scholarly journals Evaluation of the Xpert MTB/RIF Ultra Assay for Direct Detection ofMycobacterium tuberculosisComplex in Smear-Negative Extrapulmonary Samples

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Daniel Perez-Risco ◽  
David Rodriguez-Temporal ◽  
Ivan Valledor-Sanchez ◽  
Fernando Alcaide
Keyword(s):  
Nontuberculous Mycobacteria ◽  
Direct Detection ◽  
Extrapulmonary Tuberculosis ◽  
High Sensitivity ◽  
Rapid Diagnosis ◽  
Clinical Samples ◽  
Content Type ◽  
Smear Negative ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTThe rapid detection ofMycobacterium tuberculosiscomplex (MTUBC) in clinical samples is essential for successful treatment. New techniques such as real-time PCR have been developed in order to facilitate rapid diagnosis, but their sensitivity is low in extrapulmonary specimens, due to the low bacillary load in such samples. A next-generation assay has recently been developed to try to overcome this limitation. The aim of this study was to analyze the effectiveness of the Xpert MTB/RIF Ultra (GX-Ultra) for the detection of MTUBC DNA in 108 smear-negative extrapulmonary specimens that were MTUBC culture positive. In addition, 40 extrapulmonary culture-negative samples and 20 samples with nontuberculous mycobacteria were tested to evaluate the specificity of the assay. All samples were collected between May 1999 and May 2017. The GX-Ultra detected DNA of MTUBC in 82 extrapulmonary specimens that were MTUBC culture positive (75.9% sensitivity; 95% confidence interval [CI], 66.6 to 83.4%). The assay was negative for all clinical specimens that were MTUBC culture negative and the samples with nontuberculous mycobacteria (100% specificity). Furthermore, two (1.8%) samples presented mutations related to rifampin resistance. The highest sensitivity was obtained in samples of lymph nodes (94.1%) and nonsterile fluids (93.7%), followed by tissue specimens (86.6%), stool material (80%), abscess aspirates (64.7%), and sterile fluids (60.5%). Pleural fluids, one of the least optimal samples for detecting DNA of MTUBC, were GX-Ultra positive in 10/21 (47.6%) of cases. In summary, GX-Ultra showed excellent specificity and high sensitivity in paubacillary specimens, making it a useful tool for rapid diagnosis of extrapulmonary tuberculosis.

scholarly journals Culture-Independent Detection of Nontuberculous Mycobacteria in Clinical Respiratory Samples

2016 ◽  
Vol 54 (9) ◽  
pp. 2395-2398 ◽  
Author(s):  
Gianny P. Scoleri ◽  
Jocelyn M. Choo ◽  
Lex E. X. Leong ◽  
Thomas R. Goddard ◽  
Lisa Shephard ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Nontuberculous Mycobacteria ◽  
Direct Detection ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Culture Independent ◽  
Quantitative Pcr Assay ◽  
Single Reaction

Culture-based detection of nontuberculousMycobacteria(NTM) in respiratory samples is time consuming and can be subject to overgrowth by nonmycobacterial bacteria. We describe a single-reaction TaqMan quantitative PCR assay for the direct detection of NTM species in clinical samples that is specific, sensitive, and robust.

scholarly journals Direct Detection of Shigella in Stool Specimens by Use of a Metagenomic Approach

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Jie Liu ◽  
Mathieu Almeida ◽  
Furqan Kabir ◽  
Sadia Shakoor ◽  
Shahida Qureshi ◽  
...  
Keyword(s):  
Direct Detection ◽  
Positive Culture ◽  
Accurate Method ◽  
Diagnostic Methods ◽  
Metagenomic Sequencing ◽  
Illumina Hiseq ◽  
Content Type ◽  
Sequence Composition ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTThe underestimation ofShigellaspecies as a cause of childhood diarrhea disease has become increasingly apparent with quantitative PCR (qPCR)-based diagnostic methods versus culture. We sought to confirm qPCR-based detection ofShigellavia a metagenomics approach. Three groups of samples were selected from diarrheal cases from the Global Enteric Multicenter Study: nineShigellaculture-positive and qPCR-positive (culture+qPCR+) samples, nine culture-negative but qPCR-positive (culture−qPCR+) samples, and nine culture-negative and qPCR-negative (culture−qPCR−) samples. Fecal DNA was sequenced using paired-end Illumina HiSeq, whereby 3.26 × 108± 5.6 × 107high-quality reads were generated for each sample. We used Kraken software to compare the read counts specific to “Shigella” among the three groups. The proportions ofShigella-specific nonhuman sequence reads between culture+qPCR+(0.65 ± 0.42%) and culture−qPCR+(0.55 ± 0.31%) samples were similar (Mann-Whitney U test,P= 0.627) and distinct from the culture−qPCR−group (0.17 ± 0.15%,P< 0.05). The read counts of sequences previously targeted byShigella/enteroinvasiveEscherichia coli(EIEC) qPCR assays, namely,ipaH,virA,virG,ial,ShET2, andipaH3, were also similar between the culture+qPCR+and culture−qPCR+groups and distinct from the culture−qPCR−groups (P< 0.001). Kraken performed well versus other methods: its precision and recall ofShigellawere excellent at the genus level but variable at the species level. In summary, metagenomic sequencing indicates thatShigella/EIEC qPCR-positive samples are similar to those ofShigellaculture-positive samples inShigellasequence composition, thus supporting qPCR as an accurate method for detectingShigella.

