Impact of line probe assay-based molecular testing on individualized treatment in patients with rifampicin-resistant tuberculosis: data from the prospective INNOVA4TB cohort study in Ukraine.

BACKGROUND: Ukraine remains a high World Health Organization priority country for drug-resistant tuberculosis (TB). Rifampicin-resistant TB (RR-TB) has a more protracted, more complicated, and more expensive treatment. In 2021, Ukraine reported 4025 RR-TB cases - 5.4 times more (751) than all 30 European Union/ European Economic Area countries together. OBJECTIVES: The objective of the study was to determine the diagnostic accuracy of line probe assay (LPA), AID Autoimmun Diagnostika GmbH, for detecting resistance to anti-TB drugs and its clinical application for selecting treatment regimens. DESIGN: A prospective observational cohort study. METHODS: From May 2019 to June 2020, we consecutively enrolled patients with active TB hospitalized at the Regional Phthisiopulmonology Center (Vinnytsia, Ukraine), aged between 18 and 82 years. The LPA was performed in the Genetic Research Laboratory at National Pirogov Memorial Medical University, Vinnytsia, Ukraine. RESULTS: A total of 84 clinical specimens and 97 culture isolates from 126 TB patients were tested during the study. Accuracy (95% confidence interval) of LPA for clinical samples in comparison with phenotypic drug susceptibility test (DST) was 80.1 (68.5-89.0) for isoniazid (H), 74.7 (62.4-84.6) for rifampicin (R), 74.4 (62.5-84.1) for ethambutol, 71.4 (41.9-91.6) for streptomycin, 84.6 (62.4-96.5) for prothionamide/ethionamide, and 84.6 (73.6-92.3) for levofloxacin (Lfx), respectively. We found a significantly higher sensitivity of LPA for H, R, and Lfx for the culture isolates compared to clinical specimens (p < 0.05). LPA detected different mutations in 6 out of 17 (35.5%) patients susceptible to R by Xpert. A shorter treatment regimen with an injectable agent demonstrated a low suitability rate of 5% (8/156) in a cohort of RR-TB patients from Ukraine. CONCLUSION: Initial LPA testing accurately identifies resistance to anti-TB drugs and facilitates the selection of an appropriate treatment regimen, minimizing exposure to empirical therapy.

Study about the impact of rapid resistance detection on the treatment of patients with tuberculosis in Ukraine written by healthcare and biomedical professionals to better understand how we can improve the results of treatment and to prevent spreading of resistant bacteriaWhy was the study done? Ukraine has over 4000 patients with tuberculosis (TB) resistant to at least one drug (rifampicin) - five times that of all 30 European Union/European Economic Area countries combined. Unfortunately, only about 60% of such patients have been successfully treated in 2019. At that time, the majority of people suffering from tuberculosis in Ukraine, after checking resistance to rifampicin, initially received standard combinations of the first-line or second-line anti-TB medicines before the result of traditionally used tests (usually few weeks later) became available to individualize the treatment. Alternatively, the sputum could be transported to some overloaded reference laboratories located hundreds of km away from the treatment places.What did the researchers do? The INNOVA4TB team implemented rapid diagnostics of drug resistance in routine practice, guiding key antibiotics use in TB patients. A total of 181 samples from 126 individuals were tested during 2019-2020.What did the researchers find? This new diagnostic technology accurately detected resistance to 9 anti-TB drugs in sputum samples. It could be helpful to select appropriate TB treatment regimens, reducing time for decision from 1 month up to 2 days. Recommended at the study time 9-month shorter standardized treatment regimen with injectable agent was suitable only for 5% of patients for whom it was indicated in Vinnytsia region of Ukraine.What do the findings mean? The study has demonstrated successful implementation of the new molecular diagnostic technology from scratch in a country with restricted resources and limited TB laboratory capacity. This test can facilitate optimal distribution of available wards among patients with different profiles of resistance and correct choice between treatment options.

KLF15-Cyp3a11 Axis Regulates Rifampicin-Induced Liver Injury.

