Abstract:Nucleic acid amplification tests for Mycobacterium tuberculosis (MTB) detection from sputum are highly sensitive and specific with smear microscopy positive specimens, but their sensitivity with smear-negative/culture-positive specimens is much lower; therefore, these tests cannot rule out a tuberculosis diagnosis. Co-extraction of PCR inhibitors may be a cause of decreased test sensitivity. Here the design and early validation of a MTB screening assay with sample preparation and qPCR methods designed to speci… Show more
“…The sequence specific capture probes and qPCR oligonucleotides were optimized for Tm, and the amount of NaCl was increased from 250 mM to 500 mM to enhance sequence specific capture of MTB DNA 11 . To estimate the limit of detection, 1 ml sputum specimens were contrived to contain 20, 10 or 5 cfu/ml of MTB H37Ra which has 17 copies of IS 6110
16 , and 20 replicates of each concentration were assayed.…”
Section: Resultsmentioning
confidence: 99%
“…The limit of detection of the test was estimated to be 20 cfu/ml which is significantly lower than the Xpert® MTB/RIF assay, and no amplification was observed from a panel of 6 NTM species. In a preliminary field study of 60 de-identified blinded sputa, a test sensitivity of 96% and specificity of 100% was observed when compared to the Xpert® MTB/RIF assay 11 . The DNA capture probes and qPCR primers have been optimized for greater analytical sensitivity, and we hypothesize that this novel diagnostic test, hereby entitled XtracTB Assay, will result in improved sensitivity with SSM− specimens over current TB diagnostics.Figure 1Schematic of sequence specific capture work flow: six steps, Thin, Lyse & Melt, Hybridize, Bind, Wash and Elute are indicated with corresponding times and temperatures detailed.
…”
Section: Introductionmentioning
confidence: 99%
“…Human DNA, co-extracted with MTB DNA, is a key amplification inhibitor found in sputum 10, 11 . We developed a protocol to selectively purify mycobacterial DNA utilizing a sputum thinning and MTB lysis step combined with MTB DNA sequence specific capture 11 to circumvent this problem.…”
Section: Introductionmentioning
confidence: 99%
“…Human DNA, co-extracted with MTB DNA, is a key amplification inhibitor found in sputum 10, 11 . We developed a protocol to selectively purify mycobacterial DNA utilizing a sputum thinning and MTB lysis step combined with MTB DNA sequence specific capture 11 to circumvent this problem. One ml of sputum is thinned by enzymatic and surfactant treatment followed by a heat step that kills the bacteria and initiates the specific capture reaction by completely denaturing the DNA in the specimen (Fig.…”
Nucleic acid amplification tests are increasingly used to diagnose tuberculosis (TB) due to their speed and sensitivity compared to sputum smear microscopy. However, these tests fail to equal culture’s sensitivity with sputum smear microscopy negative specimens and therefore cannot be used to rule out TB disease. For molecular tests to match culture’s sensitivity, they must detect ≤10 genomic copies of Mycobacterium tuberculosis (MTB) DNA, the limit of detection of culture, process ≥1 ml of sputum ensuring sufficient number of MTB are in the reaction, and efficiently remove sputum associated inhibitors from this large sample. Here we report the preliminary characterization of XtracTB Assay, a MTB testing protocol designed for inclusion in either an integrated point-of-care platform or a high throughput automated central laboratory system. The test combines DNA sequence specific sample prep to reduce the co-extraction of qPCR inhibitors with the amplification of two MTB specific loci (IS6110 and senX3-regX3) to increase test sensitivity and minimize the likelihood of false negatives. The analytical sensitivity of the XtracTB Assay was 5 genomic copies/ml of sputum rivaling that of culture. Furthermore, 142 valid test results yield clinical sensitivity of 94.9% (95% CI: 90.1–99.9) and specificity of 100% (95% CI: 90.0–100.0).
“…The sequence specific capture probes and qPCR oligonucleotides were optimized for Tm, and the amount of NaCl was increased from 250 mM to 500 mM to enhance sequence specific capture of MTB DNA 11 . To estimate the limit of detection, 1 ml sputum specimens were contrived to contain 20, 10 or 5 cfu/ml of MTB H37Ra which has 17 copies of IS 6110
16 , and 20 replicates of each concentration were assayed.…”
Section: Resultsmentioning
confidence: 99%
“…The limit of detection of the test was estimated to be 20 cfu/ml which is significantly lower than the Xpert® MTB/RIF assay, and no amplification was observed from a panel of 6 NTM species. In a preliminary field study of 60 de-identified blinded sputa, a test sensitivity of 96% and specificity of 100% was observed when compared to the Xpert® MTB/RIF assay 11 . The DNA capture probes and qPCR primers have been optimized for greater analytical sensitivity, and we hypothesize that this novel diagnostic test, hereby entitled XtracTB Assay, will result in improved sensitivity with SSM− specimens over current TB diagnostics.Figure 1Schematic of sequence specific capture work flow: six steps, Thin, Lyse & Melt, Hybridize, Bind, Wash and Elute are indicated with corresponding times and temperatures detailed.