Evaluation of the Enhanced Amplified Mycobacterium Tuberculosis Direct Test for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens

1999 ◽  
Vol 123 (11) ◽  
pp. 1101-1103 ◽  
Author(s):  
Michael B. Smith ◽  
John S. Bergmann ◽  
Michelle Onoroto ◽  
Greg Mathews ◽  
Gail L. Woods
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nontuberculous Mycobacteria ◽  
Direct Detection ◽  
Direct Test ◽  
Tuberculosis Complex ◽  
Predictive Values ◽  
Smear Negative ◽  
Sensitivity Specificity ◽  
Culture Positive ◽  
Respiratory Specimens

Abstract Objective.—To evaluate the performance of the enhanced Mycobacterium Tuberculosis Direct Test (E-MTD), for the direct detection of M tuberculosis complex (MTBC) in respiratory specimens. Design.—Two hundred seventy-four respiratory specimens from 151 patients in respiratory isolation were tested with the E-MTD, and the results were compared with the results of mycobacterial smear, culture, and the earlier form of the test, MTD-1. Results.—Forty-one specimens were culture positive for mycobacteria (20 MTBC and 21 nontuberculous mycobacteria), 23 of which were smear positive (16 MTBC, 7 nontuberculous mycobacteria). Twenty-four specimens were positive by E-MTD, and 21 were positive by MTD-1. Of the 20 MTBC culture-positive specimens, 19 were positive by the E-MTD and 19 were positive by the MTD-1. The remaining specimens were MTBC negative by all methods. After resolution of discrepancies, the sensitivity, specificity, and positive and negative predictive values were 95.2%, 100%, 100%, 99.6% for the MTD-1 and 95.2%, 98.8%, 87.0%, and 99.6%, for the E-MTD. For the E-MTD smear-positive and smear-negative specimens, these same values were 93.8%, 100%, 100%, and 87.5% and 100%, 98.8%, 62.5%, and 100%, respectively. Conclusion.—The results suggest that the E-MTD is a reliable method for the direct detection of MTBC in smear-positive respiratory specimens.

scholarly journals Clinical Evaluation of the BDProbeTec Strand Displacement Amplification Assay for Rapid Diagnosis of Tuberculosis

1998 ◽  
Vol 36 (9) ◽  
pp. 2766-2768 ◽  
Author(s):  
John S. Bergmann ◽  
Gail L. Woods
Keyword(s):  
Nontuberculous Mycobacteria ◽  
Direct Detection ◽  
Rapid Diagnosis ◽  
Becton Dickinson ◽  
Strand Displacement Amplification ◽  
Predictive Values ◽  
Amplification Assay ◽  
Smear Negative ◽  
Sensitivity Specificity ◽  
Respiratory Specimens

The reliability of the BDProbeTec MTB Test (Becton Dickinson, Sparks, Md.) for direct detection of Mycobacterium tuberculosis in respiratory specimens was evaluated by comparing results to those of conventional mycobacterial culture, with the BACTEC TB 460 and Middlebrook 7H11 biplates. Patients known to have tuberculosis were excluded from analysis. Of 523 specimens from 277 patients, 53 grew a mycobacterium: 24 specimens of M. tuberculosis and 29 specimens of nontuberculous mycobacteria. After initial testing, 42 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 95.8, 96.2, 54.8, and 99.8%, respectively. After resolution of discrepancies, 28 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 100, 99.2, 85.7, and 100%, respectively. These same values were 100, 80.8, 93.4, and 100%, respectively, for smear-positive samples and 100, 99.4, 75.0, and 100%, respectively, for smear-negative specimens.