Rifampicin (RFP) has demonstrated potent antibacterial effects in the treatment of pulmonary tuberculosis. However, the serious adverse effects on the liver intensively limit the clinical usage of the drug. Deacetylation greatly reduces the toxicity of RFP but also retains its curative activity. Here, we found that Krüppel-like factor 15 (KLF15) repressed the expression of the major RFP detoxification enzyme Cyp3a11 in mice via both direct and indirect mechanisms. Knockout of hepatocyte KLF15 induced the expression of Cyp3a11 and robustly attenuated the hepatotoxicity of RFP in mice. In contrast, overexpression of hepatic KLF15 exacerbated RFP-induced liver injury as well as mortality. More importantly, the suppression of hepatic KLF15 expression strikingly restored liver functions in mice even after being pretreated with overdosed RFP. Therefore, this study identified the KLF15-Cyp3a11 axis as a novel regulatory pathway that may play an essential role in the detoxification of RFP and associated liver injury. SIGNIFICANCE STATEMENT: Rifampicin has demonstrated antibacterial effects in the treatment of pulmonary tuberculosis. However, the serious adverse effects on the liver limit the clinical usage of the drug. Permanent depletion and transient inhibition of hepatic KLF15 expression significantly induced the expression of Cyp3a11 and robustly attenuated mouse hepatotoxicity induced by RFP. Overall, our studies show the KLF15-Cyp3a11 axis was identified as a novel regulatory pathway that may play an essential role in the detoxification of RFP and associated liver injury.

Enhanced active case finding of drug-resistant tuberculosis in Namibia: a protocol for the hotspots, hospitals, and households (H3TB) study.

INTRODUCTION: Namibia is a high tuberculosis (TB)-burden country with an estimated incidence of 460/100 000 (around 12 000 cases) per year. Approximately 4.5% of new cases and 7.9% of previously treated TB cases are multidrug resistant (MDR) and 47% of patients with MDR-TB are HIV coinfected. Published data suggest a clustering of MDR-TB transmission in specific areas. Identifying transmission clusters is key to implementing high-yield and cost-effective interventions. This includes knowing the yield of finding TB cases in high-transmission zones (eg, community hotspots, hospitals or households) to deliver community-based interventions. We aim to identify such transmission zones for enhanced case finding and evaluate the effectiveness of this approach. METHODS AND ANALYSIS: H3TB is an observational cross-sectional study evaluating MDR-TB active case finding strategies. Sputum samples from MDR-TB cases in three regions of Namibia will be evaluated by whole genome sequencing (WGS) in addition to routine sputum investigations (Xpert MTB/RIF, culture and drug susceptibility testing). We will collect information on household contacts, use of community spaces and geographical map intersections between participants, synthesising these data to identify transmission hotspots. We will look at the feasibility, acceptability, yield and cost of case finding strategies in these hotspots, and in households of patients with MDR-TB and visitors of hospitalised patients with MDR-TB. A compartmental transmission dynamic model will be constructed to evaluate the impact and cost-effectiveness of the strategies if scaled. ETHICS AND DISSEMINATION: Ethics approval was obtained. Participants will give informed consent. H3TB will capitalise on a partnership with the Ministry of Health and Social Services to follow up individuals diagnosed with MDR-TB and integrate WGS data with innovative contact network mapping, to allow enhanced case finding. Study data will contribute towards a systems approach to TB control. Equally important, it will serve as a role model for similar studies in other high-incidence settings.

Diagnostic accuracy of Xpert MTB/RIF Ultra for childhood tuberculosis in West Africa - a multicenter pragmatic study.

OBJECTIVE: To evaluate the performance of Xpert Mycobacterium Tuberculosis/rifampicin (MTB/RIF) Ultra (Ultra) for diagnosis of childhood tuberculosis (TB) within public health systems. METHODS: In this cross-sectional study, children aged <15 years with presumptive pulmonary TB were consecutively recruited and evaluated for TB at tertiary-level hospitals in Benin, Mali, and Ghana. Bivariate random-effects models were used to determine the pooled sensitivity and specificity of Ultra against culture. We also estimated its diagnostic yield against a composite microbiological reference standard (cMRS) of positive culture or Ultra. RESULTS: Overall, 193 children were included in the analyses with a median (interquartile range) age of 4.0 (1.1-9.2) years, 88 (45.6%) were female, and 36 (18.7%) were HIV-positive. Thirty-one (16.1%) children had confirmed TB, 39 (20.2%) had unconfirmed TB, and 123 (63.7%) had unlikely TB. The pooled sensitivity and specificity of Ultra verified by culture were 55.0% (95% confidence interval [CI]: 28.0-79.0%) and 95.0% (95% CI: 88.0-98.0%), respectively. Against the cMRS, the diagnostic yield of Ultra and culture were 67.7% (95% CI: 48.6-83.3%) and 70.9% (95% CI: 51.9-85.8%), respectively. CONCLUSION: Ultra has suboptimal sensitivity in children with TB that were investigated under routine conditions in tertiary-level hospitals in three West African countries.

