Dataset Information

An electrochemical biosensor for the detection of Mycobacterium tuberculosis DNA from sputum and urine samples.

ABSTRACT: Tuberculosis (TB) is a major global public health problem with high mortality and morbidity. In low-middle income countries (LMIC) a large number of respiratory symptomatic cases that require TB screening per year demands more accurate, fast and affordable testing for TB diagnostics. Sputum smear is the initial screening test in LMICs, however, its sensitivity is limited in patients with low sputum bacilli load. The same limitation is observed in the currently available molecular tests. We designed, standardized and evaluated an electrochemical biosensor that detects the highly specific DNA insertion element 6110 (IS6110). A PCR amplified DNA product is hybridized on the surface of the working electrode built on FTO-Glass with immobilized specific DNA probes, after which cyclic voltammetry is performed with an Ag/AgCl reference electrode and a platinum counter electrode. The response of the sensor was measured by the ratio (cathodic peak current of the hybridized sensor) / (cathodic peak current of the non-hybridized sensor). We tested the biosensor, using positive hybridization control sequences, genomic DNA extracted from M. tuberculosis strains and sputum of TB patients, and extracted DNA from the urine of healthy controls spiked with M. tuberculosis DNA. This biosensor was effective for the detection of M. tuberculosis DNA with a detection limit of 16 fM in sputum sample and 1 fM in spiked urine samples. The low cost and the relatively brief duration of the assay make this an important TB screening tool in the fight against tuberculosis.

SUBMITTER: Ramos-Sono D

PROVIDER: S-EPMC7592764 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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An electrochemical biosensor for the detection of Mycobacterium tuberculosis DNA from sputum and urine samples.

Ramos-Sono Daniel D Laureano Raúl R Rueda Daniel D Gilman Robert H RH La Rosa Adolfo A Ruiz Jesús J León Raúl R Sheen Patricia P Zimic Mirko M

PloS one 20201028 10

Tuberculosis (TB) is a major global public health problem with high mortality and morbidity. In low-middle income countries (LMIC) a large number of respiratory symptomatic cases that require TB screening per year demands more accurate, fast and affordable testing for TB diagnostics. Sputum smear is the initial screening test in LMICs, however, its sensitivity is limited in patients with low sputum bacilli load. The same limitation is observed in the currently available molecular tests. We desig ...[more]

PMID: 33112923

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Project description:BackgroundThe use of early morning sputum samples (EMS) to diagnose tuberculosis (TB) can result in treatment delay given the need for the patient to return to the clinic with the EMS, increasing the chance of patients being lost during their diagnostic workup. However, there is little evidence to support the superiority of EMS over spot sputum samples. In this new analysis of the REMoxTB study, we compare the diagnostic accuracy of EMS with spot samples for identifying Mycobacterium tuberculosis pre- and post-treatment.MethodsPatients who were smear positive at screening were enrolled into the study. Paired sputum samples (one EMS and one spot) were collected at each trial visit pre- and post-treatment. Microscopy and culture on solid LJ and liquid MGIT media were performed on all samples; those missing corresponding paired results were excluded from the analyses.ResultsData from 1115 pre- and 2995 post-treatment paired samples from 1931 patients enrolled in the REMoxTB study were analysed. Patients were recruited from South Africa (47%), East Africa (21%), India (20%), Asia (11%), and North America (1%); 70% were male, median age 31 years (IQR 24-41), 139 (7%) co-infected with HIV with a median CD4 cell count of 399 cells/μL (IQR 318-535). Pre-treatment spot samples had a higher yield of positive Ziehl-Neelsen smears (98% vs. 97%, P = 0.02) and LJ cultures (87% vs. 82%, P = 0.006) than EMS, but there was no difference for positivity by MGIT (93% vs. 95%, P = 0.18). Contaminated and false-positive MGIT were found more often with EMS rather than spot samples. Surprisingly, pre-treatment EMS had a higher smear grading and shorter time-to-positivity, by 1 day, than spot samples in MGIT culture (4.5 vs. 5.5 days, P < 0.001). There were no differences in time to positivity in pre-treatment LJ culture, or in post-treatment MGIT or LJ cultures. Comparing EMS and spot samples in those with unfavourable outcomes, there were no differences in smear or culture results, and positive results were not detected earlier in Kaplan-Meier analyses in either EMS or spot samples.ConclusionsOur data do not support the hypothesis that EMS samples are superior to spot sputum samples in a clinical trial of patients with smear positive pulmonary TB. Observed small differences in mycobacterial burden are of uncertain significance and EMS samples do not detect post-treatment positives any sooner than spot samples.

Project description:Next-Generation Sequencing (NGS) is widely used to investigate genomic variation. In several studies, the genetic variation of Mycobacterium tuberculosis has been analyzed in sputum samples without previous culture, using target enrichment methodologies for NGS. Alignments obtained by different programs generally map the sequences under default parameters, and from these results, it is assumed that only Mycobacterium reads will be obtained. However, variants of interest microorganism in clinical samples can be confused with a vast collection of reads from other bacteria, viruses, and human DNA. Currently, there are no standardized pipelines, and the cleaning success is never verified since there is a lack of rigorous controls to identify and remove reads from other sputum-microorganisms genetically similar to M. tuberculosis. Therefore, we designed a bioinformatic pipeline to process NGS data from sputum samples, including several filters and quality control points to identify and eliminate non-M. tuberculosis reads to obtain a reliable genetic variant report. Our proposal uses the SURPI software as a taxonomic classifier to filter input sequences and perform a mapping that provides the highest percentage of Mycobacterium reads, minimizing the reads from other microorganisms. We then use the filtered sequences to perform variant calling with the GATK software, ensuring the mapping quality, realignment, recalibration, hard-filtering, and post-filter to increase the reliability of the reported variants. Using default mapping parameters, we identified reads of contaminant bacteria, such as Streptococcus, Rhotia, Actinomyces, and Veillonella. Our final mapping strategy allowed a sequence identity of 97.8% between the input reads and the whole M. tuberculosis reference genome H37Rv using a genomic edit distance of three, thus removing 98.8% of the off-target sequences with a Mycobacterium reads loss of 1.7%. Finally, more than 200 unreliable genetic variants were removed during the variant calling, increasing the report's reliability.

Dataset Information

An electrochemical biosensor for the detection of Mycobacterium tuberculosis DNA from sputum and urine samples.

Publications

An electrochemical biosensor for the detection of Mycobacterium tuberculosis DNA from sputum and urine samples.

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