Performance and Utilization of a Laboratory-Developed Nucleic Acid Amplification Test (NAAT) for the Diagnosis of Pulmonary and Extrapulmonary Tuberculosis in a Low-Prevalence Area

2020 ◽  
Vol 154 (1) ◽  
pp. 115-123
Author(s):  
Sanchita Das ◽  
Kathy A Mangold ◽  
Nirav S Shah ◽  
Lance R Peterson ◽  
Richard B Thomson ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Direct Detection ◽  
Bacterial Load ◽  
Extrapulmonary Tuberculosis ◽  
Nucleic Acid Amplification ◽  
Benefit Patient ◽  
Smear Negative ◽  
Low Prevalence ◽  
Culture Positive ◽  
Global Health Problem

Abstract Objectives Tuberculosis (TB) is a significant global health problem. In low-prevalence areas and low clinical suspicion, nucleic acid amplification tests (NAAT) for direct detection of Mycobacterium tuberculosis complex (MTBC) can speed therapy initiation and infection control. An NAAT assay (TBPCR) targeting MTBC IS6110 is used for detecting MTBC in our low-prevalence population. Methods Fifteen-year review of patient records identified 146 patients with culture-positive pulmonary tuberculosis (PTB) or extrapulmonary tuberculosis (EPTB). Laboratory-developed TBPCR was retrospectively compared with standard stain and cultures for PTB and EPTB diagnoses. Results TBPCR assay was used in 57% of patients with PTB and 33% of patients with EPTB. TBPCR detected 88.4% of all TB (smear-positive, 97%; smear-negative, 79%) with 100% specificity. Low bacterial load was indicated in TBPCR-negative PTB (P = .002) and EPTB (P &lt; .008). Conclusions TBPCR performance was optimum but significantly underused. Guidelines are proposed for mandated use of TBPCR that capture patients with clinically suspected PTB. Focused TBPCR use in low prevalence populations will benefit patient care, infection prevention, and public health.

scholarly journals Multiplex Real-Time PCR-shortTUB Assay for Detection of the Mycobacterium tuberculosis Complex in Smear-Negative Clinical Samples with Low Mycobacterial Loads

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Fernando Alcaide ◽  
Rocío Trastoy ◽  
Raquel Moure ◽  
Mónica González-Bardanca ◽  
Antón Ambroa ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Extrapulmonary Tuberculosis ◽  
Pcr Amplification ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Major Health Problem ◽  
Content Type ◽  
Smear Negative

ABSTRACT Tuberculosis (TB) remains a major health problem worldwide. Control of TB requires rapid, accurate diagnosis of active disease. However, extrapulmonary TB is very difficult to diagnose because the clinical specimens have very low bacterial loads. Several molecular methods involving direct detection of the Mycobacterium tuberculosis complex (MTBC) have emerged in recent years. Real-time PCR amplification simultaneously combines the amplification and detection of candidate sequences by using fluorescent probes (mainly TaqMan or Molecular Beacons) in automated systems. The multiplex real-time PCR-short assay is performed using locked nucleic acid (LNA) probes (length, 8 to 9 nucleotides) in combination with CodUNG to detect multiple pathogens in clinical samples. In this study, we evaluated the performance of this novel multiplex assay for detecting the MTBC in comparison with that of the conventional culture-based method. The multiplex real-time PCR-shortTUB assay targets two genes, whiB3 (redox-responsive transcriptional regulator) and pstS1 (phosphate-specific transporter), yielding limits of detection (LOD) of 10 copies and 100 copies, respectively, and amplification efficiencies of 92% and 99.7%, respectively. A total of 94 extrapulmonary samples and pulmonary samples with low mycobacterial loads (all smear negative; 75 MTBC culture positive) were analyzed using the test, yielding an overall sensitivity of 88% and a specificity of 95%. For pleural fluid and tissues/biopsy specimens, the sensitivity was 83% and 85%, respectively. In summary, this technique could be implemented in routine clinical microbiology testing to reduce the overall turnaround time for MTBC detection and may therefore be a useful tool for the diagnosis of extrapulmonary tuberculosis and diagnosis using pulmonary samples with low mycobacterial loads.

scholarly journals Evaluation of the performance of clinical predictors in estimating the probability of pulmonary tuberculosis among smear-negative cases in Northern Ethiopia: a cross-sectional study

BMJ Open ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. e037913
Author(s):  
Mala George ◽  
Geert-Jan Dinant ◽  
Efrem Kentiba ◽  
Teklu Teshome ◽  
Abinet Teshome ◽  
...  
Keyword(s):  
Pulmonary Tuberculosis ◽  
Cross Sectional Study ◽  
Clinical Suspicion ◽  
Secondary Outcome ◽  
Northern Ethiopia ◽  
Sectional Study ◽  
Cross Sectional ◽  
Smear Negative ◽  
Culture Positive ◽  
Culture Negative

