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ABSTRACTThe Epistem Genedrive assay rapidly detects the Mycobacterium tuberculosiscomplex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spiking M. tuberculosismc26030 cells into distilled water and M. tuberculosis-negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Genedrive to that of the Xpert MTB/RIF assay using M. tuberculosiscultures as the reference standard. The Genedrive assay had an LOD of 1 pg/µl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 104CFU/ml and 2.5 × 105CFU/ml for cells spiked into water and sputum, respectively. False-positive rpoBprobe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containing Mycobacterium abscessus, Mycobacterium gordonae, or Mycobacterium thermoresistibile. In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Genedrive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Genedrive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay.

ABSTRACTThe Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection of Mycobacterium tuberculosisin sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions of M. tuberculosisinto sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained from M. tuberculosisculture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in which M. tuberculosiswas detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%; P= 0.03) and for smear-negative specimens (67% versus 58%; P= 0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increased M. tuberculosisdetection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.

ABSTRACTSmear microscopy has suboptimal sensitivity, and there is a need to improve its performance since it is commonly used to diagnose tuberculosis (TB). We prospectively evaluated the diagnostic accuracy of the small membrane filtration (SMF) method, an approach that uses a vacuum manifold and is designed to concentrate bacilli onto a filter that can be examined microscopically. We enrolled hospitalized adults suspected to have pulmonary TB in Kampala, Uganda. We obtained a clinical history and three spontaneously expectorated sputum specimens for smear microscopy (direct, concentrated, and SMF), MGIT (mycobacterial growth indicator tube) 960 and Lowenstein-Jensen (LJ) cultures, and Xpert MTB/RIF testing. We performed per-specimen (primary) and per-patient analyses. From October 2012 to June 2013, we enrolled 212 patients (579 sputum specimens). The participants were mostly female (63.2%), and 81.6% were HIV infected; their median CD4 cell count was 47 cells/µl. Overall, 19.0%, 20.4%, 27.1%, 25.2%, and 25.9% of specimens tested positive by direct smear, concentrated smear, MGIT culture, LJ culture, and Xpert test, respectively. In the per-specimen analysis, the sensitivity of the SMF method (48.5%; 95% confidence interval [CI], 37.4 to 59.6) was lower than those of direct smear (60.9%; 51.4 to 70.5 [P= 0.0001]) and concentrated smear (63.3%; 53.6 to 73.1 [P< 0.0001]). Subgroup analyses showed that SMF performed poorly in specimens having a low volume or low bacterial load. The SMF method performed poorly compared to standard smear techniques and was sensitive to sample preparation techniques. The optimal laboratory SMF protocol may require striking a fine balance between sample dilution and filtration failure rate.

ABSTRACTGenoType MTBDRplus is a molecular assay for detection of Mycobacterium tuberculosisand drug resistance. Assay performance as applied directly to consecutive unselected sputum samples has not been established. The objective of this study was to determine the accuracy of the MTBDRplus test for direct detection of M. tuberculosis(in sputum) and for drug resistance in consecutively submitted sputum samples. In this cross-sectional study in South Africa, one sputum specimen from each person suspected of having pulmonary tuberculosis was tested by smear microscopy, direct MTBDRplus, and Mycobacterial Growth Indicator Tube (MGIT) culture with MGIT drug susceptibility testing. MGIT results were the reference standard. We tested 2,510 sputum samples, and 529 (21.1%) were positive for M. tuberculosisby MGIT. Direct MTBDRplus identified M. tuberculosisin 256 of 529 specimens (sensitivity, 48.4%; 95% confidence interval [CI], 44.1, 52.7). The sensitivity of MTBDRplus for M. tuberculosisdetection by sputum smear status was as follows: smear negative, 13.7% (95% CI, 9.8, 18.4); smear scanty, 46.2% (95% CI, 19.2, 74.9); smear 1+, 69.1% (95% CI, 55.2, 80.9); smear 2+, 86.3% (95% CI, 73.7, 94.3); smear 3+, 89.8% (95% CI, 83.7, 94.2). Direct MTBDRplus testing was negative for 1,594/1,612 sputum samples that were culture negative for M. tuberculosis(specificity, 98.9%; 95% CI, 98.2, 99.3). For specimens positive for M. tuberculosisby MTBDRplus, this assay's sensitivity and specificity for rifampin resistance were 85.7% (95% CI, 57.2, 98.2) and 96.6% (95% CI, 93.2, 98.6) and for isoniazid resistance they were 62.1% (95% CI, 42.3, 79.3) and 97.9% (95% CI, 94.8, 99.4). For sputum testing, the sensitivity of MTBDRplus is directly related to the specimen's bacillary burden. Our results support recommendations that the MTBDRplus test not be used for direct testing of smear-negative or paucibacillary sputum samples.

