Evaluation of GenoFlow DR-MTB array test for detection of rifampin and isoniazid resistance in mycobacterium tuberculosis

The aim of this study was to evaluate the GenoFlow DR-MTB array test (DiagCor Bioscience, Hong Kong) on 70 cultured isolates and 50 sputum specimens. The GenoFlow array test showed good sensitivity and specificity compared to the phenotypic Bactec 460TB. This array accurately detected mutations in rpoB, katG, and inhA associated with resistance to rifampin and isoniazid

Study of CD27 and CCR4 Markers on Specific CD4+ T-Cells as Immune Tools for Active and Latent Tuberculosis Management

The immunological characterization of different cell markers has opened the possibility of considering them as immune tools for tuberculosis (TB) management, as they could correlate with TB latency/disease status and outcome. CD4+ T-cells producing IFN-γ+ with a low expression of CD27 have been described as an active TB marker. In addition, there are unknown homing receptors related to TB, such as CCR4, which might be useful for understanding TB pathogenesis. The aim of our study is focused on the assessment of several T-cell subsets to understand immune-mechanisms in TB. This phenotypic immune characterization is based on the study of the specific immune responses of T-cells expressing CD27 and/or CCR4 homing markers. Subjects enrolled in the study were: (i) 22 adult patients with active TB, and (ii) 26 individuals with latent TB infection (LTBI). Blood samples were drawn from each patient. The expression of CD27 and/or CCR4 markers were analyzed within CD4+ T-cells producing: (i) IFN-γ+, (ii) TNF-α+, (iii) TNF-α+IFN-γ+, and (iv) IFN-γ+ and/or TNF-α+. The percentage of CD27− within all CD4+ T-cell populations analyzed was significantly higher on active TB compared to LTBI after PPD or ESAT-6/CFP-10 stimulation. As previously reported, a ratio based on the CD27 median fluorescence intensity (MFI) was also explored (MFI of CD27 in CD4+ T-cells over MFI of CD27 in IFN-γ+CD4+ T-cells), being significantly increased during disease (p < 0.0001 after PPD or ESAT-6/CFP-10 stimulation). This ratio was also assessed on the other CD4+ T-cells functional profiles after specific stimulation, being significantly associated with active TB. Highest diagnostic accuracies for active TB (AUC ≥ 0.91) were achieved for: (i) CD27 within IFN-γ+TNF-α+CD4+ T-cells in response to ESAT-6/CFP-10, (ii) CD27 and CCR4 markers together within IFN-γ+CD4+ T-cells in response to PPD, and (iii) CD27 MFI ratio performed on IFN-γ+TNF-α+CD4+ T-cells after ESAT-6/CFP-10 stimulation. The lowest diagnostic accuracy was observed when CCR4 marker was evaluated alone (AUC ≤ 0.77). CD27 and CCR4 expression detection could serve as a good method for immunodiagnosis. Moreover, the immunological characterization of markers/subset populations could be a promising tool for understanding the biological basis of the disease

Accessory gene regulator (Agr) functionality in Staphylococcus aureus derived from lower respiratory tract infections

Altres ajuts: This work has been funded by the project PI13/01418 which is part of "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluacioón and "Fondo Europeo de Desarrollo Regional"(FEDER).D. Domínguez-Villanueva is funded by "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluación and "Fondo Europeo de Desarrollo Regional"(FEDER). M. Gomes-Fernandes is funded by CAPES Foundation, Ministry of Education of Brazil (Brasılia, Brazil). Maisem Laabei was supported by a joint ERS/SEPAR fellowship (LTRF2015).This work also received a grant from the Spanish Society of Pneumology and Thoracic Surgery (SEPAR054/2011).Objective. Characterization of Staphylococcus aureus clinical isolates derived from lower respiratory tract infections (LRTIs), and correlation between the functionality of the accessory gene regulator (Agr) and genotypic and phenotypic characteristics, clinical variables and clinical outcome. Methods. S aureus isolates derived from LRTIs and control groups (nasal carriage and bacteraemia) were genotyped using StaphyType DNA microarray. Agr activity was evaluated using the CAMP synergistic haemolysis assay and the Vesicle Lysis Test (VLT). Discordant strains were analysed by quantitative reverse- transcriptase real-time PCR (qRT-PCR). Results. Agr was functional in 79.7% and 84.5% of strains according to the CAMP and VLT assays respectively. Higher concordance with RNAIII expression measured by qRT-PCR was observed with the VLT assay (76.2%) compared with the CAMP assay (23.8%). No statistically significant differences were observed in Agr functionality between the study groups, nor the phenotypical/genotypical bacterial characteristics. No association between increased mortality/respiratory complications and Agr function was observed. Conclusions. Agr activity was high (82.2%) in isolates from LRTIs suggesting the importance of this global regulator in lower respiratory tract colonisation and infection. However, equally high Agr activity was observed in isolates derived from nasal carriage and bacteraemia, contradictory to previous observations. Agr functionality measured by the VLT assay was superior to CAMP assay

