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A pre-clinical validation plan to evaluate analytical sensitivities of molecular diagnostics such as BD MAX MDR-TB, Xpert MTB/Rif Ultra and FluoroType MTB.

ABSTRACT: Rapid diagnosis of tuberculosis (TB) and antibiotic resistances are imperative to initiate effective treatment and to stop transmission of the disease. A new generation of more sensitive, automated molecular TB diagnostic tests has been recently launched giving microbiologists more choice between several assays with the potential to detect resistance markers for rifampicin and isoniazid. In this study, we determined analytical sensitivities as 95% limits of detection (LoD95) for Xpert MTB/Rif Ultra (XP-Ultra) and BD-MAX MDR-TB (BD-MAX) as two representatives of the new test generation, in comparison to the conventional FluoroType MTB (FT-MTB). Test matrices used were physiological saline solution, human and a mucin-based artificial sputum (MUCAS) each spiked with Mycobacterium tuberculosis in declining culture- and qPCR-controlled concentrations. With BD-MAX, XP-Ultra, and FT-MTB, we measured LoD95TB values of 2.1 cfu/ml (CI95%: 0.9-23.3), 3.1 cfu/ml (CI95%: 1.2-88.9), and 52.1 cfu/ml (CI95%: 16.7-664.4) in human sputum; of 6.3 cfu/ml (CI95%: 2.9-31.8), 1.5 cfu/ml (CI95%: 0.7-5.0), and 30.4 cfu/ml (CI95%: 17.4-60.7) in MUCAS; and of 2.3 cfu/ml (CI95%: 1.1-12.0), 11.5 cfu/ml (CI95%: 5.6-47.3), and 129.1 cfu/ml (CI95%: 82.8-273.8) in saline solution, respectively. LoD95 of resistance markers were 9 to 48 times higher compared to LoD95TB. BD-MAX and XP-Ultra have an equal and significantly increased analytical sensitivity compared to conventional tests. MUCAS resembled human sputum, while both yielded significantly different results than normal saline. MUCAS proved to be suitable for quality control of PCR assays for TB diagnostics.

SUBMITTER: Beutler M

PROVIDER: S-EPMC6946130 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A pre-clinical validation plan to evaluate analytical sensitivities of molecular diagnostics such as BD MAX MDR-TB, Xpert MTB/Rif Ultra and FluoroType MTB.

Beutler Markus M Plesnik Sara S Mihalic Marina M Olbrich Laura L Heinrich Norbert N Schumacher Samuel S Lindner Michael M Koch Ina I Grasse Wolfgang W Metzger-Boddien Christoph C Hofmann-Thiel Sabine S Hoffmann Harald H

PloS one 20200107 1

Rapid diagnosis of tuberculosis (TB) and antibiotic resistances are imperative to initiate effective treatment and to stop transmission of the disease. A new generation of more sensitive, automated molecular TB diagnostic tests has been recently launched giving microbiologists more choice between several assays with the potential to detect resistance markers for rifampicin and isoniazid. In this study, we determined analytical sensitivities as 95% limits of detection (LoD95) for Xpert MTB/Rif Ul ...[more]

PMID: 31910223

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Project description:The aim of this study was to evaluate the performance of Xpert MTB/RIF Ultra (Ultra) compared with its predecessor, Xpert MTB/RIF (Xpert), in the diagnosis of tuberculosis (TB) in a low TB incidence country. Retrospective analysis was performed on 689 clinical samples received between 2015 and 2018, on which Xpert was performed, and on 715 samples, received between 2018 and 2020, on which Ultra was performed. Samples were pulmonary (n = 830) and extrapulmonary (n = 574) in nature, and a total of 264 were culture positive for Mycobacterium tuberculosis complex (MTBC). The diagnostic performance of both assays was analyzed using culture as the reference standard. The sensitivity of Ultra for culture positive (smear positive and smear negative) MTBC samples, was 93.2% (110/118) compared with 82.2% (120/146) for Xpert (P = 0.0078). In smear negative-culture positive samples, Ultra had a sensitivity of 74.2% (23/31) versus 36.11% (13/36) for Xpert (P = 0.0018). Specificity of both assays was comparable at 94.8% (566/597) for Ultra and 95.8% (520/543) for Xpert (P = 0.4475). The sensitivity of Ultra and Xpert assays among exclusively pulmonary samples was 95.3% (82/86) and 90.3% (84/93), respectively (P = 0.1955), and 87.5% (28/32) and 67.9% (36/53), respectively, among extrapulmonary samples (P = 0.0426). Ultra showed improved performance compared with Xpert in a low TB incidence setting, particularly in smear negative and extrapulmonary MTBC disease. The specificity of Ultra was lower than Xpert, however, this was not statistically significant. IMPORTANCE The study demonstrates the improved sensitivity of the Ultra compared with the Xpert, particularly in smear negative TB disease, for both pulmonary and extrapulmonary samples in a low TB incidence setting. Cycle threshold (Ct) value for both assays was found to positively correlate with time to TB culture positivity, suggesting that Ct and semiquantitative results could be used as indicators of sample MTBC bacillary burden, and thus, perhaps, of transmission potential. This may have implications for the designation of patient isolation precautions.

Project description:BackgroundClinical tuberculosis diagnosis and empiric treatment have traditionally been common among patients with negative bacteriologic test results. Increasing availability of rapid molecular diagnostic tests, including Xpert MTB/RIF and the new Xpert Ultra cartridge, may alter the role of empiric treatment.MethodsWe prospectively enrolled outpatients age > = 15 who were evaluated for pulmonary tuberculosis at three health facilities in Kampala, Uganda. Using sputum mycobacterial culture, interviews, and clinical record abstraction, we estimated the accuracy of clinical diagnosis relative to Xpert and sputum culture and assessed the contribution of clinical diagnosis to case detection.ResultsOver a period of 9 months, 99 patients were diagnosed with pulmonary tuberculosis and subsequently completed sputum culture; they were matched to 196 patients receiving negative tuberculosis evaluations in the same facilities. Xpert was included in the evaluation of 291 (99%) patients. Compared to culture, Xpert had a sensitivity of 92% (95% confidence interval 83-97%) and specificity of 95% (92-98%). Twenty patients with negative Xpert were clinically diagnosed with tuberculosis and subsequently had their culture status determined; two (10%) were culture-positive. Considering all treated patients regardless of Xpert and culture data completeness, and considering treatment initiations before a positive Xpert (N = 4) to be empiric, 26/101 (26%) tuberculosis treatment courses were started empirically. Compared to sputum smear- or Xpert-positive patients with positive cultures, empirically-treated, Xpert-negative patients with negative cultures had higher prevalence of HIV (67% versus 37%), shorter duration of cough (median 4 versus 8 weeks), and lower inflammatory markers (median CRP 7 versus 101 mg/L).ConclusionJudged against sputum culture in a routine care setting of high HIV prevalence, the accuracy of Xpert was high. Clinical judgment identified a small number of additional culture-positive cases, but with poor specificity. Although clinicians should continue to prescribe tuberculosis treatment for Xpert-negative patients whose clinical presentations strongly suggest pulmonary tuberculosis, they should also carefully consider alternative diagnoses.

Project description:BACKGROUND:Diagnosis of TB in pediatric population poses several challenges. A novel initiative was implemented in several major cities of India aimed at providing upfront access to free-of-cost Xpert MTB/RIF to presumptive pediatric TB cases. This paper aims to describe the experience of implementing this large initiative and assess feasibility of the intervention in high TB burden settings. METHODS:Data were drawn from the pediatric TB project implemented in 10 major cities of India between April 2014 and March 2018. In each city, providers, both public and private, were engaged and linked with a high throughput Xpert MTB/RIF lab (established in that city) through rapid specimen transportation and electronic reporting system. Rates and proportions were estimated to describe the characteristics of this cohort. RESULTS:Of the total 94,415 presumptive pediatric TB cases tested in the project, 6,270 were diagnosed positive for MTB (6.6%) on Xpert MTB/RIF (vs 2% on smear microscopy). Among MTB positives, 545 cases were rifampicin resistant (8.7%). The median duration between collection of specimens and reporting of results was 0 days (same day) and >89% cases were initiated on treatment. Approximately 50% of the specimens tested were non-sputum. The number of providers/facilities engaged under the project increased >10-fold (from 124 in Q2'14 to 1416 in Q1'18). CONCLUSION:This project, which was one of the largest initiatives globally among pediatric population, demonstrated the feasibility of sustaining rapid and upfront access to free-of-cost Xpert MTB/RIF testing. The project underscores the efficiency of this rapid diagnostic assay in tackling several challenges in pediatric TB diagnosis, identifies opportunities for further interventions as well as brings to light scope for effective engagement with healthcare providers. The findings have facilitated a policy decision by National TB Programme mandating the use of Xpert MTB/RIF as a primary diagnostic tool for TB diagnosis in children, which is being scaled-up.

Project description:BackgroundXpert MTB/RIF Ultra (Xpert Ultra) might have higher sensitivity than its predecessor, Xpert MTB/RIF (Xpert), but its role in tuberculous meningitis diagnosis is uncertain. We aimed to compare Xpert Ultra with Xpert for the diagnosis of tuberculous meningitis in HIV-uninfected and HIV-infected adults.MethodsIn this prospective, randomised, diagnostic accuracy study, adults (≥16 years) with suspected tuberculous meningitis from a single centre in Vietnam were randomly assigned to cerebrospinal fluid testing by either Xpert Ultra or Xpert at baseline and, if treated for tuberculous meningitis, after 3-4 weeks of treatment. Test performance (sensitivity, specificity, and positive and negative predictive values) was calculated for Xpert Ultra and Xpert and compared against clinical and mycobacterial culture reference standards. Analyses were done for all patients and by HIV status.FindingsBetween Oct 16, 2017, and Feb 10, 2019, 205 patients were randomly assigned to Xpert Ultra (n=103) or Xpert (n=102). The sensitivities of Xpert Ultra and Xpert for tuberculous meningitis diagnosis against a reference standard of definite, probable, and possible tuberculous meningitis were 47·2% (95% CI 34·4-60·3; 25 of 53 patients) for Xpert Ultra and 39·6% (27·6-53·1; 21 of 53) for Xpert (p=0·56); specificities were 100·0% (95% CI 92·0-100·0; 44 of 44) and 100·0% (92·6-100·0; 48 of 48), respectively. In HIV-negative patients, the sensitivity of Xpert Ultra was 38·9% (24·8-55·1; 14 of 36) versus 22·9% (12·1-39·0; eight of 35) by Xpert (p=0·23). In HIV co-infected patients, the sensitivities were 64·3% (38·8-83·7; nine of 14) for Xpert Ultra and 76·9% (49·7-91·8; ten of 13) for Xpert (p=0·77). Negative predictive values were 61·1% (49·6-71·5) for Xpert Ultra and 60·0% (49·0-70·0) for Xpert. Against a reference standard of mycobacterial culture, sensitivities were 90·9% (72·2-97·5; 20 of 22 patients) for Xpert Ultra and 81·8% (61·5-92·7; 18 of 22) for Xpert (p=0·66); specificities were 93·9% (85·4-97·6; 62 of 66) and 96·9% (89·5-91·2; 63 of 65), respectively. Six (22%) of 27 patients had a positive test by Xpert Ultra after 4 weeks of treatment versus two (9%) of 22 patients by Xpert.InterpretationXpert Ultra was not statistically superior to Xpert for the diagnosis of tuberculous meningitis in HIV-uninfected and HIV-infected adults. A negative Xpert Ultra or Xpert test does not rule out tuberculous meningitis. New diagnostic strategies are urgently required.FundingWellcome Trust and the Foundation for Innovative New Diagnostics.

Project description:BackgroundClinical interpretation of trace results by Xpert MTB/RIF Ultra assay (Ultra) used as an initial diagnostic test for tuberculosis (TB) may be challenging. The aim of the study was to evaluate the frequency and epidemiology of trace readouts in routine clinical practice in a low TB prevalence setting and to propose guidance on how to manage patients with trace calls considering the data available (clinical, radiological, bacteriological etc.).Materials and methodsA retrospective, observational, monocentric study was conducted at IRCCS Azienda Ospedaliero-Universitaria of Bologna, Italy between November 2017-December 2020. Presumptive TB patients with at least one Ultra trace result during diagnostic workup before treatment were included in the study. Patients with ongoing anti-TB treatment at the time of the trace call result or with no clinical data available were excluded from the study.ResultsFifty-nine presumptive TB patients with Ultra trace readouts were included in the study (mean age 37.0 years, 61% males). Four patients had a history of TB in the last 2 years. Twenty-five (42.4%) of the 59 samples with trace results were respiratory material. 57/59 (96.6%) patients started anti-TB treatment soon after obtaining trace results, based on clinical, radiological or other information available, while for two patients with a recent history of TB the trace result did not lead to anti-TB treatment. Culture was positive for M. tuberculosis for 31/59 (52.5%) samples with trace calls: 13/25 (52.0%) were respiratory samples and 18/33 (54.5%) non-respiratory samples. The clinical and/or radiological findings of 47/57 (82.4%) patients given anti-TB therapy improved during treatment.ConclusionIn low TB incidence settings, Ultra trace calls in presumptive TB patients should be considered as true-positive and treatment should be started promptly, except in cases of recent history of TB, where careful evaluation of other diagnostic criteria is necessary before starting anti-TB treatment. A decisional algorithm for clinical management is proposed.

Project description:BACKGROUND:Xpert MTB/RIF, the first automated molecular test for tuberculosis, is transforming the diagnostic landscape in high-burden settings. This study assessed the impact of up-front Xpert MTB/RIF testing on detection of pulmonary tuberculosis (PTB) and rifampicin-resistant PTB (DR-TB) cases in India. METHODS:This demonstration study was implemented in 18 sub-district level TB programme units (TUs) in India in diverse geographic and demographic settings covering a population of 8.8 million. A baseline phase in 14 TUs captured programmatic baseline data, and an intervention phase in 18 TUs had Xpert MTB/RIF offered to all presumptive TB patients. We estimated changes in detection of TB and DR-TB, the former using binomial regression models to adjust for clustering and covariates. RESULTS:In the 14 study TUs, which participated in both phases, 10,675 and 70,556 presumptive TB patients were enrolled in the baseline and intervention phase, respectively, and 1,532 (14.4%) and 14,299 (20.3%) bacteriologically confirmed PTB cases were detected. The implementation of Xpert MTB/RIF was associated with increases in both notification rates of bacteriologically confirmed TB cases (adjusted incidence rate ratio [aIRR] 1.39; CI 1.18-1.64), and proportion of bacteriological confirmed TB cases among presumptive TB cases (adjusted risk ratio (aRR) 1.33; CI 1.6-1.52). Compared with the baseline strategy of selective drug-susceptibility testing only for PTB cases at high risk of drug-resistant TB, Xpert MTB/RIF implementation increased rifampicin resistant TB case detection by over fivefold. Among, 2765 rifampicin resistance cases detected, 1055 were retested with conventional drug susceptibility testing (DST). Positive predictive value (PPV) of rifampicin resistance detected by Xpert MTB/RIF was 94.7% (CI 91.3-98.1), in comparison to conventional DST. CONCLUSION:Introduction of Xpert MTB/RIF as initial diagnostic test for TB in public health facilities significantly increased case-notification rates of all bacteriologically confirmed TB by 39% and rifampicin-resistant TB case notification by fivefold.

Dataset Information

A pre-clinical validation plan to evaluate analytical sensitivities of molecular diagnostics such as BD MAX MDR-TB, Xpert MTB/Rif Ultra and FluoroType MTB.

Publications

A pre-clinical validation plan to evaluate analytical sensitivities of molecular diagnostics such as BD MAX MDR-TB, Xpert MTB/Rif Ultra and FluoroType MTB.

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