ObjectivesTo evaluate the performance of the predictors in estimating the probability of pulmonary tuberculosis (PTB) when all versus only significant variables are combined into a decision model (1) among all clinical suspects and (2) among smear-negative cases based on the results of culture tests.DesignA cross-sectional study.SettingTwo public referral hospitals in Tigray, Ethiopia.ParticipantsA total of 426 consecutive adult patients admitted to the hospitals with clinical suspicion of PTB were screened by sputum smear microscopy and chest radiograph (chest X-ray (CXR)) in accordance with the Ethiopian guidelines of the National Tuberculosis and Leprosy Program. Discontinuation of antituberculosis therapy in the past 3 months, unproductive cough, HIV positivity and unwillingness to give written informed consent were the basis of exclusion from the study.Primary and secondary outcome measuresA total of 354 patients were included in the final analysis, while 72 patients were excluded because culture tests were not done.ResultsThe strongest predictive variables of culture-positive PTB among patients with clinical suspicion were a positive smear test (OR 172; 95% CI 23.23 to 1273.54) and having CXR lesions compatible with PTB (OR 10.401; 95% CI 5.862 to 18.454). The regression model had a good predictive performance for identifying culture-positive PTB among patients with clinical suspicion (area under the curve (AUC) 0.84), but it was rather poor in patients with a negative smear result (AUC 0.64). Combining all the predictors in the model compared with only the independent significant variables did not really improve its performance to identify culture-positive (AUC 0.84–0.87) and culture-negative (AUC 0.64–0.69) PTB.ConclusionsOur finding suggests that predictive models based on clinical variables will not be useful to discriminate patients with culture-negative PTB from patients with culture-positive PTB among patients with smear-negative cases.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

scholarly journals Comparative Evaluation of Enteric Bacterial Culture and a Molecular Multiplex Syndromic Panel in Children with Acute Gastroenteritis

2019 ◽  
Vol 57 (6) ◽  
Author(s):  
Thomas Kellner ◽  
Brendon Parsons ◽  
Linda Chui ◽  
Byron M. Berenger ◽  
Jianling Xie ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Rectal Swab ◽  
Bacterial Pathogen ◽  
Positive Culture ◽  
Content Type ◽  
Negative Culture ◽  
Percent Agreement ◽  
Stool Samples ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTAlthough enteric multianalyte syndromic panels are increasingly employed, direct comparisons with traditional methods and the inclusion of host phenotype correlations are limited. Luminex xTAG gastrointestinal pathogen panel (GPP) and culture results are highly concordant. However, phenotypic and microbiological confirmatory testing raises concerns regarding the accuracy of the GPP, especially forSalmonellaspp. A total of 3,089 children with gastroenteritis submitted stool specimens, rectal swab specimens, and clinical data. The primary outcome was bacterial pathogen detection agreement for shared targets between culture and the Luminex xTAG GPP. Secondary analyses included phenotype assessment, additional testing of GPP-negative/culture-positive isolate suspensions with the GPP, and in-house and commercial confirmatory nucleic acid testing of GPP-positive/culture-negative extracts. The overall percent agreement between technologies was >99% for each pathogen.Salmonellaspp. were detected in specimens from 64 participants: 12 (19%) by culture only, 9 (14%) by GPP only, and 43 (67%) by both techniques. Positive percent agreement forSalmonellaspp. was 78.2% (95% confidence interval [CI], 64.6%, 87.8%). Isolate suspensions from the 12 participants with specimens GPP negative/culture positive forSalmonellatested positive by GPP. Specimens GPP positive/culture negative forSalmonellaoriginated in younger children with less diarrhea and more vomiting. GPP-positive/culture-negative specimen extracts tested positive using additional assays for 0/2Campylobacter-positive specimens, 0/4Escherichia coliO157-positive specimens, 0/9Salmonella-positive specimens, and 2/3Shigella-positive specimens. For both rectal swab and stool samples, the median cycle threshold (CT) values, determined using quantitative PCR, were higher for GPP-negative/culture-positive samples than for GPP-positive/culture-positive samples (for rectal swabs, 36.9 [interquartile range {IQR}, 33.7, 37.1] versus 30.0 [IQR, 26.2, 33.2], respectively [P = 0.002]; for stool samples, 36.9 [IQR, 33.7, 37.1] versus 29.0 [IQR, 24.8, 30.8], respectively [P = 0.001]). GPP and culture have excellent overall agreement; however, for specific pathogens, GPP is less sensitive than culture and, notably, identifies samples false positive forSalmonellaspp.

scholarly journals Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

2017 ◽  
Vol 55 (6) ◽  
pp. 1755-1766 ◽  
Author(s):  
Divya Anthwal ◽  
Rakesh Kumar Gupta ◽  
Manpreet Bhalla ◽  
Shinjini Bhatnagar ◽  
Jaya Sivaswami Tyagi ◽  
...  
Keyword(s):  
High Resolution ◽  
Direct Detection ◽  
Melting Curve ◽  
Curve Analysis ◽  
High Resolution Melt ◽  
Melt Curve Analysis ◽  
Content Type ◽  
Plasmid Library ◽  
Mdr Tb ◽  
Smear Negative

ABSTRACT Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB , katG , and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH- katG , and INH- inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.

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