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ABSTRACTGiven the increases in drug-resistant tuberculosis, laboratory capacities for drug susceptibility testing are being scaled up worldwide. A laboratory must decide among several endorsed methodologies. We evaluated 87 Mycobacterium tuberculosisisolates for concordance of susceptibility results across six methods: the L-J proportion method, MGIT 960 SIRE AST, Gene/Xpert MTB/RIF, GenoType MTBDRplusline probe assay, MycoTB MIC plate, and a laboratory-developed mycobacteriophage quantitative PCR (qPCR)-based method. Most (80%) isolates were multidrug resistant. Of the culture-based methods, the mycobacteriophage qPCR method was fastest, the L-J proportion method was the slowest, and the MGIT method required the most repeat testing (P< 0.05). For isoniazid (INH), 82% of isolates were susceptible by all methods or resistant by all methods, whereas for rifampin (RIF), ethambutol (EMB), and streptomycin (STR), such complete concordance was observed in 77%, 50%, and 51% of isolates, respectively (P< 0.05 for INH or RIF versus EMB or STR). The discrepancies of EMB and STR stemmed largely from diminished concordance of the MGIT EMB results (kappa coefficient range, 0.26 to 0.30) and the L-J STR result (kappa range, 0.35 to 0.45) versus other methods. Phage qPCR and the MycoTB MIC plate were the only methods that yielded second-line susceptibilities and revealed significant quantitative correlations for all drugs except cycloserine, as well as moderate to excellent kappa coefficients for all drugs except for para-aminosalicylic acid. In summary, the performance of M. tuberculosissusceptibility testing differs by platform and by drug. Laboratories should carefully consider these factors before choosing one methodology, particularly in settings where EMB and STR results are clinically important.

ABSTRACTThe noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolyticaparasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolyticaDNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolyticaDNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolyticaDNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolyticaDNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolyticaDNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolyticaDNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.

ABSTRACTPyrazinamide (PZA) is a frontline antituberculosis (anti-TB) drug used in both first- and second-line treatment regimens. However, due to complex laboratory requirements, the PZA susceptibility test is rarely performed, leading to a scarcity of data on susceptibility to PZA. Bangladesh is a country with a burden of high rates of both TB and multidrug-resistant TB (MDR-TB), but to our knowledge, published data on rates of PZA susceptibility (PZAs), especially among MDR-TB patients, are limited. We aimed to analyze the PZA susceptibility patterns of Mycobacterium tuberculosisisolates from MDR-TB patients and to correlate the pncAmutation with PZA resistance in Bangladesh. A total of 169 confirmed MDR M. tuberculosisisolates from a pool of specimens collected in a nationwide surveillance study were included in this analysis. All the isolates were tested for phenotypic PZA susceptibility in Bactec mycobacterial growth indicator tube (MGIT) culture medium, and the pncAgene was sequenced. We also correlated different types of clinical information and treatment outcomes with PZA susceptibility. We found that 45% of isolates were phenotypically PZA resistant. Sequencing of the pncAgene revealed a high concordance (82.2%) between the pncAgene sequence and the phenotypic assay results. A total of 64 different mutations were found, and 9 isolates harbored multiple mutations. We detected 27 new pncAmutations. We did not find any significant correlation between the different clinical categories, the genetic lineage, or treatment outcome group and PZA susceptibility. Considering the turnaround time, sequencing would be the more feasible option to determine PZA susceptibility, and further studies to investigate the MIC of PZA should be conducted to determine an effective dose of the drug.

ABSTRACTGenotypic methods for drug susceptibility testing of Mycobacterium tuberculosisare desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncAgenes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes.IMPORTANCEMultidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics.