Detection Of Rifampicin Resistance Research Articles

Detection of mycobacterium tuberculosis and rifampicin resistance with Xpert® MTB/RIF analysis in a tertiary hospital

Detection of mycobacterium tuberculosis and rifampicin resistance with Xpert® MTB/RIF analysis in a tertiary hospital

Diagnostic performance of Xpert MTB/RIF ultra in detecting Mycobacterium tuberculosis and Rifampicin Resistance in AFB Smear-negative Pulmonary and Extrapulmonary Tuberculosis samples in Malaysia

Rapid and highly accurate diagnostic tools are critically needed to diagnose Mycobacterium tuberculosis and rifampicin resistance in AFB smear-negative samples. In this study, we evaluated the diagnostic performance of Xpert MTB/RIF Ultra (Ultra) as a rapid test to diagnose tuberculosis in smear-negative cases in Malaysia. A retrospective study of 1960 smear-negative pulmonary and extrapulmonary samples obtained from patients was conducted. Culture was used as the reference standard for the study. The overall sensitivity and specificity of Ultra on the tested samples were 88.7 % and 77.2 %, respectively, while the PPV was 32.3 % and the NPV was 98.2 %. Ultra showed slightly higher sensitivity in pulmonary (89.9 %) compared to extrapulmonary samples (86.1 %). The overall accuracy of Ultra was 78.5 % (kappa=0.37; 95 %CI: 0.32,0.42). Ultra showed good diagnostic accuracy for detecting MTB and rifampicin resistance in various AFB smear-negative samples. Ultra also had excellent capability in rifampicin resistance detection.

Truenat: Can it be A Game Changer in Extrapulmonary Tuberculosis?

Tuberculosis is a leading health problem worldwide, with India accounting for the majority of cases. Owing to the diverse clinical presentation and paucibacillary nature of extrapulmonary tuberculosis, it is tough to diagnosis by routine microbiological methods. Newer chip/cartridge based nucleic amplification tests (NAATs) like Truenat and Xpert have proved to be game changer in diagnosis of pulmonary tuberculosis. However, role of Truenat in extrapulmonary tuberculosis is still to be evaluated. This study was undertaken to evaluate the diagnostic yield of truant in various extrapulmonary tuberculosis. The study was conducted during 01 June 2021 to 31 July 2022. Of the total 1481 samples received during study, 761 (52%) were extrapulmonary samples. The highest yield of 36% was found in pus aspirate, followed by a 12% yield in pleural fluid. Overall yield of 12% in various extrapulmonary samples is encouraging. Further studies are required for evaluating Truenat role in rapid diagnosis of EPTB and rifampicin resistance detection for better patient care.

A comparative study of MassARRAY and GeneXpert assay in detecting rifampicin resistance in tuberculosis patients' clinical specimens.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potent tool for detecting drug resistance in tuberculosis (TB); however, concerns about its reliability have been raised. In this study, we assessed the reliability of MassARRAY (Sequenom, Inc.), which is a MALDI-TOF MS-based method, by comparing it to the well-established GeneXpert assay (Cepheid) as a reference method. A retrospective study was conducted using laboratory data retrieved from Henan Chest Hospital (Zhengzhou, China). To ensure a rigorous evaluation, we adopted a comprehensive assessment approach by integrating multiple outcomes of the Xpert assay across various specimen types. Among the 170 enrolled TB cases, MassARRAY demonstrated significantly higher sensitivity (85.88%, 146 of 170) compared to the Xpert assay (76.62%, 118 of 154) in TB diagnosis (p < 0.05). The concordance in detecting rifampicin resistance between MassARRAY and the combined outcomes of the Xpert assay was 90%, while it was 97.37% (37 of 38) among smear-positive cases and 89.06% (57 of 64) among culture-positive cases. When compared to the phenotypic susceptibility outcomes of the 12 included drugs, consistency rates of 81.8 to 93.9% were obtained, with 87.9% for multiple drug resistance (MDR) identification. MassARRAY demonstrates high reliability in detecting rifampicin resistance, and these findings may offer a reasonable basis for extrapolation to other drugs included in the test panel.

Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

Mutational analysis in drug resistant Mycobacterium tuberculosis in Western Uttar Pradesh

BackgroundMulti-drug resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes. This study was carried out with the aim to determine the pattern of drug resistance against Mycobacterium tuberculosis towards first line ATT (anti-tubercular treatment) in sputum smear–positive patients using Line Probe Assay (LPA). MethodsA cross sectional prospective study was carried out in a tertiary care Hospital of Meerut. A total of 898 sputum samples (on spot and early morning) collected from 449 suspected pulmonary tuberculosis patients as per RNTCP guidelines were screened by microscopy. Decontamination was done by N-acetyl-l-cysteine and sodium hydroxide. Then smear positive samples were subjected to 1st line drug susceptibility testing (DST) using LPA GenoType® MTBDRplus (HAIN Life Science) assay, a molecular method which allows rapid detection of Rifampicin (Rif) and Isoniazid (INH) resistance. ResultsThe overall burden of MDR TB in this geographical area was 7.9 %. Mono-resistance with Rif alone was around 2.8 %. However, the mono-resistance with INH (inhA gene) and INH (katG gene) was 2.8 % and 1.1 % respectively. Drug resistance of Rif was due to mutations in rpoB gene while resistances to INH were more commonly due to mutation in inhA gene followed by katG gene. TB was more commonly seen in the age group of 30–59 years (43.8 %) and predominantly in males. ConclusionTuberculosis positivity rate is high in Western Uttar Pradesh. Burden of MDR TB in Western Uttar Pradesh was similar to National data. Line probe assay can be used as a primary method to diagnose multi drug resistant TB as done in present study which can help in earlier initiation of correct therapy.

Using ultrasound guided needle biopsy in conjunction with GeneXpert MTB/RIF to diagnose epididymal tuberculosis

This study aimed to investigate the diagnostic utility of percutaneous ultrasound-guided needle biopsy conjunction with GeneXpert MTB/RIF for epididymal tuberculosis. A retrospective analysis was conducted on the pathological and laboratory examinations of 20 patients with epididymal lesions undergoing ultrasound guided biopsy at Shandong Public Health Clinical Center. Laboratory examination included acid-fast staining, Mycobacterium tuberculosis culture by BACTEC MGIT 960, and GeneXpert MTB/RIF test. Diagnosis and complications were comprehensively analyzed. Among the 20 patients, 15 had epididymal tuberculosis and 5 had non-epididymal tuberculosis. Ten patients had granulomatous inflammation and necrotic tissues. The sensitivity and specificity of acid-fast staining, Mycobacterium tuberculosis culture, and GeneXpert MTB/RIF for the diagnosis of epididymis tuberculosis were 26.67% and 100.00%, 33.33% and 100.00%, and 86.67% and 100.00%, respectively. The diagnostic value analysis of the 3 detection techniques indicated that the GeneXpert MTB/RIF technique (Kappa = 0.765, Area under the curve (AUC) = 0.933) was superior to Mycobacterium tuberculosis culture (Kappa = 0.200, AUC = 0.667) and acid-fast staining (Kappa = 0.154, AUC = 0.633). Ultrasound-guided percutaneous biopsy is a safe procedure. The GeneXpert MTB/RIF test has high sensitivity, specificity, and superior AUC value, which are of great value in the diagnosis of epididymal tuberculosis and rifampicin resistance detection.

Diagnosis of Tuberculosis by Conventional and Molecular Methods in Our Laboratory: A 4-Year Assessment

Objective: The aim of this study was to compare direct microscopy, culture and Polymerase Chain Reaction (PCR) methods and to present the antibiotic resistance profile of the last 4 years comparatively by conventional and molecular methods.&#x0D; Materials and Methods: Bacterial culture, EZN and PCR methods were applied to all samples. Direct rapid resistance test was performed for EZN positive samples. &#x0D; Results: 968 patients were included in the study. Culture was positive in 81 (8%), PCR in 78 (8%) and EZN in 39 (46%) patients. PCR performed on the same day in both respiratory and other samples showed very good agreement with culture, while EZN staining showed moderate agreement. It was observed that the rapid resistance test detected rifampicin resistance which was not detected in culture, and in the case of INH, culture antibiogram and rapid resistance test were fully compatible. Application of the rapid resistance test to every patient with positive EZN staining resulted in very early detection of resistance. &#x0D; Conclusion: It was concluded that PCR tests are useful in the rapid diagnosis of tuberculosis and resistance in suspicious clinical samples.