Usefulness of Xpert MTB/RIF Ultra for rapid diagnosis of extrapulmonary tuberculosis in Tunisia.

Extrapulmonary tuberculosis (EPTB) remains a challenging diagnosis. The purpose of this study was to assess the accuracy of Xpert MTB/RIF Ultra (Cepheid, USA) for rapid diagnosis of EPTB in Tunisia. Eight hundred and forty-seven extrapulmonary samples collected from 2017 to 2021, were subjected to Xpert MTB/RIF Ultra. Microscopy and culture were performed for all the specimens. The accuracy of Xpert Ultra was evaluated in comparison to the culture. Xpert Ultra diagnosed EPTB with a global sensitivity of 80.66% (74.3-85.75) and specificity of 70.87% (67.31-74.20). The molecular test was most accurate when performed in cerebrospinal fluids, bones and joints and cutaneous specimens showing a sensitivity of 100% and a specificity ranging from 70.60 to 91.11%. In lymph node samples comprising aspirates and biopsies, the sensitivity of Xpert Ultra was high 87.50% (77.23-93.53), however, the specificity was 51.08% (44.67-57.46). For pleural samples, the Xpert Ultra sensitivity was 77.50% (68.34-84.68) ranging from 71.43 to 80% in pleural biopsies and fluids respectively. The specificity in all pleural specimens was 79.56% (74.40-83.91). Xpert Ultra showed promise in the diagnosis of EPTB. The performances varied according to the site of the disease. The test may be more valuable if used in combination with other diagnostic modalities.

Xpert MTB/RIF Ultra for the rapid diagnosis of extrapulmonary tuberculosis in a clinical setting of high tuberculosis prevalence country and interpretation of 'trace' results.

To evaluate the diagnostic performance of Xpert MTB/RIF Ultra (Ultra) for the diagnosis of extrapulmonary tuberculosis (EPTB) from different types of extrapulmonary specimens in comparison with culture and composite microbiological reference standard (CRS). A total of 240 specimens were prospectively collected from presumptive EPTB patients between July 2021-January 2022 and tested by Ultra, Xpert, culture and acid-fast bacilli (AFB) smear microscopy. Out of 240 specimens, 35.8 %, 20.8 %, 11.3 %, and 7.1 % were detected as Mycobacterium tuberculosis complex by Ultra, Xpert, culture and AFB microscopy, respectively. An additional 15.0 % cases were detected by Ultra compared to Xpert MTB/RIF (Xpert) assay. A total of 28 (11.7 %) cases were identified as 'trace' category by Ultra with indeterminate rifampicin resistance result; of which 36.4 % were clinically confirmed as EPTB. Compared to culture, the sensitivity and specificity of Ultra and Xpert were 100 % and 72.3 %; 92.6 % and 88.3 %, respectively. In comparison with CRS, these were respectively: 98.9 % and 100 %; 57.5 % and 100 %. For individual category of specimens, sensitivity of Ultra was 100 % with varying specificity. We found that Ultra was highly sensitive for the rapid diagnosis of EPTB and has extensive potential over current diagnostics in high TB burden countries, but 'trace' results should be interpreted with caution.

A meta-analysis of the accuracy of Xpert MTB/RIF in diagnosing intestinal tuberculosis.

