scholarly journals Improving the Sensitivity of the Xpert MTB/RIF Assay on Sputum Pellets by Decreasing the Amount of Added Sample Reagent: a Laboratory and Clinical Evaluation

2015 ◽  
Vol 53 (4) ◽  
pp. 1258-1263 ◽  
Author(s):  
Nila J. Dharan ◽  
Danielle Amisano ◽  
Gerald Mboowa ◽  
Willy Ssengooba ◽  
Robert Blakemore ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Limit Of Detection ◽  
Smear Microscopy ◽  
Content Type ◽  
Clinical Specimens ◽  
Test Sensitivity ◽  
Smear Negative ◽  
Almost All ◽  
Culture Positive ◽  
Xpert Assay

The Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection ofMycobacterium tuberculosisin sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions ofM. tuberculosisinto sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained fromM. tuberculosisculture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in whichM. tuberculosiswas detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%;P= 0.03) and for smear-negative specimens (67% versus 58%;P= 0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increasedM. tuberculosisdetection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.

scholarly journals Evaluation of the Abbott RealTi m e MTB and RealTi m e MTB INH/RIF Assays for Direct Detection of Mycobacterium tuberculosis Complex and Resistance Markers in Respiratory and Extrapulmonary Specimens

2016 ◽  
Vol 54 (12) ◽  
pp. 3022-3027 ◽  
Author(s):  
Sabine Hofmann-Thiel ◽  
Nikolay Molodtsov ◽  
Uladzimir Antonenka ◽  
Harald Hoffmann
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Nucleic Acid Amplification ◽  
Content Type ◽  
Clinical Specimens ◽  
Different Types ◽  
Resistance Markers ◽  
Smear Negative

The Abbott RealTi m e MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTi m e MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDR plus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culture-negative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities.

scholarly journals Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons

2007 ◽  
Vol 56 (10) ◽  
pp. 1356-1362 ◽  
Author(s):  
Sagarika Haldar ◽  
Soumitesh Chakravorty ◽  
Manpreet Bhalla ◽  
Shyamasree De Majumdar ◽  
Jaya Sivaswami Tyagi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Beacon ◽  
Cost Effective ◽  
Nucleic Acid Amplification ◽  
Smear Microscopy ◽  
Direct Smear ◽  
Effective Manner ◽  
Directly Observed Treatment ◽  
Smear Negative ◽  
Culture Positive

The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the ‘gold’ standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a cost-effective manner.

scholarly journals Analytical and Clinical Evaluation of the Epistem Genedrive Assay for Detection of Mycobacterium tuberculosis

2016 ◽  
Vol 54 (4) ◽  
pp. 1051-1057 ◽  
Author(s):  
Shubhada Shenai ◽  
Derek T. Armstrong ◽  
Eloise Valli ◽  
David L. Dolinger ◽  
Lydia Nakiyingi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evaluation ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Performance Standards ◽  
World Health ◽  
Smear Microscopy ◽  
Respiratory Pathogens ◽  
Tuberculosis Case ◽  
Content Type

The Epistem Genedrive assay rapidly detects theMycobacterium tuberculosiscomplex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spikingM. tuberculosismc26030 cells into distilled water andM. tuberculosis-negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Genedrive to that of the Xpert MTB/RIF assay usingM. tuberculosiscultures as the reference standard. The Genedrive assay had an LOD of 1 pg/μl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 104CFU/ml and 2.5 × 105CFU/ml for cells spiked into water and sputum, respectively. False-positiverpoBprobe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containingMycobacterium abscessus,Mycobacterium gordonae, orMycobacterium thermoresistibile. In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Genedrive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Genedrive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay.

scholarly journals Use of Transrenal DNA for the Diagnosis of Extrapulmonary Tuberculosis in Children: a Case of Tubercular Otitis Media

