Raquel Moure

Rapid Detection of Mycobacterium tuberculosis Complex and Rifampin Resistance in Smear-Negative Clinical Samples by Use of an Integrated Real-Time PCR Method

ABSTRACT 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.

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

ABSTRACT 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.

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.

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.

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.

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‐γ 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 ≥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. Liver Transpl 17:1205–1211, 2011.

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

ABSTRACT 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.

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.

Silent Mutation in rpoB Detected from Clinical Samples with Rifampin-Susceptible Mycobacterium tuberculosis

We read with great interest the note of Alonso et al. ([1][1]) concerning the existence of a silent mutation in rpoB in Mycobacterium tuberculosis strains. In this note, the authors describe a phenomenon already observed by several study groups. The misassignment of phenotypic resistance due to the