Dataset Information

T CELL EPITOPES OF THE ESXA FULL GENE OF MYCOBACTERIUM TUBERCULOSIS FROM SPUTUM OF MDR-TB PATIENTS.

ABSTRACT: Background:In 2015, World Health Organization (WHO) discovered 10.4 million tuberculosis (TB) cases around the world. Multidrug-resistant tuberculosis (MDR-TB) became a threat because it has high mortality number. There were 480,000 new MDR-TB cases in 2015. Based on those problems, diagnostic development to detect M. tuberculosis rapidly and accurately is needed. The importance of detecting epitope expression of esxA full gene because there was a potential of complexity over the protein structure and might affect the protein concentration. By knowing epitope prediction, there's an expectation that it can help the development of TB diagnostic. This research was aimed to determine the T cell epitope prediction of esxA full gene from MDR-TB patients. Material and Methods:Total of 24 MDR-TB sputum isolate from TB patients at Dr. Soetomo Hospital were collected from September to December 2016. Samples were confirmed as MDR-TB using GeneXpert and Bactec MGIT 960. Those samples tested using PCR targeted 580 bp of esxA gene and sequencing. Gene sequence was aligned against wild type using Bioedit program version 7.2.5 and NCBI BLAST. T cell epitope prediction was analyzed by GENETYX version 10. Results:Epitope predictions that could be obtained were IEAAAS, ASAIQG, VTSIHS, TKLAAA, VTGMFA based IAd Pattern Position and EAAAS based Rothbard/Taylor Pattern Position. Those prediction epitopes can determine the severity of disease, therefore full gene of esxA could be used as diagnostic target. Conclusion:This research discovered five specific T cell epitope prediction based on IAd Pattern Position and one epitope prediction according to Rothbard/Taylor Pattern Position.

SUBMITTER: Dewi DNSS

PROVIDER: S-EPMC6085733 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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T CELL EPITOPES OF THE <i>ESXA</i> FULL GENE OF <i>MYCOBACTERIUM TUBERCULOSIS</i> FROM SPUTUM OF MDR-TB PATIENTS.

Dewi Desak Nyoman Surya Suameitria DNSS Soedarsono Mertaniasih Ni Made NM

African journal of infectious diseases 20180618 2

<h4>Background</h4>In 2015, World Health Organization (WHO) discovered 10.4 million tuberculosis (TB) cases around the world. Multidrug-resistant tuberculosis (MDR-TB) became a threat because it has high mortality number. There were 480,000 new MDR-TB cases in 2015. Based on those problems, diagnostic development to detect <i>M. tuberculosis</i> rapidly and accurately is needed. The importance of detecting epitope expression of <i>esxA</i> full gene because there was a potential of complexity ov ...[more]

PMID: 30109288

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Project description:BackgroundMixed M. tuberculosis (MTB) infection occurs when one is infected with more than one clonally distinct MTB strain. This form of infection can assist MTB strains to acquire additional mutations, facilitate the spread of drug-resistant strains, and boost the rate of treatment failure. Hence, the presence of mixed MTB infection could affect the performance of some rapid molecular diagnostic tests such as Line Probe Assay (LPA) and GeneXpert MTB/RIF (Xpert) assays.MethodsThis was a cross-sectional study that used sputum specimens collected from participants screened for STREAM 2 clinical trial between October 2017 and October 2019. Samples from 62 MTB smear-positive patients and rifampicin-resistant patients from peripheral health facilities were processed for Xpert and LPA as screening tests for eligibility in the trial. From November 2020, processed stored sputum samples were retrieved and genotyped to determine the presence of mixed-MTB strain infection using a standard 24-locus Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem-Repeat (MIRU-VNTR). Samples with at least 20/24 MIRU-VNTR loci amplified were considered for analysis. Agar proportional Drug Susceptibility Test (DST) was performed on culture isolates of samples that had discordant results between LPA and Xpert. The impact of the presence of mixed-MTB strain on Xpert and LPA test interpretation was analyzed.ResultsA total of 53/62 (85%) samples had analyzable results from MIRU-VNTR. The overall prevalence of mixed-MTB infection was 5/53 (9.4%). The prevalence was highest among male's 3/31 (9.7%) and among middle-aged adults, 4/30 (33.3%). Lineage 4 of MTB contributed 3/5 (60.0%) of the mixed-MTB infection prevalence. Having mixed MTB strain infection increased the odds of false susceptible Xpert test results (OR 7.556, 95% CI 0.88-64.44) but not for LPA. Being HIV-positive (P = 0.04) independently predicted the presence of mixed MTB infection.ConclusionsThe presence of mixed-MTB strain infection may affect the performance of the GeneXpert test but not for LPA. For patients with high pre-test probability of rifampicin resistance, an alternative rapid method such as LPA should be considered.

Project description:BackgroundIncreasing incidence of multidrug-resistant Mycobacterium tuberculosis infections is hampering global tuberculosis control efforts. Kuwait is a low-tuberculosis-incidence country, and ~ 1% of M. tuberculosis strains are resistant to rifampicin and isoniazid (MDR-TB). This study detected mutations in seven genes predicting resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin in MDR-TB strains. Sequence data were combined with spoligotypes for detecting local transmission of MDR-TB in Kuwait.MethodsNinety-three MDR-TB strains isolated from 12 Kuwaiti and 81 expatriate patients and 50 pansusceptible strains were used. Phenotypic drug susceptibility was determined by MGIT 460 TB/960 system. Mutations conferring resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin were detected by genotype MTBDRplus assay and/or PCR sequencing of three rpoB regions, katG codon 315 (katG315) + inhA regulatory region, pncA, three embB regions and rpsL + rrs-500-900 regions. Spoligotyping kit was used, spoligotypes were identified by SITVIT2, and phylogenetic tree was constructed by using MIRU-VNTRplus software. Phylogenetic tree was also constructed from concatenated sequences by MEGA7 software. Additional PCR sequencing of gidB and rpsA was performed for cluster isolates.ResultsPansusceptible isolates contained wild-type sequences. Mutations in rpoB and katG and/or inhA were detected in 93/93 and 92/93 MDR-TB strains, respectively. Mutations were also detected for pyrazinamide resistance, ethambutol resistance and streptomycin resistance in MDR-TB isolates in pncA, embB and rpsL + rrs, respectively. Spoligotyping identified 35 patterns with 18 isolates exhibiting unique patterns while 75 isolates grouped in 17 patterns. Beijing genotype was most common (32/93), and 11 isolates showed nine orphan patterns. Phylogenetic analysis of concatenated sequences showed unique patterns for 51 isolates while 42 isolates grouped in 16 clusters. Interestingly, 22 isolates in eight clusters by both methods were isolated from TB patients typically within a span of 2 years. Five of eight clusters were confirmed by additional gidB and rpsA sequence data.ConclusionsOur study provides the first insight into molecular epidemiology of MDR-TB in Kuwait and identified several potential clusters of local transmission of MDR-TB involving 2-6 subjects which had escaped detection by routine surveillance studies. Prospective detection of resistance-conferring mutations can identify possible cases of local transmission of MDR-TB in low MDR-TB settings.