2014 ◽  
Vol 53 (1) ◽  
pp. 336-338 ◽  
Author(s):  
Roberta Petrucci ◽  
Giulia Lombardi ◽  
Ilaria Corsini ◽  
Francesca Visciotti ◽  
Antonio Pirodda ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Otitis Media ◽  
Extrapulmonary Tuberculosis ◽  
Smear Microscopy ◽  
Test Results ◽  
Content Type ◽  
Dna Test ◽  
Smear Negative ◽  
Tuberculosis In Children

The diagnosis of tuberculosis (TB) is difficult in children, especially for smear-negative pulmonary and extrapulmonary TB, which are common at this age. We report an 11-year-old girl with TB otitis media with negative smear microscopy and Xpert MTB/RIF but positiveMycobacterium tuberculosis-specific transrenal DNA (Tr-MTB-DNA) test results and culture forM. tuberculosis.

scholarly journals Discordance between Xpert MTB/RIF Assay and Bactec MGIT 960 Culture System for Detection of Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Country with a Low Tuberculosis (TB) Incidence

2015 ◽  
Vol 53 (4) ◽  
pp. 1351-1354 ◽  
Author(s):  
Eiman Mokaddas ◽  
Suhail Ahmad ◽  
Hanaa S. Eldeen ◽  
Noura Al-Mutairi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Culture System ◽  
Content Type ◽  
Rifampin Resistance ◽  
Xpert Assay

Among 452 samples that were positive by the Xpert MTB/RIF (Xpert) assay and MGIT 960 system (MGIT), 440 and 10Mycobacterium tuberculosissamples were detected as rifampin susceptible and rifampin resistant, respectively. Two isolates that were rifampin susceptible by the MGIT system were rifampin resistant by the Xpert assay.rpoBsequencing identified a silent (CTG521TTG) mutation in one isolate and a missense (GAC516TAC) mutation in another. The detection of rifampin resistance is imperfect with both the Xpert assay and MGIT system. Any discordant rifampin resistance results should be confirmed by sequencing of therpoBgene.

scholarly journals Comparison of Proportion and Resistance Ratio Methods for Drug Susceptibility Testing of Mycobacterium tuberculosis Isolated from Patients Attending National Tuberculosis Centre, Nepal

2010 ◽  
Vol 5 (1) ◽  
pp. 13-20
Author(s):  
S Acharya ◽  
P Ghimire ◽  
DK Khadka ◽  
S Nepali
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fluorescence Microscopy ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Resistance Ratio ◽  
National Tuberculosis ◽  
Smear Negative ◽  
Culture Positive ◽  
Good Agreement

Background: Tuberculosis (TB) is among the most serious infectious cause of global morbidity and mortality. Emergence of Multi-drug resistant tuberculosis (MDR-TB) is posing an increased threat to TB control programs. Drug susceptibility testing (DST) of Mycobacterium tuberculosis (M. tuberculosis) isolates is important for tackling such problems. Setting: National Tuberculosis Centre (NTC), Thimi, Bhaktapur, Nepal. Objectives: Comparative evaluation of two in vitro DST methods in determining susceptibility of M. tuberculosis isolates from patients attending NTC, to front-line anti-TB drugs: (Isoniazid-INH, Rifampicin-RFP, Streptomycin-SM, and Ethambutol-EMB). Methodology: This study was conducted from Sep 2006-Jun 2007. A total of 862 sputum samples (diagnosis or follow up cases) collected from patients (type of patients or their categories was not differentiated in this study) attending NTC bacteriology lab for sputum direct smear microscopy were analyzed using fluorescence microscopy. All smear positive samples, smear negative samples requested for culture were cultured. All culture positive samples confirmed as M. tuberculosis by biochemical tests were processed for DST by both proportion (PR) and resistance ratio (RR) methods. Results: Out of 862 sputum samples analyzed, 226 (26.2%) samples were positive for Acid Fast Bacilli (AFB) by fluorescence microscopy. Among 323 samples 226 smear positive samples and 97 smear negative samples requested for culture), 221 (68.4%) were culture positive, 92 (28.5%) were culture negative and 10 (3.1%) were contaminated. Out of 221 isolates of M. tuberculosis, 57.5% were resistant to one or more drugs by the PR method and 56.6% by the RR method. Similarly, MDR isolates were 29.9% and 29% by PR and RR methods respectively. On correlation analysis using Mc Nemar Chi-square test, no significant difference between the two tests were observed (p>0.05). The results showed high agreement between both methods and agreement rates to INH, RFP, SM and EMB were 93.2%, 93.7%, 93.2% and 94.1% respectively. Similarly, the agreement rates between both methods using kappa analysis showed kappa (k) value of 0.86, 0.85, 0.86 and 0.84 for INH, RFP, SM and EMB respectively, which is believed to be good agreement between both methods (k=0.80 to 1.00: Very good agreement). Conclusion: In conclusion, this study showed that both the Proportion and Resistance ratio methods are equally good for determining drug susceptibility of M. tuberculosis. Keywords: Mycobacterium tuberculosis; Drug Susceptibility Testing; Proportion Method; Resistance Ratio Method. DOI: 10.3126/saarctb.v5i1.3078 SAARC J. Tuber. Lung Dis. HIV/AIDS 2008 Vol.5(1) 13-20

scholarly journals Clinical Evaluation of COBAS TaqMan PCR for the Detection ofMycobacterium tuberculosisandM. aviumComplex

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Satoshi Ikegame ◽  
Yoritake Sakoda ◽  
Nao Fujino ◽  
Kazuhito Taguchi ◽  
Masayuki Kawasaki ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Observational Study ◽  
Careful Consideration ◽  
Pcr Analysis ◽  
Test Results ◽  
Clinical Specimens ◽  
Cobas Taqman ◽  
Taqman Pcr ◽  
Smear Negative ◽  
Pcr Test

A retrospective observational study was performed to determine the sensitivity and limitation of PCR test for the detection ofMycobacterium tuberculosisandM. aviumcomplex. We obtained clinical specimens collected from the respiratory tract, culturedM. tuberculosisorM. aviumcomplex, and performed PCR analysis. A total of 299 samples (M. tuberculosis, 177;M. avium, 35;M. intracellulare, 87) were analyzed by COBAS TaqMan PCR from April 2007 to March 2011. The PCR positivity rates were 50–55%, 70–100%, 88–98%, and 100% in smear-negative, smear 1+, 2+, and 3+ groups, respectively. The PCR positivity of tuberculosis in smear 1+ was 80.6%, which was statistically significantly (P<0.001) lower than that of smear 2+ (97.3%). From January 2005 to March 2007, we collected an additional 138 samples (M. tuberculosis, 74;M. avium, 21; M. intracellulare, 43), which were analyzed by COBAS Amplicor PCR. The PCR positivity rates obtained using COBAS TaqMan PCR and COBAS Amplicor PCR were not significantly different. The sensitivity of PCR test for mycobacteria is not sufficient in case of smear 1+. Careful consideration must be given to the interpretation of negative PCR test results in smear 1+, because smear-positive tuberculosis is the criterion for isolation.

scholarly journals Monitoring Tuberculosis Drug Activity in Live Animals by Using Near-Infrared Fluorescence Imaging

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Raphael Sommer ◽  
Stewart T. Cole
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Near Infrared ◽  
Imaging System ◽  
Limit Of Detection ◽  
Bacterial Load ◽  
Genetically Engineered ◽  
Fluorescent Imaging ◽  
Effective Vaccine ◽  
Content Type ◽  
Immunodeficient Mice

