Background Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect. Methods We assessed the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), with fully integrated sample processing in 1730 patients with suspected drug-sensitive or multidrug-resistant pulmonary tuberculosis. Eligible patients in Peru, Azerbaijan, South Africa, and India provided three sputum specimens each. Two specimens were processed with N-acetyl-l-cysteine and sodium hydroxide before microscopy, solid and liquid culture, and the MTB/RIF test, and one specimen was used for direct testing with microscopy and the MTB/RIF test. Results Among culture-positive patients, a single, direct MTB/RIF test identified 551 of 561 patients with smear-positive tuberculosis (98.2%) and 124 of 171 with smear-negative tuberculosis (72.5%). The test was specific in 604 of 609 patients without tuberculosis (99.2%). Among patients with smear-negative, culture-positive tuberculosis, the addition of a second MTB/RIF test increased sensitivity by 12.6 percentage points and a third by 5.1 percentage points, to a total of 90.2%. As compared with phenotypic drug-susceptibility testing, MTB/RIF testing correctly identified 200 of 205 patients (97.6%) with rifampin-resistant bacteria and 504 of 514 (98.1%) with rifampin-sensitive bacteria. Sequencing resolved all but two cases in favor of the MTB/RIF assay. Conclusions The MTB/RIF test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than 2 hours with minimal hands-on time. (Funded by the Foundation for Innovative New Diagnostics.)

SummaryBackgroundThe Xpert MTB/RIF test (Cepheid, Sunnyvale, CA, USA) can detect tuberculosis and its multidrug-resistant form with very high sensitivity and specificity in controlled studies, but no performance data exist from district and subdistrict health facilities in tuberculosis-endemic countries. We aimed to assess operational feasibility, accuracy, and effectiveness of implementation in such settings.MethodsWe assessed adults (≥18 years) with suspected tuberculosis or multidrug-resistant tuberculosis consecutively presenting with cough lasting at least 2 weeks to urban health centres in South Africa, Peru, and India, drug-resistance screening facilities in Azerbaijan and the Philippines, and an emergency room in Uganda. Patients were excluded from the main analyses if their second sputum sample was collected more than 1 week after the first sample, or if no valid reference standard or MTB/RIF test was available. We compared one-off direct MTB/RIF testing in nine microscopy laboratories adjacent to study sites with 2–3 sputum smears and 1–3 cultures, dependent on site, and drug-susceptibility testing. We assessed indicators of robustness including indeterminate rate and between-site performance, and compared time to detection, reporting, and treatment, and patient dropouts for the techniques used.FindingsWe enrolled 6648 participants between Aug 11, 2009, and June 26, 2010. One-off MTB/RIF testing detected 933 (90·3%) of 1033 culture-confirmed cases of tuberculosis, compared with 699 (67·1%) of 1041 for microscopy. MTB/RIF test sensitivity was 76·9% in smear-negative, culture-positive patients (296 of 385 samples), and 99·0% specific (2846 of 2876 non-tuberculosis samples). MTB/RIF test sensitivity for rifampicin resistance was 94·4% (236 of 250) and specificity was 98·3% (796 of 810). Unlike microscopy, MTB/RIF test sensitivity was not significantly lower in patients with HIV co-infection. Median time to detection of tuberculosis for the MTB/RIF test was 0 days (IQR 0–1), compared with 1 day (0–1) for microscopy, 30 days (23–43) for solid culture, and 16 days (13–21) for liquid culture. Median time to detection of resistance was 20 days (10–26) for line-probe assay and 106 days (30–124) for conventional drug-susceptibility testing. Use of the MTB/RIF test reduced median time to treatment for smear-negative tuberculosis from 56 days (39–81) to 5 days (2–8). The indeterminate rate of MTB/RIF testing was 2·4% (126 of 5321 samples) compared with 4·6% (441 of 9690) for cultures.InterpretationThe MTB/RIF test can effectively be used in low-resource settings to simplify patients' access to early and accurate diagnosis, thereby potentially decreasing morbidity associated with diagnostic delay, dropout and mistreatment.FundingFoundation for Innovative New Diagnostics, Bill & Melinda Gates Foundation, European and Developing Countries Clinical Trials Partnership (TA2007.40200.009), Wellcome Trust (085251/B/08/Z), and UK Department for International Development.