The molecular methodologies used in our laboratories have allowed us to define a group of Nocardia isolates from clinical samples which resemble the type strain of Nocardia veterana. Three patient isolates and the type strain of N. veterana gave identical and distinctive restriction fragment length polymorphisms (RFLPs) for an amplified portion of the 16S rRNA gene. These three isolates and the N. veterana type strain also gave identical RFLPs for an amplified portion of the 65-kDa heat shock protein gene, but this pattern was identical to that obtained for the Nocardia nova type strain. Sequence analysis of both a 1,359-bp region of the 16S rRNA gene and a 441-bp region of the heat shock protein gene of the patient isolates showed 100% identities with the same regions of the N. veterana type strain. DNA-DNA hybridization of the DNA of one of the patient isolates with the DNA of the N. veterana type strain showed a relative binding ratio of 82%, with 0% divergence, confirming that the isolate was N. veterana. Biochemical and susceptibility testing showed no significant differences among the patient isolates and the N. veterana type strain. Significantly, the results of antimicrobial susceptibility testing obtained for our isolates were similar to those obtained for N. nova, indicating that susceptibility testing alone cannot discriminate between these species. We present two case studies which show that N. veterana is a causative agent of pulmonary disease in immunocompromised patients residing in North America. We also describe difficulties encountered in using 16S rRNA gene sequences alone for discrimination of N. veterana from the related species Nocardia africana and N. nova because of the very high degree of 16S rRNA gene similarity among them.

ABSTRACTThe molecular methodologies used in our laboratories have allowed us to define a group of Nocardiaisolates from clinical samples which resemble the type strain of Nocardia veterana. Three patient isolates and the type strain of N. veteranagave identical and distinctive restriction fragment length polymorphisms (RFLPs) for an amplified portion of the 16S rRNA gene. These three isolates and the N. veteranatype strain also gave identical RFLPs for an amplified portion of the 65-kDa heat shock protein gene, but this pattern was identical to that obtained for the Nocardia novatype strain. Sequence analysis of both a 1,359-bp region of the 16S rRNA gene and a 441-bp region of the heat shock protein gene of the patient isolates showed 100% identities with the same regions of the N. veteranatype strain. DNA-DNA hybridization of the DNA of one of the patient isolates with the DNA of the N. veteranatype strain showed a relative binding ratio of 82%, with 0% divergence, confirming that the isolate was N. veterana. Biochemical and susceptibility testing showed no significant differences among the patient isolates and the N. veteranatype strain. Significantly, the results of antimicrobial susceptibility testing obtained for our isolates were similar to those obtained for N. nova, indicating that susceptibility testing alone cannot discriminate between these species. We present two case studies which show that N. veteranais a causative agent of pulmonary disease in immunocompromised patients residing in North America. We also describe difficulties encountered in using 16S rRNA gene sequences alone for discrimination of N. veteranafrom the related species Nocardia africanaand N. novabecause of the very high degree of 16S rRNA gene similarity among them.