ABSTRACT Worldwide, tuberculosis (TB) is the leading cause of death due to infection with a single pathogenic agent, Mycobacterium tuberculosis. In the absence of an effective vaccine, new, more powerful antibiotics are required to halt the growing spread of multidrug-resistant strains and to shorten the duration of TB treatment. However, assessing drug efficacy at the preclinical stage remains a long and fastidious procedure that delays the progression of drugs down the pipeline and towards the clinic. In this investigation, we report the construction, optimization, and characterization of genetically engineered near-infrared (NIR) fluorescent reporter strains of the pathogens Mycobacterium marinum and Mycobacterium tuberculosis that enable the direct visualization of bacteria in infected zebrafish and mice, respectively. Fluorescence could be measured precisely in infected immunodeficient mice, while its intensity appeared to be below the limit of detection in immunocompetent mice, probably because of the lower bacterial load obtained in these animals. Furthermore, we show that the fluorescence level accurately reflects the bacterial load, as determined by CFU enumeration, thus enabling the efficacy of antibiotic treatment to be assessed in live animals in real time. The NIR fluorescent imaging system disclosed here is a valuable resource for TB research and can serve to accelerate drug development.

scholarly journals Feasibility and Performance of Loop-Mediated Isothermal Amplification Assay in the Diagnosis of Pulmonary Tuberculosis in Decentralized Settings in Eastern China

2019 ◽  
Vol 2019 ◽  
pp. 1-4
Author(s):  
Zhongdong Wang ◽  
Haiyan Sun ◽  
Zhisheng Ren ◽  
Bai Xue ◽  
Jie Lu ◽  
...  
Keyword(s):  
Eastern China ◽  
Isothermal Amplification ◽  
Smear Microscopy ◽  
Loop Mediated Isothermal Amplification ◽  
Resource Limited ◽  
Control And Prevention ◽  
Amplification Assay ◽  
Smear Negative ◽  
And Performance ◽  
Culture Positive

Early diagnosis is essential for the control and prevention of tuberculosis (TB). The objective of this study was to investigate the feasibility and performance of loop-mediated isothermal amplification (LAMP) in the diagnosis of pulmonary TB in county-level microscopy centers in Qingdao, Eastern China. A total of 523 presumptive TB patients were consecutively recruited between July 2017 and April 2018, and 22 patients were excluded from the analysis. Of 102 culture-positive cases, TB-LAMP identified 91 cases, demonstrating a sensitivity of 89.2%. In comparison, the sensitivity of routine smear microscopy was 69.6% (71/102), which was significantly lower than that of TB-LAMP (P=0.001). In addition, TB-LAMP sensitivities in smear-positive and smear-negative samples were 98.6% and 67.7%, respectively. In conclusion, our data demonstrate that TB-LAMP outperforms conventional smear microscopy in TB diagnosis, which could be used as an alternative method for smear microscopy in resource-limited settings in China.

scholarly journals Molecular detection of Mycobacterium tuberculosis in poor-quality cough specimens

2020 ◽  
Vol 69 (9) ◽  
pp. 1179-1182
Author(s):  
Tondani A. Mboneni ◽  
Owen O. Eales ◽  
Ntsoaki L. Mosina ◽  
P. Bernard Fourie
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Molecular Detection ◽  
Molecular Transport ◽  
Poor Quality ◽  
Rt Pcr ◽  
Missed Opportunities ◽  
Content Type ◽  
Clinical Specimens ◽  
Enhanced Sensitivity

Clinical specimens unfit for laboratory processing represent missed opportunities for diagnosing tuberculosis. Poor-quality cough specimens (n=61) from presumptive tuberculosis cases were cultured and GeneXpert MTB/RIF (Xpert) successfully performed on samples transferred by flocked swab into PrimeStore molecular transport medium (PS-MTM). Mycobacterium tuberculosis was grown in culture from 13 (21.3 %) and Xpert reported 15 (24.2 %) positive, of which 10 concordant. RT-PCR of PS-MTM samples showed enhanced sensitivity; three positives were missed by Xpert, five by culture and three more detected for a total of 21 positives (34.4 %).

Sign in / Sign up

Export Citation Format

Share Document