Current nucleic acid amplification methods to detect

Bacterial 16S ribosomal RNA (rRNA) genes contain nine "hypervariable regions" (V1-V9) that demonstrate considerable sequence diversity among different bacteria. Species-specific sequences within a given hypervariable region constitute useful targets for diagnostic assays and other scientific investigations. No single region can differentiate among all bacteria; therefore, systematic studies that compare the relative advantage of each region for specific diagnostic goals are needed. We characterized V1-V8 in 110 different bacterial species including common blood borne pathogens, CDC-defined select agents and environmental microflora. Sequence similarity dendrograms were created for hypervariable regions V1-V8, and for selected combinations of regions or short segments within individual hypervariable regions that might be appropriate for DNA probing and real-time PCR. We determined that V1 best differentiated among Staphylococcus aureus and coagulase negative Staphylococcus sp. V2 and V3 were most suitable for distinguishing all bacterial species to the genus level except for closely related enterobacteriaceae. V2 best distinguished among Mycobacterium species and V3 among Haemophilus species. The 58 nucleotides-long V6 could distinguish among most bacterial species except enterobacteriaceae. V6 was also noteworthy for being able to differentiate among all CDC-defined select agents including Bacillus anthracis, which differed from B. cereus by a single polymorphism. V4, V5, V7 and V8 were less useful targets for genus or species-specific probes. The hypervariable sequence-specific dendrograms and the "MEGALIGN" files provided online will be highly useful tools for designing specific probes and primers for molecular assays to detect pathogenic bacteria, including select agents.

Escherichia coli O157:H7, a toxin-producing food and waterborne bacterial pathogen, has been linked to large outbreaks of gastrointestinal illness for more than two decades. E. coli O157 causes a wide range of clinical illness that varies by outbreak, although factors that contribute to variation in disease severity are poorly understood. Several recent outbreaks involving O157 contamination of fresh produce (e.g., spinach) were associated with more severe disease, as defined by higher hemolytic uremic syndrome and hospitalization frequencies, suggesting that increased virulence has evolved. To test this hypothesis, we developed a system that detects SNPs in 96 loci and applied it to >500 E. coli O157 clinical strains. Phylogenetic analyses identified 39 SNP genotypes that differ at 20% of SNP loci and are separated into nine distinct clades. Differences were observed between clades in the frequency and distribution of Shiga toxin genes and in the type of clinical disease reported. Patients with hemolytic uremic syndrome were significantly more likely to be infected with clade 8 strains, which have increased in frequency over the past 5 years. Genome sequencing of a spinach outbreak strain, a member of clade 8, also revealed substantial genomic differences. These findings suggest that an emergent subpopulation of the clade 8 lineage has acquired critical factors that contribute to more severe disease. The ability to detect and rapidly genotype O157 strains belonging to such lineages is important and will have a significant impact on both disease diagnosis and treatment guidelines.pathogens ͉ polymorphisms ͉ population genetics E nterohemorrhagic Escherichia coli (EHEC) includes a diverse population of Shiga toxin-producing E. coli that causes outbreaks of food and waterborne disease (1-3). EHEC often resides in bovine reservoirs and is transmitted via many food vehicles including cooked meat, such as hamburger (4) and salami (5), and raw vegetables, such as lettuce (6, 7) and spinach (8). In North America, E. coli O157:H7 is the most common EHEC serotype contributing to Ͼ75,000 human infections (9) and 17 outbreaks (3) per year.It is not clear why outbreaks of EHEC O157 vary dramatically in the severity of illness and the frequency of the most serious complication, hemolytic uremic syndrome (HUS) (10-12). The 1993 outbreak in western North America (4) and the large 1996 outbreak in Japan (13) had low rates of hospitalization and HUS (14, 15), whereas the 2006 North American spinach outbreak (8) had high rates of both hospitalization (Ͼ50%) and HUS (Ͼ10%). One hypothesis is that outbreak strains differ in virulence as a result of variation in the presence and expression of different Shiga toxin (Stx) gene combinations (16)(17)(18)(19).To assess the genetic diversity and variability in virulence among E. coli O157 strains, we developed a real-time PCR system for identifying synonymous and nonsynonymous mutations as SNPs (20-23). Although molecular subtyping methods, such as pulsedfield gel electrophoresis (PFGE), ...