Project description:Beijing strains of Mycobacterium tuberculosis (lineage 2) have been associated with drug-resistance and transmission of tuberculosis worldwide. Most of the Beijing strains identified in the Colombian Pacific coast have exhibited a multidrug resistant (MDR) phenotype. We sought to evaluate the clonality and sublineage of Beijing strains circulating in Southwestern Colombia. Thirty-seven Beijing strains were identified through spoligotyping out of 311 clinical isolates collected in 9 years from 2002-2010. Further analysis by MIRU-VNTR 24 loci was conducted for the Beijing strains. For sublineage classification, deletions of RD105, RD207, and RD131 and point mutations at fbpB, mutT2, and acs were evaluated. Drug-resistance associated mutations to first- and second-line anti-TB drugs were also evaluated. Additionally, two Beijing strains were Illumina-whole genome sequenced (one MDR and one drug-susceptible). Among the 37 Beijing strains characterized, 36 belonged to the SIT190 type from which 28 were MDR, four pre-extensively drug resistant (XDR) TB, and four XDR-TB. The remaining strain was SIT1 and drug susceptible. MIRU-VNTR analysis allowed the identification of three Beijing clusters and two unique strains. Beijing strains were confirmed as "modern" sublineage. The mutations rpoB S531L and katG S315T were the most common among MDR strains. Moreover, the two strains evaluated by whole genome sequencing (WGS) shared most of the genetic features with the sublineage 2.2.1 "modern" Beijing previously characterized from Asian strains. WGS analysis of the MDR strain revealed the presence of eight SNPs previously reported in other MDR "Beijing-like" strains from Colombia. The presence of "modern" Beijing strains in Southwestern Colombia, most of them with MDR phenotype, suggests a different origin of this M. tuberculosis sublineage compared to other Beijing strains found in neighboring South American countries. This work may serve as a genetic baseline to study the evolution and spread of M. tuberculosis Beijing strains in Colombia, which play an important role in the propagation of MDR-TB.

Project description:Diagnostics that more accurately detect and quantify viable Mycobacterium tuberculosis (Mtb) in the sputum of patients undergoing therapy are needed. Current culture- and molecular-based tests have shown limited efficacy for monitoring treatment response in TB patients, either due to the presence of viable sub-populations of Mtb which fail to grow under standard culture conditions (termed differentially detectable/culturable Mtb, DD Mtb) or the prolonged half-life of Mtb DNA in sputum. Here, we report an optimized RNA-based method for detecting and quantifying viable Mtb from patient sputum during the course of therapy. We first empirically derived a novel RNA extraction protocol from sputum that improves recovery of Mtb RNA while almost completely eliminating contamination from Mtb DNA and host nucleic acids. Next, we identified five Mtb 16S rRNA primer sets with varying limits of detection that were capable of distinguishing between live versus dead H37Rv Mtb. This combined protocol was then tested on sputa from a longitudinal cohort of patients receiving therapy for drug sensitive (DS) or drug resistant (DR) TB with first-line or second-line regimens, respectively. Results were compared with that of culture, including CFU, BACTEC MGIT, and a limiting dilution assay capable of detecting DD Mtb. The five 16S rRNA primer sets positively identified nearly all (range 94-100%) culture positive sputa, and a portion (19-37%) of culture negative sputa. In comparison, ten highly expressed Mtb mRNAs showed positivity in 72-86% of culture positive sputa, and in 0-13% of culture negative sputa. Two of the five 16S rRNA primer sets were able to positively identify 100% of culture positive sputa, and when tested on culture negative sputa from the DS cohort at 2 months post-initiation of therapy, identified 40% of samples as positive; a percentage that is in line with expected treatment failure rates when first-line therapy is discontinued early. These two primer sets also detected 16S rRNA in 13-20% of sputa at 6 months post-initiation of therapy in the DR cohort. Cycle threshold values for 16S rRNA showed a strong correlation with Mtb numbers as determined by culture (R > 0.87), including as Mtb numbers declined during the course of treatment with first-line and second-line regimens. The optimized molecular assay outlined here may have utility for monitoring treatment response in TB patients.

Dataset Information

T CELL EPITOPES OF THE ESXA FULL GENE OF MYCOBACTERIUM TUBERCULOSIS FROM SPUTUM OF MDR-TB PATIENTS.

Publications

T CELL EPITOPES OF THE <i>ESXA</i> FULL GENE OF <i>MYCOBACTERIUM TUBERCULOSIS</i> FROM SPUTUM OF MDR-TB PATIENTS.

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