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Diagnosis of pediatric pulmonary tuberculosis by stool PCR.

ABSTRACT: Pediatric pulmonary tuberculosis diagnosis is difficult because young children are unable to expectorate sputum samples. Testing stool for tuberculosis DNA from swallowed sputum may diagnose pulmonary tuberculosis. Hospitalized children with suspected tuberculosis had stool, nasopharyngeal, and gastric aspirates cultured that confirmed pulmonary tuberculosis in 16/236 patients. Twenty-eight stored stools from these 16 children were used to evaluate stool polymerase chain reaction (PCR) for tuberculosis diagnosis compared with 28 stool samples from 23 healthy control children. Two DNA extraction techniques were used: fast-DNA mechanical homogenization and Chelex-resin chemical extraction. DNA was tested for tuberculosis DNA with a hemi-nested IS6110 PCR. PCR after Fast-DNA processing was positive for 6/16 culture-proven tuberculosis patients versus 5/16 after Chelex extraction (sensitivity 38% and 31%, respectively). All controls were negative (specificity 100%). If sensitivity can be increased, stool PCR would be a rapid, non-invasive, and relatively bio-secure initial test for children with suspected pulmonary tuberculosis.

SUBMITTER: Wolf H

PROVIDER: S-EPMC2912504 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Diagnosis of pediatric pulmonary tuberculosis by stool PCR.

Wolf Hilary H Mendez Melissa M Gilman Robert H RH Sheen Patricia P Soto Giselle G Velarde Angie K AK Zimic Mirko M Escombe A Roderick AR Montenegro Sonia S Oberhelman Richard A RA Evans Carlton A CA

The American journal of tropical medicine and hygiene 20081201 6

Pediatric pulmonary tuberculosis diagnosis is difficult because young children are unable to expectorate sputum samples. Testing stool for tuberculosis DNA from swallowed sputum may diagnose pulmonary tuberculosis. Hospitalized children with suspected tuberculosis had stool, nasopharyngeal, and gastric aspirates cultured that confirmed pulmonary tuberculosis in 16/236 patients. Twenty-eight stored stools from these 16 children were used to evaluate stool polymerase chain reaction (PCR) for tuber ...[more]

PMID: 19052299

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Project description:The WHO has endorsed the use of stool samples for diagnosis of tuberculosis (TB) in children, and targeted next-generation sequencing (tNGS) of stool has been shown to support diagnosis and provide information about drug susceptibility (DS). Optimizing extraction of DNA from stool for sequencing is critical to ensure high diagnostic sensitivity and accurate DS information. Human stool samples were spiked with various concentrations of Mycobacterium bovis bacillus Calmette-Guérin (BCG), and DNA was extracted from the samples using four different DNA extraction kits. Each sample was subjected to quantitative PCR for identifying Mycobacterium tuberculosis complex bacteria and underwent further analysis to assess the overall DNA yield, fragment length, and purity. This same process was performed with 10 pediatric participants diagnosed with pulmonary TB, and the samples underwent tNGS. The FastDNA spin kit for soil showed the best results on model samples spiked with known quantities of BCG, compared to the other extraction methods evaluated. For clinical samples, the FastDNA and PowerFecal Pro DNA (PowerFecal) kits both showed an increase in the overall DNA quantity, M. tuberculosis-specific DNA quantity, and successful targeted sequencing when testing was performed on stool samples, compared to the two other kits. Three samples extracted via PowerFecal and three samples extracted via FastDNA (from different patients) provided successful sequencing data, with an average depth of coverage of the rpoB region for FastDNA of 298 (range, 107 to 550) and for PowerFecal of 310 (range, 182 to 474), results that were comparable to one another (P = 0.946). The PowerFecal Pro and FastDNA spin kits were superior for extracting DNA from pediatric stool samples for tNGS. IMPORTANCE This is the first study to compare Mycobacterium tuberculosis DNA extraction techniques from pediatric stool samples for use with sequencing technologies. It provides an important starting point for other researchers to isolate quality DNA for this purpose to further the field and to continue to optimize protocols and approaches.