Direct susceptibility testing for multi drug resistant tuberculosis: A meta-analysis

<p>Abstract</p> <p>Background</p> <p>One of the challenges facing the tuberculosis (TB) control programmes in resource-limited settings is lack of rapid techniques for detection of drug resistant TB, particularly multi drug resistant tuberculosis (MDR TB). Results obtained with the conventional indirect susceptibility testing methods come too late to influence a timely decision on patient management. More rapid tests directly applied on sputum samples are needed. This study compared the sensitivity, specificity and time to results of four direct drug susceptibility testing tests with the conventional indirect testing for detection of resistance to rifampicin and isoniazid in <it>M. tuberculosis</it>. The four direct tests included two in-house phenotypic assays – Nitrate Reductase Assay (NRA) and Microscopic Observation Drug Susceptibility (MODS), and two commercially available tests – Genotype^®MTBDR and Genotype^®MTBDR<it>plus </it>(Hain Life Sciences, Nehren, Germany).</p> <p>Methods</p> <p>A literature review and meta-analysis of study reports was performed. The Meta-Disc software was used to analyse the reports and tests for sensitivity, specificity, and area under the summary receiver operating characteristic (sROC) curves. Heterogeneity in accuracy estimates was tested with the Spearman correlation coefficient and Chi-square.</p> <p>Results</p> <p>Eighteen direct DST reports were analysed: NRA – 4, MODS- 6, Genotype MTBDR^®– 3 and Genotype^®MTBDR<it>plus </it>– 5. The pooled sensitivity and specificity for detection of resistance to rifampicin were 99% and 100% with NRA, 96% and 96% with MODS, 99% and 98% with Genotype^®MTBDR, and 99% and 99% with the new Genotype^®MTBDR<it>plus</it>, respectively. For isoniazid it was 94% and 100% for NRA, 92% and 96% for MODS, 71% and 100% for Genotype^®MTBDR, and 96% and 100% with the Genotype^®MTBDR<it>plus</it>, respectively. The area under the summary receiver operating characteristic (sROC) curves was in ranges of 0.98 to 1.00 for all the four tests. Molecular tests were completed in 1 – 2 days and also the phenotypic assays were much more rapid than conventional testing.</p> <p>Conclusion</p> <p>Direct testing of rifampicin and isoniazid resistance in <it>M. tuberculosis </it>was found to be highly sensitive and specific, and allows prompt detection of MDR TB.</p

Evaluation of Interferon-Gamma Release Assays in the Diagnosis of Recent Tuberculosis Infection in Health Care Workers