OBJECTIVE: A detection method with high efficiency and accuracy is urgently needed in clinical work. The purpose of our study was to determine the diagnostic accuracy of the Xpert MTB/RIF assay for intestinal tuberculosis (ITB). METHODS: We searched PubMed and 4 other databases from their establishment to July 19, 2022, for published essays of diagnostic performance in which Xpert MTB/RIF was used to test patients with clinically suspected ITB. An assessment of the quality of the selected literature was conducted using QUADAS-2. We built forest plots by MetaDiSc software. RESULTS: The pooled Xpert MTB/RIF sensitivity was 48%, and the specificity was 99%. Moreover, the positive likelihood ratio for ITB diagnosis was 21.61. The negative likelihood ratio was 0.54. There were substantial variations between the study estimates of sensitivity (I2 = 87.6%) and specificity (I2 = 82.4%). CONCLUSION: Intestinal TB is detected with limited diagnostic sensitivity by Xpert MTB/RIF but with high specificity. An Xpert-positive result may facilitate the rapid identification of ITB cases. Nevertheless, a negative result has less certainty in excluding the disease.

The new Xpert Mycobacterium tuberculosis/rifampicin (MTB/Rif) Ultra assay in comparison to Xpert MTB/Rif assay for diagnosis of tuberculosis in children and adolescents.

BACKGROUND: Microbiological diagnosis of pediatric tuberculosis (TB) using conventional microbiological techniques has been challenging due to paucibacillary nature of the disease. Molecular methods using cartridge-based tests like Xpert, have immensely improved diagnosis. A novel next-generation cartridge test, Xpert Ultra, incorporates two additional molecular targets and claims to have much lower detection limit. We attempted to compare the two techniques in presumptive pediatric TB patients. OBJECTIVES: The aim of this study was to compare the diagnostic performance of Xpert MTB/Rif Ultra with Xpert MTB/Rif for the detection of pediatric TB. STUDY DESIGN: This is an observational comparative analytical study. METHODS: Children under 15 years of age with presumptive TB were enrolled. Appropriate specimens were obtained (sputum, induced sputum or gastric aspirate for suspected pulmonary TB, cerebrospinal fluid for suspected tubercular meningitis and pleural fluid for suspected tubercular pleural effusion), subjected to smear microscopy, mycobacterial culture, Xpert and Xpert ultra tests and other appropriate diagnostic investigations. RESULTS: Out of 130 enrolled patients, 70 were diagnosed with TB using a composite reference standard (CRS). The overall sensitivity of Xpert was 64.29% [95% confidence interval (CI) 51.93-75.93%] and that of Xpert Ultra was 80% (95% CI 68.73-88.61%) with 100% overall specificity for both. The sensitivity of Xpert and Xpert Ultra in pulmonary specimens (n = 112) was 66.67% and 79.37% and in extrapulmonary specimens (n = 18) was 42.86% and 85.71%, respectively. CONCLUSION: Our study found Ultra to be more sensitive than Xpert for the detection of Mycobacterium tuberculosis in children. Our findings support the use of Xpert Ultra as initial rapid molecular diagnostic test in children under evaluation for TB.

Tuberculosis Variant with Rifampin Resistance Undetectable by Xpert MTB/RIF, Botswana.

GeneXpert MTB/RIF, a tool widely used for diagnosing tuberculosis, has limitations for detecting rifampin resistance in certain variants. We report transmission of a pre-extensively drug-resistant variant in Botswana that went undetected by GeneXpert. The public health impact of misdiagnosis emphasizes the need for comprehensive molecular testing to identify resistance and guide treatment.

Evaluation of Xpert MTB/RIF Ultra for the Diagnosis of Extrapulmonary Tuberculosis: A Retrospective Analysis in Saudi Arabia.

The incidence of extrapulmonary tuberculosis (EPTB) in low- and middle-income countries, as well as, high-income countries has increased over the last two decades. The acid-fast bacillus (AFB) smear test is easy to perform and cost-effective with a quick turnaround time but the test has low sensitivity. Culture remains the gold standard for detecting TB; however, it has low sensitivity and slow bacterial growth patterns, as it may take up to 6 to 8 weeks to grow. Therefore, a rapid detection tool is crucial for the early initiation of treatment and ensuring an improved therapeutic outcome. Here, the Xpert Ultra system was developed as a nucleic acid amplification technique to accelerate the detection of MTB in paucibacillary clinical samples and endorsed by the World Health Organization. From March 2020 to August 2021, Xpert Ultra was evaluated for its sensitivity and specificity against EPTB and compared with those of the routinely used Xpert, culture, and AFB tests in 845 clinical samples in Saudi Arabia. The results indicate the overall sensitivity and specificity of Xpert Ultra to be 91% and 95%, respectively, compared with the Xpert (82% and 99%, respectively) and AFB smear (18% and 100%, respectively) tests. The results also indicated that despite the low microbial loads that were categorized as trace, very low, or low on Xpert Ultra, yet, complete detection was achieved with some sample types (i.e., 100% detection). Consequently, Xpert Ultra has great potential to replace conventional diagnostic approaches as a standard detection method for EPTB.

