Multiplex Real-Time PCR-shortTUB Assay for Detection of the Mycobacterium tuberculosis Complex in Smear-Negative Clinical Samples with Low Mycobacterial Loads.

Tuberculosis (TB) remains a major health problem worldwide. Control of TB requires rapid, accurate diagnosis of active disease. However, extrapulmonary TB is very difficult to diagnose because the clinical specimens have very low bacterial loads. Several molecular methods involving direct detection of the Mycobacterium tuberculosis complex (MTBC) have emerged in recent years. Real-time PCR amplification simultaneously combines the amplification and detection of candidate sequences by using fluorescent probes (mainly TaqMan or Molecular Beacons) in automated systems. The multiplex real-time PCR-short assay is performed using locked nucleic acid (LNA) probes (length, 8 to 9 nucleotides) in combination with CodUNG to detect multiple pathogens in clinical samples. In this study, we evaluated the performance of this novel multiplex assay for detecting the MTBC in comparison with that of the conventional culture-based method. The multiplex real-time PCR-shortTUB assay targets two genes, whiB3 (redox-responsive transcriptional regulator) and pstS1 (phosphate-specific transporter), yielding limits of detection (LOD) of 10 copies and 100 copies, respectively, and amplification efficiencies of 92% and 99.7%, respectively. A total of 94 extrapulmonary samples and pulmonary samples with low mycobacterial loads (all smear negative; 75 MTBC culture positive) were analyzed using the test, yielding an overall sensitivity of 88% and a specificity of 95%. For pleural fluid and tissues/biopsy specimens, the sensitivity was 83% and 85%, respectively. In summary, this technique could be implemented in routine clinical microbiology testing to reduce the overall turnaround time for MTBC detection and may therefore be a useful tool for the diagnosis of extrapulmonary tuberculosis and diagnosis using pulmonary samples with low mycobacterial loads.

Lab-on-Chip-Based Platform for Fast Molecular Diagnosis of Multidrug-Resistant Tuberculosis.

We evaluated the performance of the molecular lab-on-chip-based VerePLEX Biosystem for detection of multidrug-resistant tuberculosis (MDR-TB), obtaining a diagnostic accuracy of more than 97.8% compared to sequencing and MTBDRplus assay for Mycobacterium tuberculosis complex and rifampin and isoniazid resistance detection on clinical isolates and smear-positive specimens. The speed, user-friendly interface, and versatility make it suitable for routine laboratory use.

Characterization of the embB gene in Mycobacterium tuberculosis isolates from Barcelona and rapid detection of main mutations related to ethambutol resistance using a low-density DNA array.

OBJECTIVES: Ethambutol resistance has mostly been related to mutations in the embB gene. The objective of the present study was to characterize the embB gene in a collection of ethambutol-resistant and ethambutol-susceptible isolates of Mycobacterium tuberculosis complex (MTBC) from Barcelona, and to develop a DNA microarray for the rapid detection of embB mutations in our area. METHODS: Fifty-three ethambutol-resistant and 702 ethambutol-susceptible isolates of MTBC were sequenced in internal 982-1495 bp fragments of the embB gene. In addition, a low-cost, low-density array was designed to include the embB codons identified as being most frequently mutated in our area (LD-EMB array). RESULTS: The global prevalence of embB mutations found among the ethambutol-resistant isolates was 77.4% (41/53). Substitutions in embB306 were the most common [53.7% (22/41)], followed by substitutions in embB406 [26.8% (11/41)]. The presence of mutations in embB406 was related to higher levels of ethambutol resistance and to multidrug resistance. Among unrelated isolates (from 24-locus MIRU-VNTR genotyping), the percentage of embB-mutated isolates was 72.9% (27/37)--59.3% (16/27) in embB306 and 25.9% (7/27) in embB406. None of the ethambutol-susceptible isolates studied showed a mutation in codon 306 or 406. The LD-EMB array showed 100% sensitivity and specificity in identifying the main embB substitutions in our area. CONCLUSIONS: Mutations at codons 306 and 406 of embB have a relevant role in resistance to ethambutol in our area. The LD-EMB array developed in this study would appear to be a good molecular test for rapid detection of ethambutol resistance.