ABSTRACTA decision analysis was conducted to evaluate the cost-effectiveness of programs in which the Amplified Mycobacterium Tuberculosis Direct test (MTD) (Gen-Probe) is used to rapidly exclude Mycobacterium tuberculosiscomplex as a cause of disease in smear-positive respiratory specimens. MTD sensitivity, specificity, and probability of inhibition for smear-positive specimens were estimated from literature reports. Costs and laboratory performance characteristics were determined from review of records and practices at an urban hospital in the mid-Atlantic United States. In the base case, 31.4% of smear-positive specimens were assumed to be culture positive for M. tuberculosis. Under these conditions, the marginal cost of the MTD testing program was estimated as $338 per smear-positive patient, or $494 per early exclusion of tuberculosis based on negative MTD results. By comparison, the cost of respiratory isolation ($27.77/day) and drugs ($5.66/day) averted by MTD testing was estimated at $201 per early tuberculosis exclusion. MTD testing was therefore not cost-effective in this scenario. Sensitivity analysis revealed that cost-effectiveness estimates are sensitive to the number of smear-positive specimens processed annually, the relative prevalence of M. tuberculosisin smear-positive specimens, and the marginal daily cost of respiratory isolation. A decision tool is therefore presented for assessing the cost-effectiveness of MTD under various combinations of those three variables. While routine MTD testing of smear-positive specimens is not expected to be cost-saving for most individual hospitals, centralized reference laboratories may be able to implement MTD in a cost-effective manner across a wide range of situations.

ABSTRACTRapid diagnostic tests for tuberculosis (TB) are needed to facilitate early treatment of TB and prevention of Mycobacterium tuberculosistransmission. The ICT Tuberculosis test is a rapid, card-based immunochromatographic test for detection of antibodies directed against M. tuberculosisantigens. The objective of the study was to evaluate the performance of the ICT Tuberculosis test for the diagnosis of active pulmonary TB (PTB) with whole blood, plasma, and serum from patients suspected of having PTB and from asymptomatic controls in a setting with a high prevalence of PTB. Seventy patients suspected of having PTB (and who were later confirmed to have or not to have PTB by use of M. tuberculosisculture as the “gold standard”) and 42 controls were studied. Twenty-one controls were neither vaccinated with Mycobacterium bovisbacillus Calmette-Gue´rin (BCG) nor tuberculin skin test (TST) positive (group A controls), and 21 controls were TST positive and/or had previously been vaccinated with BCG (group B controls). Study subjects were drawn from one hospital and one primary health care unit in Rio de Janeiro City, Brazil. One version of the test (ICT-1) was evaluated by using whole blood, plasma, and serum samples. Sera obtained for this study were frozen and later tested with a manufacturer-modified version of the test (ICT-2). Among the patients suspected of having PTB, the sensitivities of the ICT-1 with whole blood, serum, and plasma were 83, 65, and 70%, respectively, and the specificities were 46, 67, and 56%, respectively. Among the group A controls, the specificities of ICT-1 with the three specimen types were 95, 100, and 95%, respectively. Among the group B controls, the specificities of ICT-1 with the three specimen types were 71, 86, and 86%, respectively. Among the patients suspected of having PTB, the sensitivity of ICT-2 was 70% and the specificity was 65%. Among the group A controls, the specificity of ICT-2 was 95%, and among the group B controls, the specificity of ICT-2 was 81%. With a 29% observed prevalence of PTB among patients suspected of having PTB, the positive predictive values of the ICT tests ranged from 39 to 50% and the negative predictive values ranged from 82 to 87%. The ICT Tuberculosis tests were not sufficiently predictive to warrant their widespread use as routine diagnostic tests for PTB in this setting. However, further evaluation of these tests in specific epidemiologic settings may be warranted.