…”
Section: Introductionmentioning
confidence: 99%
“…Human DNA, co-extracted with MTB DNA, is a key amplification inhibitor found in sputum 10, 11 . We developed a protocol to selectively purify mycobacterial DNA utilizing a sputum thinning and MTB lysis step combined with MTB DNA sequence specific capture 11 to circumvent this problem.…”
Section: Introductionmentioning
confidence: 99%
“…Human DNA, co-extracted with MTB DNA, is a key amplification inhibitor found in sputum 10, 11 . We developed a protocol to selectively purify mycobacterial DNA utilizing a sputum thinning and MTB lysis step combined with MTB DNA sequence specific capture 11 to circumvent this problem. One ml of sputum is thinned by enzymatic and surfactant treatment followed by a heat step that kills the bacteria and initiates the specific capture reaction by completely denaturing the DNA in the specimen (Fig.…”
Nucleic acid amplification tests are increasingly used to diagnose tuberculosis (TB) due to their speed and sensitivity compared to sputum smear microscopy. However, these tests fail to equal culture’s sensitivity with sputum smear microscopy negative specimens and therefore cannot be used to rule out TB disease. For molecular tests to match culture’s sensitivity, they must detect ≤10 genomic copies of Mycobacterium tuberculosis (MTB) DNA, the limit of detection of culture, process ≥1 ml of sputum ensuring sufficient number of MTB are in the reaction, and efficiently remove sputum associated inhibitors from this large sample. Here we report the preliminary characterization of XtracTB Assay, a MTB testing protocol designed for inclusion in either an integrated point-of-care platform or a high throughput automated central laboratory system. The test combines DNA sequence specific sample prep to reduce the co-extraction of qPCR inhibitors with the amplification of two MTB specific loci (IS6110 and senX3-regX3) to increase test sensitivity and minimize the likelihood of false negatives. The analytical sensitivity of the XtracTB Assay was 5 genomic copies/ml of sputum rivaling that of culture. Furthermore, 142 valid test results yield clinical sensitivity of 94.9% (95% CI: 90.1–99.9) and specificity of 100% (95% CI: 90.0–100.0).
“…Reflex genotypic drug resistance testing of patients with a positive TB test is an alternative strategy to up-front resistance testing for all patients. Possible strategies to improve sensitivity include nucleic acid extraction from the full sample volume, use of DNA baits to preferentially extract M. tuberculosis DNA [13], use of all of the extracted nucleic acid in the amplification reaction, nested amplification and detection of multiple and multicopy targets.…”
Background
Xpert MTB/RIF (Xpert MTB/RIF) and Xpert MTB/RIF Ultra (Xpert Ultra), the newest version, are the only World Health Organization (WHO)‐recommended rapid tests that simultaneously detect tuberculosis and rifampicin resistance in persons with signs and symptoms of tuberculosis, at lower health system levels. A previous Cochrane Review found Xpert MTB/RIF sensitive and specific for tuberculosis (
). Since the previous review, new studies have been published. We performed a review update for an upcoming WHO policy review.
Objectives
To determine diagnostic accuracy of Xpert MTB/RIF and Xpert Ultra for tuberculosis in adults with presumptive pulmonary tuberculosis (PTB) and for rifampicin resistance in adults with presumptive rifampicin‐resistant tuberculosis.
Search methods
We searched the Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, Web of Science, Latin American Caribbean Health Sciences Literature, Scopus, the WHO International Clinical Trials Registry Platform, the International Standard Randomized Controlled Trial Number Registry, and ProQuest, to 11 October 2018, without language restriction.
Selection criteria
Randomized trials, cross‐sectional, and cohort studies using respiratory specimens that evaluated Xpert MTB/RIF, Xpert Ultra, or both against the reference standard, culture for tuberculosis and culture‐based drug susceptibility testing or MTBDR
plus
for rifampicin resistance.
Data collection and analysis
Four review authors independently extracted data using a standardized form. When possible, we also extracted data by smear and HIV status. We assessed study quality using QUADAS‐2 and performed meta‐analyses to estimate pooled sensitivity and specificity separately for tuberculosis and rifampicin resistance. We investigated potential sources of heterogeneity. Most analyses used a bivariate random‐effects model. For tuberculosis detection, we first estimated accuracy using all included studies and then only the subset of studies where participants were unselected, i.e. not selected based on prior microscopy testing.
Main results
We identified in total 95 studies (77 new studies since the previous review): 86 studies (42,091 participants) evaluated Xpert MTB/RIF for tuberculosis and 57 studies (8287 participants) for rifampicin resistance. One study compared Xpert MTB/RIF and Xpert Ultra on the same participant specimen.
Tuberculosis detection
Of the total 86 studies, 45 took place in high tuberculosis burden and 50 in high TB/HIV burden countries. Most studies had low risk of bias.
Xpert MTB/RIF pooled sensitivity and specificity (95% credible Interval (CrI)) were 85% (82% to 88%) and 98% (97% to 98%), (70 studies, 37,237 unselected participants; high‐certainty evidence). We found similar accuracy when ...
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