Xpert MTB/RIF Ultra on contaminated liquid cultures for tuberculosis and rifampicin-resistance detection: a diagnostic accuracy evaluation.

BACKGROUND: Xpert MTB/RIF Ultra (Ultra) is a widely used rapid front-line tuberculosis and rifampicin-susceptibility testing. Mycobacterium Growth Indicator Tube (MGIT) 960 liquid culture is used as an adjunct but is vulnerable to contamination. We aimed to assess whether Ultra can be used on to-be-discarded contaminated cultures. METHODS: We stored contaminated MGIT960 tubes (growth-positive, acid-fast bacilli [AFB]-negative) originally inoculated at a high-volume laboratory in Cape Town, South Africa, to diagnose patients with presumptive pulmonary tuberculosis. Patients who had no positive tuberculosis results (smear, Ultra, or culture) at contamination detection and had another, later specimen submitted within 3 months of the contaminated specimen were selected. We evaluated the sensitivity and specificity of Ultra on contaminated growth from the first culture for tuberculosis (next-available non-contaminated culture result reference standard) and rifampicin resistance (vs MTBDRplus on a later isolate). We calculated potential time-to-diagnosis improvements and also evaluated the immunochromatographic MPT64 TBc assay. FINDINGS: Between June 1 and Aug 31, 2019, 36 684 specimens from 26 929 patients were processed for diagnostic culture. 2402 (7%) cultures from 2186 patients were contaminated. 1068 (49%) of 2186 patients had no other specimen submitted. After 319 exclusions, there were 799 people with at least one repeat specimen submitted; of these, we included in our study 246 patients (31%) with a culture-positive repeat specimen and 429 patients (54%) with a culture-negative repeat specimen. 124 patients (16%) with a culture-contaminated repeat specimen were excluded. When Ultra was done on the initial contaminated growth, sensitivity was 89% (95% CI 84-94) for tuberculosis and 95% (75-100) for rifampicin-resistance detection, and specificity was 95% (90-98) for tuberculosis and 98% (93-100) for rifampicin-resistance detection. If our approach were used the day after contamination detection, the time to tuberculosis detection would improve by a median of 23 days (IQR 13-45) and provide a result in many patients who had none. MPT64 TBc had a sensitivity of 5% (95% CI 0-25). INTERPRETATION: Ultra on AFB-negative growth from contaminated MGIT960 tubes had high sensitivity and specificity, approximating WHO criteria for sputum test target product performance and exceeding drug susceptibility testing. Our approach could mitigate negative effects of culture contamination, especially when repeat specimens are not submitted. FUNDING: The European & Developing Countries Clinical Trials Partnership, National Institutes of Health.

Newer TB diagnostics: An update.

In recent years, nucleic-acid amplification tests (NAATs), which are highly specific and sensitive, have helped to transform the TB diagnostic landscape. According to the WHO 2021 Guidelines on Diagnostics, the NAATs used in TB diagnosis at the point of care (POC) include Xpert MTB/RIF a cartridge-based test manufactured by Cepheid, and Truenat a chip-based test manufactured by Molbio. Other POC tests that are expected to be implemented in near future include Xpert Omni and Xpert MTB/XDR. The use of line probe assay is involved at the level of reference labs for the detection of MTB and its resistance to first-line (Isoniazid and Rifampicin) and second-line (fluoroquinolones and second-line injectables) drugs. When the currently available NAATs detect mutations for drug resistance at a particular region of MTB sequence, the Whole genome sequencing (WGS) platform demonstrates the exceptional potential for reliable and comprehensive resistance prediction for MTB isolates, by multiple gene regions or whole genome sequence analysis allowing for accurate clinical decisions.

[Evaluation of the performance of InnowaveDX MTB/RIF for the detection of Mycobacterium tuberculosis complex and rifampicin resistance].