A molecular hybridization technique using biotinylated DNA probes was used to detect bovine herpesvirus 1 (BHV-1) nucleic acid species immobilized on nitrocellulose. Seventeen recombinant plasmids containing HindIII restriction fragments of the BHV-1 genome were compared for their ability to detect immobilized BHV-1 DNA from purified virus and infected cells. One probe, pCB2, labeled by nick translation with either 3H or biotin, detected as little as 10 pg of viral DNA. In time course experiments, BHV-1 DNA could be detected by 2 h postinfection in 10(6) infected cells. BHV-1 DNA was detected in nasal swabs and exudate from experimentally infected cattle, even when specimens had been stored for over a year. In a retrospective study of a respiratory disease outbreak in a feedlot, hybridization was compared with virus isolation for diagnosis of BHV-1 infections. The sensitivity rate was 0.68 with virus isolation as the referent standard. Blot hybridization provides a novel approach with unique applications for the diagnosis of bovine herpesvirus infections.

ABSTRACTUrinary lipoarabinomannan (LAM) detection is a promising approach for rapid diagnosis of active tuberculosis (TB). In microbiologically confirmed TB patients, quantitative LAM detection results increased progressively with bacillary burden and immunosuppression. Patients with disseminated TB and/or advanced HIV are target populations for whom urine LAM detection may be particularly useful.

ABSTRACTSpecimen dilution has been proposed as a strategy to minimize amplified Mycobacterium tuberculosisdirect (MTD) test inhibition (N. Pollock, J. Westerling, and A. Sloutsky, Am. J. Clin. Pathol. 126:142-147, 2006; A. Sloutsky, L. L. Han, and B. G. Werner, J. Clin. Microbiol. 42:1547-1551, 2004). We evaluated the impact of respiratory specimen dilution on MTD test accuracy in a public health laboratory. The difference in MTD test sensitivity between the dilution and conventional methods was 15.9% (P= 0.001) for smear microscopy-positive specimens and -3.6% (P= 0.38) for smear microscopy-negative specimens.

ABSTRACTThis prospective study evaluated the performance of the microscopic observation drug susceptibility (MODS) assay for the direct detection of Mycobacterium tuberculosisdrug resistance. MODS assay sensitivity, specificity, and positive and negative predictive values were 96.7% (95% confidence interval [95% CI], 92.1 to 98.8%), 78.4% (95% CI, 73.5 to 80.6%), 82.4% (95% CI, 78.4 to 84.2%), and 95.8% (95% CI, 89.9 to 98.5%), respectively, for isoniazid resistance and 96.0% (95% CI, 90.3 to 98.6%), 82.9% (95% CI, 78.8 to 84.7%), 80.0% (95% CI, 75.2 to 82.1%), and 96.7% (95% CI, 91.9 to 98.8%), respectively, for rifampin resistance. For both rifampin and isoniazid testing, the likelihood ratio for a negative test was =0.05, indicating that the MODS assay may be useful for ruling out drug resistance.

ABSTRACTThe drug susceptibility testing performance of a broth-based method with microscopic reading of bacillary growth, the microscopic observation drug susceptibility (MODS) assay, was compared to that of the reference 7H10 agar method of proportion by using 53 isolates of Mycobacterium tuberculosisfrom persons at risk for multidrug-resistant TB. For isoniazid (0.1 µg/ml) and rifampin (2.0 µg/ml), there was 100% agreement between MODS results read at day 11 and the reference method. Levels of agreement for ethambutol tested at 2.5 and 7.5 µg/ml were 70 and 58%, respectively. Levels of agreement for streptomycin tested at 2.0 and 6.0 µg/ml were 77 and 51%, respectively. For isoniazid and rifampin drug susceptibility testing, MODS is as accurate as and more rapid than the reference method.

ABSTRACTWe conducted a decision analysis to assess and compare four algorithms for amplified Mycobacterium tuberculosisdirect (MTD) testing of respiratory specimens in terms of cost-effectiveness. The most cost-effective strategy was one in which smear-positive specimens but not smear-negative specimens were diluted prior to MTD testing.