BACKGROUND:Health care workers (HCWs) are a group at risk of latent tuberculosis infection (LTBI). The aims of this study were to determine IFN-gamma response by QuantiFERON-TB GOLD In Tube (QFN-G-IT) and T-SPOT.TB in HCWs, comparing the results with tuberculin skin test (TST); and to analyze the capacity of IFN-gamma tests to detect recent versus remote LTBI with a prolonged stimulation test (PST). METHODOLOGY/PRINCIPAL FINDINGS:A total of 147 HCWs were enrolled; 23 of whom were BCG vaccinated. 95 HCWs (64.6%) had a previous positive TST and were not retested; and 52 HCWs had a previous negative TST or were tested for the first time. When we analysed individuals without previous positive TST, the number of positive results for T-SPOT.TB was 12/52 (23.1%); and for QFN-G-IT, 9/52 (17.3%). The global concordance (kappa) between T-SPOT.TB and QFN-G-IT with TST was 0.754 and 0.929 respectively. Of individuals with previous positive TST, T-SPOT.TB and QFN-G-IT were negative in 51.6% (49/95) and 62.1% (59/95) respectively, decreasing the concordance to 0.321 and 0.288, respectively. In non-BCG vaccinated HCWs with previous positive TST a positive IFN-gamma test was associated with degree of exposure and diameter of TST. PST was performed in 24 HCW with previous positive TST and negative IFN-gamma tests. PST was developed in 3 cell cultures stimulated with medium alone, ESAT-6 and CFP-10, respectively. In the third and sixth day of incubation period, part of the supernatants were replaced with complete medium supplemented with (rIL)-2. On day 9, ELISPOT assay was performed. In 14 samples PST was not valid due to not having enough cells. In 8 cases, the response was negative, and in 2 cases positive, suggesting that these patients were infected with Mycobacterium tuberculosis in some point in the past. CONCLUSIONS:Both IFN-gamma tests showed a similar number of positive results, and concordance between the tests was excellent. None of the tests was affected by prior BCG vaccination. IFN-gamma tests are a useful tool for detecting recent infection in HCW population

Drug resistance mutations and heteroresistance detected using the GenoType MTBDRplus assay and their implication for treatment outcomes in patients from Mumbai, India

<p>Abstract</p> <p>Background</p> <p>Only 5% of the estimated global multidrug resistant TB (MDRTB) load is currently detected. Endemic Mumbai with increasing MDR would benefit from the introduction of molecular methods to detect resistance.</p> <p>Methods</p> <p>The GenoType MTBDR<it>plus </it>assay was used to determine mutations associated with isoniazid and rifampicin resistance and their correlation with treatment outcomes. It was performed on a convenience sample comprising 88 onset and 67 fifth month isolates for which phenotypic drug susceptibility testing (DST) was determined by the Buddemeyer technique for an earlier study. Simultaneous presence of wild type and mutant bands was referred to as "mixed patterns" (heteroresistance).</p> <p>Results</p> <p>Phenotypically 41 isolates were sensitive; 11 isoniazid, 2 rifampicin, 2 pyrazinamide and 5 ethambutol monoresistant; 16 polyresistant and 78 MDR. The agreement between both methods was excellent (kappa = 0.72-0.92). Of 22 rifampicin resistant onset isolates, the predominant <it>rpoB </it>mutations were the singular lack of WT8 (n = 8) and mixed D516V patterns (n = 9). Of the 64 rifampicin resistant fifth month isolates, the most frequent mutations were in WT8 (n = 31) with a further 9 showing the S531L mutation. Mixed patterns were seen in 22 (34%) isolates, most frequently for the D516V mutation (n = 21). Of the 22 onset and 35 fifth month <it>katG </it>mutants, 13 and 12 respectively showed the S315T1 mutation with loss of the WT. Mixed patterns involving both S315T1 and S315T2 were seen in 9 and 23 isolates respectively. Seventeen of 23 and 23/35 <it>inhA </it>mutant onset and fifth month isolates showed mixed A16G profiles. Additionally, 10 fifth month isolates lacked WT2. Five onset and 6 fifth month isolates had both <it>katG </it>and <it>inhA </it>mutations. An association was noted between only <it>katG </it>but not only <it>inhA </it>resistance and poor outcome (<it>p </it>= 0.037); and additional resistance to ethambutol (<it>p </it>= 0.0033). More fifth month than onset isolates had mixed profiles for at least 1 gene (<it>p </it>= 0.000001).</p> <p>Conclusions</p> <p>The use of the assay to rapidly diagnose MDR could guide simultaneous first- and second-line DST, and reduce the delay in administering appropriate regimens. Furthermore, detection of heteroresistance could prevent inaccurate "cured" treatment outcomes documented through smear microscopy and permit more sensitive detection of neonascent resistance.</p