Objective: To evaluate the performance of Mycobacterium tuberculosis and rifampicin resistance mutation detection kit (InnowaveDX MTB/RIF, referred to as "InnowaveDX") in diagnosing tuberculosis and rifampicin resistance using sputum samples. Methods: From June 19, 2020 to May 16, 2022, patients with suspected tuberculosis were prospectively and consecutively enrolled in Hunan Provincial Tuberculosis Prevention and Control Institute, Henan Provincial Hospital of Infectious Diseases and Wuhan Jinyintan Hospital. A total of 1 328 patients with suspected tuberculosis were finally included. According to the inclusion and exclusion criteria, 1 035 pulmonary tuberculosis patients (357 were confirmed tuberculosis cases and 678 were clinically diagnosed tuberculosis cases) and 180 non-tuberculosis patients were finally included. Sputum samples were collected from all patients for routine sputum smear acid-fastness tests, mycobacterial culture and drug susceptibility testing. Moreover, the diagnostic value of Xpert®MTB/RIF (referred to as "Xpert") and InnowaveDXin detecting tuberculosis and rifampicin resistance was evaluated. Clinical diagnosis and culture results of Mycobacterium tuberculosis were used as reference standards to assess tuberculosis diagnosis, and phenotypic drug sensitivity and Xpert were used as reference standards to assess rifampicin resistance. The sensitivity, specificity, positive predictive value and negative predictive value of the two methods for tuberculosis diagnosis and rifampicin resistance were analyzed. The consistency of the two techniques was analyzed usingkappa test. Results: Taking clinical diagnosis as the reference standard, the detection sensitivity of InnowaveDX [58.0% (600/1 035)] was higher than that of Xpert [51.7% (535/1 035)] in 1035 patients with pulmonary tuberculosis, and the difference was statistically significant (P<0.001). In 270 pulmonary tuberculosis patients with culture-positive pulmonary tuberculosis identified as M.tuberculosis-complex, the positive rates of InnowaveDX and Xpert were both high [99.6%(269/270)and 98.2%(265/270), respectively] and there was no statistical difference. In culture-negative patients with pulmonary tuberculosis, the sensitivity of InnowaveDX was 38.8% (198/511), which was higher than that of Xpert (29.4%, 150/511), and the difference was statistically significant (P<0.001). Taking phenotypic drug-susceptibility testing (DST) as reference, the sensitivity of InnowaveDX to rifampicin resistance was 99.0% (95%CI: 94.7%-100.0%) and the specificity was 94.0%(95%CI: 88.5%-97.4%). With Xpert as the reference, the sensitivity and specificity of InnowaveDX were 97.1% (95%CI: 93.4%-99.1%) and 99.7% (95%CI: 98.4%-100.0%), respectively, and the kappa value was 0.97 (P<0.001). Conclusions: InnowaveDX show a high sensitivity for detecting Mycobacterium tuberculosis, especially in pulmonary tuberculosis patients with a clinical diagnosis and negative culture results. It also showed high sensitivity in detecting rifampicin resistance with DST and Xpert as reference respectively. InnowaveDX is an early and accurate diagnostic tool for TB and drug-resistant TB, particularly suitable for application in low- and middle-income countries.

GeneXpert MTB/RIF Ultra performance to detect uncommon rpoB mutations in Mycobacterium tuberculosis.

OBJECTIVE: To investigate the performance of GeneXpert MTB/RIF Ultra to accurately detect rifampicin resistance for less common rpoB mutations that potentially confer phenotypic resistance, we tested 28 such Mycobacterium tuberculosis cultures with Xpert Ultra. RESULTS: They represented 22 different (combinations of) rpoB mutations. Of 28 isolates tested, one was reported by Xpert Ultra as "No rifampicin resistance detected", 8 yielded a "Rifampicin indeterminate" result, and 19 were identified as rifampicin resistant. Overall, our results corroborate previous observations on the "Indeterminate" results for mutations at codon 432, while we add Lys446Gln as additional "Indeterminate" result and Pro439Leu as a false rifampicin-susceptible result. Furthermore, we document other uncommon point mutations and indels across the rpoB gene that are mostly correctly identified as rifampicin resistant by Xpert ultra (V3). Taken together, "Indeterminate" results in Xpert Ultra may indicate underlying rpoB mutations within the rifampicin-resistance determining region and thus increase the post-test probability of rifampicin resistance, albeit to an unknown extent.

