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Background: MTBDRplus line probe assay (LiPA) has been endorsed by the World Health Organization for pulmonary tuberculosis (TB) diagnosis. However, its value for Mycobacterium tuberculosis (MTB) detection in bronchoalveolar lavage (BAL) needs exploration. This study determined the diagnostic performance of MTBDRplus in BAL for MTB complex detection and isoniazid/rifampicin resistance in smear-negative and sputum scarce patients. Materials and Methods: Retrospective evaluation of data (January–December 2013) from patients who underwent bronchoscopy was done. Of these, patients with high TB suspicion with available data on acid-fast bacilli (AFB) smear/culture and MTBDRplus were selected. Results of MTBDRplus were compared with AFB smear/culture and drug susceptibility. Sensitivity and specificity of MTBDRplus was determined using TB culture as gold standard. Results: Data on 383 patients who underwent bronchoscopy were collected. Of these, 154 previously untreated TB suspect patients that were either smear negative on sputum microscopy or sputum scarce were selected. Out of 154 patients, 11 were smear positive and 34 patients were AFB culture positive. MTBDRplus detected MTB in 23/34 cases, the sensitivity and specificity being 67.6% and 85% (P < 0.001) versus 32.4% and 100% (P < 0.001) compared to smear microscopy. All smear-positive cases (n = 11) were detected by MTBDRplus. There were no discrepancies between phenotypic drug susceptibility testing and LiPA for isoniazid and rifampicin resistance in patients. Two cases of multidrug-resistant TB were detected. Conclusion: MTBDRplus detected TB more rapidly and accurately than smear microscopy with significant accuracy for isoniazid and rifampicin resistance. Its use in clinical practice would lead to rapid detection and effective management.

Background: This study aimed to compare the accuracy of Candida species identification by direct inoculation of positive blood culture specimens into CornMeal Tween™ (CMT) 80 agar with the conventional methods. Methods: This study was performed in two phases: Phase 1: Inoculation of direct CMT from artificially spiked blood culture bottles with identified Candida species. Phase 2: Direct inoculation of smear-positive bottles on CMT agar for identification. Routine identification method was carried out using urease production, sensitivity to cycloheximide, CMT inoculation, and colony morphology on BiGGY agar. All isolates were also evaluated for sugar assimilation on the biochemical test panel Analytical profile index (API) 20C AUX (BioMerieux). Statistical Analysis Used: The correlation between both methods was determined as percentage agreement rate using kappa score. Results: In the first phase 50 artificially spiked blood culture bottles were included in the study. The sample set consisted of 24% C albicans, 38% C tropicalis, and 22% C glabrata isolates. There was 100% agreement between the directly inoculated and CMT inoculated from colonies at 48 hours. Phase 2: The overall agreement between directly inoculated CMT and conventional method of identification at 72 h was 92%. Conclusion: Direct inoculation of CMT agar can easily differentiate common Candida species microscopically on the basis of chlamydospores, blastopores, and arrangement of pseudo hyphae. However, identification of rare species from specimens containing two different species using direct method is challenging. Therefore, conventional methods including macroscopic/microscopic characteristics and biochemical profile with the aid of API with API 20 AUX would be a better choice for such cases.