Project description:IntroductionTuberculosis (TB) continues to be a significant health burden, most commonly affecting the lungs and referred to as pulmonary TB (PTB). Diagnostic techniques of PTB primarily rely on expectorated sputum samples. However, the diagnostic yields are often hindered due to insufficient volume and quality of the sputum specimens. Moreover, some individuals are unable to provide sputum samples due to scanty sputum production or difficulty in coughing up and require an invasive procedure to obtain a respiratory sample, such as bronchoscopic or gastric aspiration. Thus, challenges in the acquisition of respiratory specimens warrant an alternate specimen. Therefore, this systematic review aims to evaluate the diagnostic accuracy of a stool specimen for the diagnosis of PTB in adults.Methods and analysisWe will search MEDLINE (Ovid), Embase (Ovid), Web of Science and Cochrane database from inception to April 2021 using a comprehensive search strategy. Two reviewers will independently perform screening, data extraction and quality assessment. The risk of bias assessment and applicability of results of eligible studies will be performed using the Quality of Diagnostic Accuracy Studies-2 tool. Bivariate random-effects models will be performed to calculate pooled sensitivity, specificity, positive likelihood ratio and negative likelihood ratio and diagnostic odds ratio along with 95% CI of stool specimen for each reported diagnostic method against any of the reference standard test (ie, mycobacterial culture or smear microscopy or Xpert assay using respiratory specimens). Heterogeneity between studies will be assessed by I2 statistics and Q statistic of the χ2 test.Ethics and disseminationThe results will be disseminated through publishing in a peer-reviewed medical journal and public presentations in relevant national and international conferences. As this is a systematic review of publicly available data, ethics approval is not required.Prospero registration numberCRD42021245203.

Project description:Tuberculosis is a rising global public health emergency. Then, it is a priority to undertake innovations in preventive, diagnostic, and therapeutic methods. Improved diagnostic methods for tuberculosis are urgently needed to address this global epidemic. These methods should be rapid, accurate, affordable, and able to detect drug-resistant tuberculosis. The benefits of these new diagnostic technics include earlier diagnosis and treatment, improved patient outcomes, and reduced economic burden. Therefore, we aimed to systematically review the diagnostic performance of droplet digital PCR (ddPCR)-a third-generation PCR-compared with quantitative Real Time-PCR (qPCR) for diagnosing pulmonary and extrapulmonary tuberculosis. We included 14 diagnostic accuracy test studies performed in Asia, Europe, and Latin America, 1,672 participants or biological samples, and 975 events (pulmonary or extrapulmonary tuberculosis). Most of the included studies had a low risk of bias (QUADAS-C tool). Sensitivity and specificity were lower for ddPCR [0.56 (95% CI 0.53-0.58) and 0.97 (95% CI 0.96-0.98), respectively] than for qPCR [0.66 (95% CI 0.60-0.71) and 0.98 (95% CI 0.97-0.99), respectively]. However, the area under the ROC curve (AUC) was higher for ddPCR than for qPCR (0.97 and 0.94, respectively). Comparing both AUCs using the Hanley & McNeil method, we found statistically significant differences (AUC difference of 4.40%, p = 0.0020). In the heterogeneity analysis, we found significant differences between both techniques according to the continent of origin of the study and the location of tuberculosis (pulmonary or extrapulmonary disease). The AUCs of both methods were similar in pulmonary tuberculosis. However, for extrapulmonary tuberculosis, the AUC was higher for ddPCR. We found some limitations: (1) significant heterogeneity of the studies, and (2) we could not perform subgroup analyses according to other relevant variables, such as the age and sex of the participants. Nonetheless, this study is the first meta-analysis that shows that ddPCR has a comparable diagnostic performance than qPCR for pulmonary tuberculosis. However, for extrapulmonary tuberculosis, ddPCR has a better discriminant capacity to differentiate between patients with and without extrapulmonary tuberculosis. We conclude that ddPCR is likely the best diagnostic technic for tuberculosis diagnosis, especially for extrapulmonary tuberculosis. More studies are still needed yet.Systematic review registrationhttps://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022382768, CRD42022382768.

Project description:BACKGROUNDThe Xpert MTB/RIF (Xpert) assay technology allows rapid and sensitive diagnosis of pulmonary tuberculosis (PTB) from sputum specimens. However, diagnosis of PTB is difficult for patients who cannot produce sputum. The objective of this study was to investigate the use of Xpert assay for successful detection of PTB using stool samples from adult subjects.METHODSBoth stool and sputum samples from known smear and Xpert positive PTB patients were collected from a TB hospital in Dhaka. Stool samples were collected from healthy individuals without TB symptoms from a slum area of Dhaka. Stool and sputum samples were decontaminated and concentrated using NALC-NaOH-Na-citrate solution and the resultant sediment was used for Xpert, acid-fast bacilli (AFB) microscopy and culture.RESULTSA total of 102 stool samples were collected from PTB patients and another 50 stool samples from healthy individuals without TB. The sensitivity of the Xpert assay for detection of M. tuberculosis in stool samples of PTB patients was 90.2% (95% CI, 82.9-95.0). All 50 stool samples from healthy individuals were negative by the assay (Specificity 100%; 95% CI, 92.9-100). Compared with the sputum culture positive results the sensitivity of the stool Xpert assay was 94.8% (95% CI, 88.5-97.8). Moreover, stool Xpert demonstrated full concordant results with the sputum culture for detection of rifampicin susceptibility. The cycle threshold values of rpoB probes obtained from Xpert assay correlated significantly with the bacilli load in the corresponding stool (Spearman correlation = -0.40, P < 0.01) and sputum (Spearman correlation = -0.77, P < 0.01) samples as determined by microscopy.CONCLUSIONSStool Xpert can be applied as a potential alternative of sputum testing for detection of M. tuberculosis and accurate determination of RIF susceptibility in adult PTB patients. The assay would be beneficial for rapid diagnosis of PTB for those adult patients who cannot expectorate sputum.