Evaluation of Xpert MTB/RIF Ultra assay for detection of Mycobacterium tuberculosis and rifampicin resistance.

Tuberculosis (TB) is a public health challenge globally, and molecular testing is recommended to expedite diagnosis. Concerns that Xpert MTB/RIF assay (Xpert) may be less sensitive when testing paucibacillary samples led to the development of the Xpert MTB/RIF Ultra assay (Ultra). We evaluated the performance of Ultra against Xpert on clinical samples sent to the national reference laboratory in Singapore. In total, 149 samples collected between January 2019 and November 2020 were analysed. Mycobacterium tuberculosis complex (MTBC) was isolated from 55 cultures. Using culture as the reference standard, Ultra demonstrated higher sensitivity (96.4% vs 85.5%) and marginally lower specificity (88.3% vs 89.4%) compared to Xpert in the full cohort. When considering only paucibacillary specimens such as extrapulmonary and smear-negative samples, similar results were obtained. Reclassifying Ultra trace results (low levels of MTB are detected but no rifampicin resistant result is detected) as negative in the full cohort led to a decrease in sensitivity by 10.9% and a marginal increase in specificity by 1.1%. In instances of low bacillary load, Ultra also identified rifampicin resistance more accurately than Xpert, when corroborated against other methods such as broth microdilution, line probe assay and whole genome sequencing (WGS). One isolate tested rifampicin-resistant using Xpert and Ultra, but was phenotypically susceptible and WGS demonstrated the presence of the silent mutation Thr444Thr. Ultra is more sensitive than Xpert in the detection of MTBC and rifampicin resistance in our local setting. Nevertheless, the results of molecular testing should still be correlated with phenotypic studies.

Diagnosis of extrapulmonary tuberculosis by GeneXpert MTB/RIF Ultra assay.

INTRODUCTION: Diagnosis of extrapulmonary tuberculosis (EPTB) is an arduous task owing to different anatomical locations, unusual clinical presentations, and sparse bacillary load in clinical specimens. Although GeneXpert® MTB/RIF is a windfall in TB diagnostics including EPTB, it yields low sensitivities but high specificities in many EPTB specimens. To further improve the sensitivity of GeneXpert®, GeneXpert® Ultra, a fully nested real-time PCR targeting IS6110, IS1081 and rpoB (Rv0664) has been endorsed by the WHO (2017), wherein melt curve analysis is utilized to detect rifampicin-resistance (RIF-R). AREA COVERED: We described the assay chemistry/work design of Xpert Ultra and evaluated its performance in several EPTB types, that is, TB lymphadenitis, TB pleuritis, TB meningitis, and so on, against the microbiological reference standard or composite reference standard. Notably, Xpert Ultra exhibited better sensitivities than Xpert, but mostly at the compensation of specificity values. Moreover, Xpert Ultra exhibited low false-negative and false-positive RIF-R results, compared with Xpert. We also detailed other molecular tests, that is, Truenat MTBTM/TruPlus, commercial real-time PCR, line probe assay, and so on, for EPTB diagnosis. EXPERT OPINION: A combination of clinical features, imaging, histopathological findings, and Xpert Ultra are adequate for definite EPTB diagnosis so as to initiate an early anti-tubercular therapy.

We discussed the assay chemistry and evaluated performance of GeneXpert®MTB/RIF Ultra to identify the TB germs and resistance to one of the potent bactericidal drugs, that is, rifampicin in different types of extrapulmonary TB (EPTB, TB in organs other than lungs) and compared its performance with its ancestor, that is, GeneXpert. We briefly outlined the other molecular tests, such as Truenat MTB^TM/Truenat MTB^TM Plus, commercial real-time PCR, line probe assay, and so on for EPTB diagnosis. While evaluating Xpert Ultra results, the 'trace call' has been introduced, whose interpretation is important. By and large, Xpert Ultra assay outperformed in TB lymphadenitis, TB pleuritis, and TB meningitis when compared with Xpert, though limited information is available on other EPTB forms. Overall, the sensitivity of Xpert Ultra has been increased in most of EPTB cases, but mostly at the cost of specificity values.

The Use of Xpert MTB/RIF Ultra Testing for Early Diagnosis of Tuberculosis: A Retrospective Study from a Single-Center Database.