Direct detection of Mycobacterium tuberculosis complex in clinical samples by a molecular method based on GenoQuick technology.

Several molecular systems for direct detection of Mycobacterium tuberculosis complex (MTBC) have recently been developed. The GenoQuick MTB assay (GQ-MTB) used in this study detected 82 of the 96 (85.4%) samples with MTBC, including 50 of 64 (78.1%) samples with negative acid-fast bacillus smears. Fifteen samples containing nontuberculous mycobacteria were also studied: 13 were GQ-MTB negative, one was positive, and one was indeterminate. GQ-MTB showed good effectiveness for the direct detection of MTBC from clinical samples.

Effectiveness of an integrated real-time PCR method for detection of the Mycobacterium tuberculosis complex in smear-negative extrapulmonary samples in an area of low tuberculosis prevalence.

Early extrapulmonary tuberculosis (EPTB) diagnosis is particularly difficult. Among 108 smear-negative extrapulmonary samples showing a positive culture for Mycobacterium tuberculosis complex (43 body fluids and 65 nonliquid specimens), 63 (58.3%) were positive with the Xpert MTB/RIF assay (GX). GX sensitivity was quite low for samples from sterile locations (especially for pleural fluids: 26.9%) but high for some nonliquid samples, like abscess aspirates (76.5%). In summary, GX may be a useful tool to be considered for EPTB diagnosis.

Rapid detection of Mycobacterium tuberculosis complex and rifampin resistance in smear-negative clinical samples by use of an integrated real-time PCR method.

Sixty-four of 85 (75.3%) smear-negative respiratory (n = 78) and nonrespiratory (n = 7) samples with positive cultures of Mycobacterium tuberculosis complex (MTC) were detected by the GeneXpert system using the Xpert MTB/RIF assay (GX). In addition, GX found rpoB mutations in all six of the rifampin-resistant strains detected. The test was negative in 20 culture-negative and 20 nontuberculous culture-positive samples (100% specificity). GX offers high potential for the diagnosis of tuberculosis due to its capacity for direct detection of MTC, its rapidity, and its simplicity.

Impaired fitness of Mycobacterium tuberculosis resistant isolates in a cell culture model of murine macrophages.

OBJECTIVES: We analysed the ability of Mycobacterium tuberculosis clinical isolates to penetrate and grow inside murine macrophages as a surrogate of fitness. METHODS: Thirty-five drug-resistant and 10 drug-susceptible M. tuberculosis isolates were studied in a murine macrophage model from the J774.2 cell line in a 6 day protocol, performing semi-quantitative counts in Middlebrook 7H11 medium. The mycobacterial penetration index (MPI) after infection and the mycobacterial growth ratio (MGR) inside the macrophages were determined to evaluate the fitness of isolates. RESULTS: Isolates with the katG S315T mutation and multidrug-resistant (MDR) isolates had a significantly lower MGR compared with drug-susceptible isolates. The MPI of the isolates with the katG S315T mutation showed a significant decrease compared with the MPI of those without this mutation. A trend to significantly lower values was also observed on comparing the MPI of the MDR isolates with that of the drug-susceptible isolates and the isolates resistant to isoniazid. CONCLUSIONS: The isoniazid-resistant and MDR isolates with mutations in the katG gene showed decreased multiplication inside murine macrophages, suggesting a lower fitness of M. tuberculosis with these resistance patterns.

Comparison of the 2-step tuberculin skin test and the quantiFERON-TB Gold In-Tube Test for the screening of tuberculosis infection before liver transplantation.