Background: Duration of digestion/decontamination has a considerable impact on yield of mycobacteria especially from sterile body fluids and pus specimens. Additionally, duration of digestion/decontamination affects the contamination rates. This study evaluates the effect of digestion/decontamination protocol for 15 and 20min versus inoculation of media directly from the sample on contamination rates as well as the yield of mycobacteria from pus and sterile fluids other than cerebrospinal fluids. Methods: Pleural fluid (n = 60), pus (n = 48) and ascitic fluid (n = 12) specimens were cultured for mycobacteria and evaluated for contamination and mycobacterial yield using three different processing methodologies: without digestion/decontamination with 5% NaOH-NALC (D/D), D/D for 15min and D/D for 20min. All samples >3 mL in volume were spun at 3000 RCF for 15min, whereas those less than 3 mL were used as is. They were simultaneously processed using the three different methods as mentioned above, and inoculated on LJ media and MGIT. Smear was made from samples treated for 20min and stained with fluorescent stain. Kinyoun staining was done on smears with dubious findings. Mycobacterial culture yield and contamination rates were recorded at 6 weeks as recommended by the Global Laboratory Initiative (GLI) laboratory manual 2014. Results: Pleural fluid and pus contamination rates were substantially lowered by increasing decontamination time from 15 to 20min, but it did not have any effect for ascitic fluid (Table 1). The 5-min difference in the decontamination procedure improved mycobacterial culture yield for pus samples by 10%, but there was no substantial effect on pleural and ascitic fluids. Prolonged decontamination did not compromise the culture yield in any of the mentioned specimens. Conclusion: In areas where specimen delay is common and sterility of collection procedure cannot be ensured, digestion/decontamination with NaOH-NALC for up to 20min can reduce contamination rates without considerably compromising mycobacterial culture yield. However, one should be alert to the possibility of decreased viability, and culture should be supplemented with molecular methods.

Background and objective: Pakistan is one of the high-tuberculosis (TB) burden countries (HBCs) with a considerable proportion of multidrug-resistant TB (MDR-TB) strains. Recently, high resistance to fluoroquinolone has been reported in both MDR and non-MDR-TB strains from Pakistan. This study aims to evaluate trends of resistance in Mycobacterium tuberculosis (MTB) against injectable second-line drugs (2010–July 2014) from Aga Khan University Laboratory, a technical partner of the Pakistan National TB Program and part of the WHO Supra-national Laboratory Network for TB based in Karachi, Pakistan. Methods: MTB strains were isolated using standard methods. Susceptibility testing was performed using the agar proportion method with drug concentrations as recommended by the Clinical Laboratory Institute Standards (CLSI). MTB H37Rv was used as a control with each batch of susceptibility testing. MDR-TB was defined as resistance to both isoniazid (0.2 μg/ml) and rifampicin (1.0 μg/ml). Results: During the study period, 14,711 MTB strains were isolated. Of these, 43.5% were MDR and 56.5% were non-MDR. Resistance pattern to amikacin, kanamycin and capreomycin over the years is shown in the table below. [INLINE:1] Conclusions: Rising resistance to injectable anti-tuberculous agents in MDR-TB isolates possibly reflects their increasing use in patient management. Presence of capreomycin resistance even amongst non-MDR strains is alarming for a country where fluoroquinolone resistance is also high and patient follow-up and drug compliance is a challenge.

Aims and objective: Despite effective treatment of pulmonary tuberculosis (TB) patients, destruction of lung parenchyma may lead to complications including repeated infections. These infections are often misdiagnosed or wrongly identified as TB recurrence, and hence are not treated effectively. The frequency and severity of these infections vary with the extent of damage, and are much more prominent in patients with post-TB bronchiectasis and fibrocavitary diseases. This presentation will focus on the epidemiology, treatment, and management of post-TB infections and challenges, and the impact of these infections on public health in high-TB-burden countries. Methods: Published literature and review articles were evaluated to address this objective. Results: Apart from conventional agents of pneumonia, patients with post-TB bronchiectasis and post-TB fibrocavitary diseases are prone to develop chronic pulmonary aspergillosis and nontuberculous mycobacterial infections. A high burden of chronic pulmonary aspergillosis has been reported in TB-endemic countries. Similarly, prior TB increases the risk of acquiring nontuberculous mycobacterial infections. Diagnosis and management of chronic pulmonary aspergillosis and nontuberculous mycobacterial infections require expertise and high-level care. Conclusion: Limited diagnostic and therapeutic capacities compounded by nonavailability of essential antimicrobials in most high-TB-burden countries pose great challenges to physicians involved in the management of these infections. These infections affect the overall outcome and lead to high cost for public health systems.