Tuberculosis (TB) is a multisystemic contagious disease produced by Mycobacterium tuberculosis complex bacteria (MTBC), with a prevalence of 65:100,000 inhabitants in Romania (six times higher than the European average). The diagnosis usually relies on the detection of MTBC in culture. Although this is a sensitive method of detection and remains the "gold standard", the results are obtained after several weeks. Nucleic acid amplification tests (NAATs), being a quick and sensitive method, represent progress in the diagnosis of TB. The aim of this study is to assess the assumption that NAAT using Xpert MTB/RIF is an efficient method of TB diagnosis and has the capacity to reduce false-positive results. Pathological samples from 862 patients with TB suspicion were tested using microscopic examination, molecular testing and bacterial culture. The results show that the Xpert MTB/RIF Ultra test has a sensitivity of 95% and a specificity of 96.4% compared with 54.8% sensitivity and 99.5% specificity for Ziehl-Neelsen stain microscopy, and an average of 30 days gained in the diagnosis of TB compared with bacterial culture. The implementation of molecular testing in TB laboratories leads to an important increase in early diagnostics of the disease and the prompter isolation and treatment of infected patients.

Evaluation of Xpert MTB/RIF assay for the diagnosis of extrapulmonary tuberculosis in Southwest China.

The purpose of this study was to determine the diagnostic efficacy of Xpert MTB/RIF assay for rapid diagnosis of Tuberculosis (TB) and detection of rifampicin (RIF) resistance in patients suspected of having EPTB, assessing it against traditional culture and drug susceptibility test (DST) by proportional method, and the ability to predict multidrug resistance TB by Xpert MTB/RIF assay. In this study, the Xpert MTB/RIF assay was applied to 1,614 extrapulmonary specimens. Compared with TB culture and Composite Reference Standard (CRS), the Xpert MTB/RIF assay had a high sensitivity and specificity for detection of EPTB. Depending on the culture method or CRS as the standard, sensitivity of the Xpert MTB/RIF assay for detection of MTB in pleural effusion, cerebrospinal fluid, thoracic drainage fluid and throat swabs specimens were lower than that of other specimens. According to the experimental results, we have reason to believe that Xpert MTB/RIF assay is a rapid and simple technique with high sensitivity and specificity for diagnosing EPTB and detecting drug resistance in variety of specimens. Xpert MTB/RIF assay combined with DST maybe identify more cases of multi-drug resistant tuberculosis (MDR-TB).

Rapid Detection of Extensive Drug Resistance by Xpert MTB/XDR Optimizes Therapeutic Decision-Making in Rifampin-Resistant Tuberculosis Patients.

The Xpert MTB/XDR assay met the critical need for etiologic diagnosis of tuberculosis and rifampin resistance in previous studies. However, its benefits in tailoring the treatment regimen and improving the outcome for patients with rifampin-resistant tuberculosis (RR-TB) require further investigation. In this study, the Xpert MTB/XDR assay was used to determine the resistance profile of second-line drugs for RR-TB patients in two registered multicenter clinical trials, TB-TRUST (NCT03867136) and TB-TRUST-plus (NCT04717908), with the aim of testing the efficacy of all-oral shorter regimens in RR-TB patients in China. Patients would receive the fluoroquinolone-based all-oral shorter regimen, the injectable-containing regimen, or the bedaquiline-based regimen depending on fluoroquinolone susceptibility by using Xpert MTB/XDR. Among the 497 patients performed with Xpert MTB/XDR, 128 (25.8%) had infections resistant to fluoroquinolones and/or second-line injectable drugs (SLIDs). A total of 371 participants were recruited for the trials, and whole-genome sequencing (WGS) was performed on all corresponding culture-positive baseline strains. Taking the WGS results as the standard, the accuracy of the Xpert MTB/XDR assay in terms of resistance detection was 95.2% to 99.0% for all drugs. A total of 33 cases had inconsistent results, 9 of which were due to resistance heterogeneity. Most of the patients (241/281, 85.8%) had sputum culture conversion at 2 months. In conclusion, the Xpert MTB/XDR assay has the potential to serve as a quick reflex test in patients with RR-TB, as detected via Xpert MTB/RIF, to provide a reliable drug susceptibility profile of the infecting Mycobacterium tuberculosis strain and to initiate optimized treatment promptly.