The ability of interferon-gamma release assays (IGRAs) to detect latent tuberculosis (TB) infection before liver transplantation (LT)is not well established. The aims of this study were (1) to compare the ability of the tuberculin skin test (TST) and the QuantiFERON-TB Gold In-Tube (QFT-IT) test (a whole-blood IGRA) to diagnose latent TB infections in patients awaiting LT and (2) to correlate the results with the severity of liver disease. We conducted a prospective, cross-sectional study of patients who were evaluated for LT between July 2008 and July 2010. The 95 patients who were included underwent the 2-step TST and the QFT-IT test. The mean Model for End-Stage Liver Disease (MELD) score was 13.8. Forty-four patients (46.3%) had positive TST results, 42 (44.2%) had positive QFT-IT results, and 2 (2.1%) had indeterminate QFT-IT results. Simultaneous TST and QFT-IT testing yielded a positivity rate of 55.8% [95% confidence interval (CI) = 45.3-65.9] with either test, and the 2-step TST yielded a positivity rate of 46.3% (95% CI = 36.1-56.8); the difference was 9.5% (P = 0.004). In an adjusted analysis, the rates for positive TST results were lower in patients with MELD scores > or = 18 [odds ratio (OR) = 0.2, 95% CI = 0.04-0.7], lower in Child-Pugh-Turcotte (CPT) class C patients versus CPT class A patients (OR = 0.1, 95% CI = 0.02-0.6), and higher in males (OR = 6.4, 95% CI = 1.9-22.0). In contrast, only being male (OR = 3.5, 95% CI = 1.1-11.0) was associated with positive QFT-IT results; no association was found with the MELD score (OR = 0.8, 95% CI = 0.2-2.8) or the CPT class (OR = 0.3; 0.05-1.4). In conclusion, the QFT-IT test is better than the TST for detecting latent TB infection in patients with more advanced liver disease. Our results support the regular use of the QFT-IT test for screening patients with end-stage liver disease for latent TB infection before LT.

Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis clinical isolates in the area of Barcelona.

OBJECTIVES: To determine the proportion and type of mutations in Mycobacterium tuberculosis isolates resistant to streptomycin, and their relationship with the level of resistance and with the epidemiological molecular pattern of the isolates. METHODS: Sixty-nine streptomycin-resistant isolates from a M. tuberculosis strain collection (1995-2005) from Barcelona were studied. The MIC of streptomycin for each isolate was determined using the proportions method with Middlebrook 7H11 medium. The entire rpsL gene and two specific fragments of the rrs gene (the 530 loop and the 912 region) were sequenced. IS6110-restriction fragment length polymorphism and spoligotyping were performed in each isolate. RESULTS: Twenty-six (26/69, 37.7%) streptomycin-resistant isolates presented a mutation in either the rpsL gene and/or the rrs530 loop, with no mutation in the rrs912 region. Seventeen (24.6%) isolates showed rpsL mutations (codons 43 and 88) associated with high MIC levels. Nine (13.0%) isolates had alterations in the rrs gene (A513T, A513C and C516T). Nineteen isolates (19/64, 29.7%) were classified into seven clusters (containing 2-5 isolates per cluster). Nineteen different spoligotype patterns were found. All the LAM3 spoligotype isolates (10/67, 14.9%) were associated with a C491T change in the rrs gene, being also observed in all LAM3 streptomycin-susceptible isolates. CONCLUSIONS: Mutations in the rpsL and rrs genes were detected in 37.7% of streptomycin-resistant M. tuberculosis isolates. High-level resistance was associated with mutations in the rpsL gene, whereas wild-type isolates showed low MIC levels. The presence of the C491T substitution in the rrs gene in streptomycin-susceptible and -resistant isolates demonstrates that this change is an epidemiological marker associated with LAM3 sublineage.

Use of the cobas 4800 system for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus.

The new cobas® Cdiff and cobas® MRSA/SA tests were compared with conventional methods for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus. The final concordance between cobas Cdiff Test and GDH/toxin gene screening was 97.62% and between cobas MRSA/SA Test and chromogenic culture, 91.30%, respectively.

GeneXpert® for smear-negative pulmonary tuberculosis: does it play a role in low-burden countries?

We performed a retrospective analysis of costs and time to treatment (TT) of 150 culture-confirmed TB cases: 100 sputum smear (SS) (+) and 50 SS(-). This group underwent GeneXpert® (GX) assay. Expenditures and TT of SS(-)/GX(+) cases were inferred from the SS(+) group. GX detected 68% of SS(-) cases.