Background: Pakistan has an annual tuberculosis (TB) incidence rate of 233 per 100,000 population and ranks sixth among the high TB burden countries worldwide. Tuberculous pleural effusion (TPE) occurs in up to 25% of TB patients. Owing to the pauci-bacillary nature of the pleural fluid, the diagnosis of TPE is a challenge. Objective: The aim of the present study is to evaluate the performance of MTBDRplus line probe assay in terms of its sensitivity and specificity for the rapid TB diagnosis in TPE and frequency of multidrug-resistant TB (MDR-TB) in TPE through the detection of rifampicin (Rif) and isoniazid (INH) resistance. Methods: This study is an ongoing prospective cross-sectional study being performed at Aga Khan University Hospital. Patients aged 18 years or more with suspected TPE were asked to participate. In addition to a preformed questionnaire which gathered information regarding clinical history and previous laboratory investigations, already tapped pleural effusion samples are tested for pleural fluid composition, TB smear and culture, MTBDRplus line probe assay and ADA analysis. Results: So far, 22 patients with a mean age of 49.81±21.75 (range: 18–86; IQR: 29.25, 68.00) years have been enrolled. Almost two-thirds are males (n = 15, 68.2%). The majority of the patient population belongs to Karachi (n = 18, 81.8%), followed by Quetta (n = 2, 9.1%), Faisalabad (n = 1, 4.5%) and Sukkur (n = 1, 4.5%). Half of the patients had a history of TB contact (n = 11, 50.0%), whereas only two (9.1%) had a past history of TB. Fifty percent (n = 11) had a right-sided pleural effusion. Effusion size was moderate in 36.4% (n = 8), mild in 27.3% (n = 6) and massive in 22.5% (n = 5). Pleural fluid MTBDRplus positively detected TB in 9.1% (n = 2) of cases. INH resistance was detected in one of these two cases. Pleural fluid culture was positive in two cases. Pleural fluid AFB smear was negative in all cases. Conclusion: Given the study is still under way, it is hoped that the results of this study may investigate the relevance, sensitivity and specificity of MTBDRplus line probe assay as a tool in the rapid detection of MTB in TPE and the early detection of MDR-TB through the detection of Rif and INH resistance. It may also provide an estimate of prevalence of drug resistant strains in TPE, which would be useful for guiding empirical anti-tuberculous therapy in TPE.

Introduction: In 2008, the World Health Organization (WHO) approved a rapid molecular test known as Line Probe Assay (LiPA) for the diagnosis of multidrug-resistant tuberculosis (MDR-TB) in pulmonary specimens. Due to lack of available data, its use in extra-pulmonary tuberculosis (EPTB) specimens has not been determined yet. Recommendations for the use of Xpert MTB/RIF in EPTB have been issued, but alternative rapid molecular tests for EPTB diagnosis need to be evaluated. Method: LiPA (GenoType® MTBDRplus 2.0 Hain Lifescience) was performed on 97 specimens of extrapulmonary origin at the clinical laboratory at Aga Khan University (Dec 2012–Jan 2014). The results were compared with TB cultures in solid and liquid medium. Results: 97 specimens were tested simultaneously for culture and LiPA, including pleural fluid (35), CSF (22), pus (17), tissue (10) and urine (3). Concentrated smear was positive for 7 while 14 were culture positive for MTB. All 7/7 smear-positive specimens were LiPA positive, while 6/7 were culture positive. Amongst the smear-negative specimens, 8/90 were culture positive and 9/90 were LiPA positive. The overall sensitivity and specificity of LiPA for the detection of MTB in the EPTB specimens was 71.4% (95% CI 41.9–91.4) and 92.8% (95% CI 84.9–97.3), respectively. The highest sensitivity (100%) was seen in urine as 2 of 3 which were culture positive were also LiPA positive. Pus samples showed sensitivity of 85.7% and specificity of 70%. Conclusion: The study shows that LiPA has good overall sensitivity and specificity compared with culture. Although the number of samples was very small, the applicability appears to be most useful in urine and pus specimens and should be explored further as a diagnostic tool in these cases.