Project description:BackgroundDiagnosis of tuberculosis (TB) in children is challenging mainly due to the difficulty of obtaining respiratory specimen and lack of sensitive diagnostic tests. The objective of this study was to evaluate the diagnostic performance of Xpert MTB/RIF (Xpert here after) for the diagnosis of pulmonary TB (PTB) from stool specimen in children.MethodsA cross-sectional study was conducted among consecutively recruited children (less than 15 years old) with presumptive PTB at Jimma Medical Center, Ethiopia. One pulmonary specimen (expectorated sputum or gastric aspirate) was collected from each participant and tested for TB by Xpert and Lowenstein-Jensen (LJ) culture. In addition, one stool specimen per child was collected and tested by Xpert after a single step, centrifuge-free stool processing method adapted from KNCV TB Foundation. Diagnostic performance of Xpert was calculated with reference to LJ culture and to a composite reference standards (CRS) comprising of confirmed TB (positive by Xpert and/or culture) and unconfirmed TB (clinical diagnosis with improvement after anti-TB treatment).ResultsA total of 178 children were enrolled; 152 of whom had complete microbiological results. Overall, TB was diagnosed in 13.2% (20/152) of the children with presumptive TB. Of these, only ten had microbiologically confirmed TB (positive Xpert and/or culture) and the remaining ten were clinically diagnosed with positive response to anti-TB treatment and were classified as unconfirmed TB. Stool Xpert had sensitivity of 100% (95%CI: 66.4-100) and specificity of 99.3% (95%CI: 96.2-100) compared to culture; however, the sensitivity was decreased to 50% (95%CI: 27.2-72.8) when compared to CRS. The Xpert on gastric aspirate had sensitivity of 77.8% (95%CI: 40-97.2) compared to culture and 40% (95%CI: 19.1-64) compared to CRS.ConclusionsThe sensitivity of Xpert for stool sample is comparable to that for gastric aspirate. Stool sample is a potential alternative to pulmonary specimen in the diagnosis of pulmonary TB in children using Xpert.

Project description:BACKGROUND. Novel rapid diagnostics for active tuberculosis (TB) are required to overcome the time delays and inadequate sensitivity of current microbiological tests that are critically dependent on sampling the site of disease. Multiparametric blood transcriptomic signatures of TB have been described as potential diagnostic tests. We sought to identify the best transcript candidates as host biomarkers for active TB, extend the evaluation of their specificity by comparison with other infectious diseases, and to test their performance in both pulmonary and extrapulmonary TB. METHODS. Support vector machine learning, combined with feature selection, was applied to new and previously published blood transcriptional profiles in order to identify the minimal TB?specific transcriptional signature shared by multiple patient cohorts including pulmonary and extrapulmonary TB, and individuals with and without HIV-1 coinfection. RESULTS. We identified and validated elevated blood basic leucine zipper transcription factor 2 (BATF2) transcript levels as a single sensitive biomarker that discriminated active pulmonary and extrapulmonary TB from healthy individuals, with receiver operating characteristic (ROC) area under the curve (AUC) scores of 0.93 to 0.99 in multiple cohorts of HIV-1-negative individuals, and 0.85 in HIV-1-infected individuals. In addition, we identified and validated a potentially novel 4-gene signature comprising CD177, haptoglobin, immunoglobin J chain, and galectin 10 that discriminated active pulmonary and extrapulmonary TB from other febrile infections, giving ROC AUCs of 0.94 to 1. CONCLUSIONS. Elevated blood BATF2 transcript levels provide a sensitive biomarker that discriminates active TB from healthy individuals, and a potentially novel 4-gene transcriptional signature differentiates between active TB and other infectious diseases in individuals presenting with fever. FUNDING. MRC, Wellcome Trust, Rosetrees Trust, British Lung Foundation, NIHR.

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Diagnosis of pediatric pulmonary tuberculosis by stool PCR.

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Diagnosis of pediatric pulmonary tuberculosis by stool PCR.

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