Aplicación de la técnica Speed Oligo al estudio de la tuberculosis

Tesis Univ. Granada. Programa Oficial de Doctorado en: Medicina Clínica y Salud Públic

Aplicación de la técnica Speed Oligo al estudio de la tuberculosis

Tesis Univ. Granada. Programa Oficial de Doctorado en: Medicina Clínica y Salud Públic

Evaluation of the speed-oligo direct Mycobacterium tuberculosis assay for molecular detection of mycobacteria in clinical respiratory specimens.

Comparative Study; Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't;We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.The study was partially financed by Junta de Andalucía (PI-0444/2008 and PI-0306-2009) and SEPAR (763-09)Ye

Preliminary evaluation of a new kit for differentiation of Mycobacterium tuberculosis complex species using Speed-Oligo MTBC.

We present the first evaluation of a novel molecular assay, the Speed-Oligo Mycobacterium tuberculosis complex (SO-MTBC), which is based on PCR combined with a dipstick for the differentiation of M. tuberculosis complex (MTBC) members. The results of this assay were compared with findings obtained using the Genotype MTBC assay. In this study, 189 strains of MTBC isolates from 2011 to 2014 were evaluated to determine the MTBC species. Most (174, 92 %) of the strains were identified as M. tuberculosissensu stricto, 7 (3.7 %) as Mycobacteriumbovis, 5 (2.6 %) as M. bovis bacillus Calmette-Guérin, 2 (1.1 %) as Mycobacteriumafricanum and 1 (0.5 %) as Mycobacteriumcaprae; no strains belonged to Mycobacteriummicroti and Mycobacteriumcanettii subsp. The concordance κ coefficient obtained was 0.96 with the results of the Genotype MTBC assay. SO-MTBC may represent a fast and easy-to-use alternative for differentiating among MTBC subspecies in laboratories with standard equipment

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old