCRs tests with dis- crepancies in results were tested in duplicate. Data analysis Epidemiological and laboratory data were entered into a computer database and analyzed by appropriate statistical software (SPSS version 10.0). The endpoints were sensitiv- ity (SE), specificity (SP), positive and and positive and negative predictive values (PPV, NPV) for detection in smear-negative subjects. For MTB DNA detection, the analyses of PCR SE, SP, PPV, NPV were performed on a per-study-subject basis, using the diagnosis of PTB as a ref- erence standard (defined above). Agreement between the PCRs duplicates was evaluated using the Kappa score, a measurement of agreement that considers the excess of the amount of agreement that could be expected by chance. For secondary analysis, using the high pretest probability (HPP) as a diagnostic test, suspects of SNPTB with high pretest probabilities were considered as positive for active PTB, and those with intermediate and low pretest proba- bilities were considered negative. Additionally, test performances of HPP in parallel with PCR as a diagnostic test were calculated using specific for- mulas: SE of HPP with PCR: SEHPP + SEPCR- (SEHPP + SEPCR), predictive values (PV) for different prevalence rates according to the literature [22]. Results Of the total of 277 SNPTB suspects enrolled, 64 (23.1%) were not included in the analysis for the following rea- sons: 63 (22.6%) had an incomplete set of clinical data (14 patients had no chest X-ray available; 48 did not fulfill the SNPTB definition, one refused to participate, and the culture of one was contaminated. Of the 213 SNPTB sus- pects included in the analysis, all with known HIV test results, 104 (48.8%) were diagnosed with active TB. Strat- ifying by HIV status, the sensitivity of acid-fast bacilli in expectorated sputum was lower in HIV seropositive than in HIV seronegative suspects of SNPTB (67% vs 41%; p = 0.01). Among the 213 SNPTB suspects, 62 (29%) had pos- itive smear acid-fast bacilli tests and were excluded. In this study, data were analyzed for 151 SNPTB suspects with negative smear tests. Overall, active PTB was diagnosed in 28.4% (43/151) of patients, HIV infection in 29.8% (45/ 151), and a history of previous PTB was referred to by 35.0% (53/151). Positive culture results occurred in 69.8% (30/43) of all TB cases, and 73.7% (14/19) of HIV seropositive TB cases. Page 3 of 10 (page number not for citation purposes)