Detection of streptomycin and quinolone resistance in Mycobacterium tuberculosis by a low-density DNA array.

In cases of multidrug-resistant tuberculosis, it is crucial to rule out resistance to second-line antituberculous (anti-TB) agents. In the present study, a low-cost low-density DNA array including four genetic regions (rrs 530 loop, rrs 1400, rpsL and gyrA) was designed for the rapid detection of the most important mutations related to anti-TB injectable drugs (mainly streptomycin) and fluoroquinolone resistance (LD-SQ array). A total of 108 streptomycin- and/or ofloxacin-resistant and 20 streptomycin- and ofloxacin-susceptible Mycobacterium tuberculosis clinical isolates were analysed with the array. The results obtained were compared with sequencing data and phenotypic susceptibility pattern. The LD-SQ array offered a good sensitivity compared to sequencing, especially among resistant strains: 92.5% (37/40) for streptomycin and 87.5% (7/8) for fluoroquinolones. Therefore, this array could be considered a good approach for the rapid detection of mutations related to streptomycin and fluoroquinolone resistance. On the other hand, there were discordant results in 16 resistant strains and six susceptible isolates, mostly concerning the gyrA region, in which the existence of polymorphisms next to informative positions might cause cross-hybridization. These discrepancies were caused by some technical limitations; consequently, the present array should be considered as a first-step prior to a forthcoming optimized version of the array.

Detection of latent tuberculosis by the tuberculin skin test and a whole-blood interferon-γ release assay, and the development of active tuberculosis in HIV-seropositive persons.

This study evaluated the QuantiFERON-TB Gold In-Tube (QFT-GIT; Cellestis, Carnegie, Australia) test and the tuberculin skin test (TST) for the detection of latent tuberculosis infection (LTBI) in HIV-infected adults. One hundred thirty-five HIV-seropositive persons and 135 controls underwent TST and QFT-GIT. HIV-infected patients who gave a positive result on either test were offered chemoprophylaxis. The prevalence of LTBI was 6.7% by TST and 9.6% by QFT-GIT (P = 0.3) in HIV-seropositive subjects, and 34.8% by TST and 21.5% by QFT-GIT (P = 0.02) among controls. TST reactivity declined sharply as CD4(+) cells fell (15.8%, 10.3%, and 0% for >500, 301-500 and ≤300 CD4(+) cells/mm(3), respectively; P = 0.002). A less pronounced fall occurred with QFT-GIT (15.8%, 13.8%, and 0% for >500, 301-500, and <100 CD4(+) cells/mm(3), respectively; P = 0.03). No cases of tuberculosis occurred during follow-up (0.26 per 100 person-years). Simultaneous testing with TST and QFT-GIT for targeting of chemoprophylaxis, early in the course of HIV infection, might minimize the risk of tuberculosis in these patients.

Diagnosis of tuberculosis infection by tuberculin skin test and a whole-blood interferon-γ release assay in patients considered for anti-tumor necrosis factor-α therapy.

To assess the performance of QuantiFERON®-TB Gold in-Tube (QFT-GIT; Cellestis, Carnegie, Australia) and tuberculin skin test (TST) in patients with immune-mediated inflammatory diseases (IMID), before anti-tumor necrosis factor-α (TNF-α) therapy, and to compare the results with those from the healthy population. Three hundred fourteen subjects (214 with IMID and 100 controls) underwent simultaneous QFT-GIT and TST. QFT-GIT was positive in 21% of IMID patients and in 16% of controls (P = 0.29). Among IMID patients, 21% tested positive by QFT-GIT and 24%, by TST (P = 0.30). Positive QFT-GIT results were not affected by immunosuppressive therapy (odds ratio, 0.78; 95% confidence interval [CI], 0.36-1.68; P = 0.52). Agreement between both tests in those patients who tested positive by one of the tests was 50% (95% CI, 37.2-62.8). QFT-GIT is useful for identifying IMID patients requiring treatment of latent tuberculosis before anti-TNF therapy. However, given the poor agreement between TST and QFT-GIT, we advocate a strategy of simultaneous testing to optimize diagnostic sensitivity.