Performance of the MeltPro TB assay as initial test for diagnosis of pulmonary tuberculosis with drug-resistance detection

BackgroundThe MeltPro TB assay (MeltPro) is a molecular rapid diagnostic test designed for detecting resistance to antituberculosis drugs. However, the performance of MeltPro as an initial diagnostic test for simultaneously detecting the presence of Mycobacterium tuberculosis (MTB) and drug resistance has not been evaluated. This study aims to assess the performance of MeltPro as initial diagnostic test for simultaneous detection of MTB and drug resistance in clinical samples from patients with presumptive pulmonary tuberculosis (PTB).MethodsA retrospective analysis was conducted on 1283 patients with presumptive PTB from two clinical centers, out of which 875 were diagnosed with PTB. The diagnostic accuracy of MeltPro, Xpert MTB/RIF (Xpert), and MGIT 960 for PTB detection was evaluated. Rifampicin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (STR), and fluoroquinolone (FQ) resistance were detected using MeltPro, with Xpert and/or the broth microdilution plate method (MYCOTB) results as references.ResultsFor the diagnosis of PTB, MeltPro showed a sensitivity of 69.0%, which was similar to Xpert (72.7%; P > 0.05) and higher than MGIT (58.1%; P < 0.001). The specificity of MeltPro was 97.1%, similar to Xpert (98.0%; P > 0.05). In smear-negative patients, MeltPro's sensitivity was 50.9%, similar to Xpert (56.5%; P > 0.05), and higher than MGIT (33.1%; P < 0.001). Based on Xpert and/or MYCOTB results, MeltPro exhibited a sensitivity and specificity of 98.3% and 99.2%, respectively, for detecting RIF resistance. Based on MYCOTB results, MeltPro's sensitivity for detecting resistance to INH, EMB, STR, and FQ was 96.4%, 89.1%, 97.5%, and 90.3%, respectively, with specificities of 96.0%, 96.0%, 95.2%, and 99.4%, respectively.ConclusionThe MeltPro TB assay could potentially be an effective alternative as the initial test for rapid diagnosis of PTB with drug-resistance detection in clinical practice.

Xpert MTB/RIF assay among the extra-pulmonary and non-sputum respiratory samples: One year experience at National Tuberculosis Reference Laboratory, Bangladesh

Background: Extra-pulmonary tuberculosis (EPTB) is a diagnostic and therapeutic challenge due to the paucity of suitable rapid diagnostic tools. Objectives: The aim was to assess the frequency of detection of Mycobacterium tuberculosis using Xpert MTB /RIF assay among the extra-pulmonary and non-sputum respiratory samples (NSRS) along with demographic profile, life style variables and proportion of Rifampicin resistant TB among those specimens. Methodology: This cross-sectional retrospective study was conducted at National Tuberculosis Reference Laboratory (NTRL) from January 2018 to December 2018 for period of 1 year. Total 3918 extra-pulmonary and non-sputum respiratory samples from suspected TB patients were enrolled. Detection of Mycobacterium tuberculosis and Rifampicin resistance was performed by Xpert MTB /RIF assay and culture in both liquid and solid media. Results: Among 3918 samples, 3152 were new cases and 766 were previously treated TB cases. A total of 853 (21.77%) MTB were detected by Xpert MTB /RIF assay. Among them, 713 (83.59%) were extra-pulmonary and 140 (16.41%) were non-sputum respiratory samples. Lymph node (LN) aspirates and tissues were the most frequent (37.9 and 9.9%) samples of EPTB. The proportion of RR cases among EPTB was 2.8% and 6.3% among new and previously treated cases. And 2.6% and 11.1% among NSRS new and previously treated cases respectively. Conclusion: This study will give an insight of EPTB and NSRS profile with a view to diagnostic and therapeutic approach in Bangladesh. Bangladesh Journal of Medical Microbiology, January 2023;17(1):21-28

Comprehensive assessment of invalid and indeterminate results in Truenat MTB-RIF testing across sites under the national TB elimination program of India.

Truenat MTB-RIF assay (Truenat), a nucleic acid amplification test (NAAT), is a real-time polymerase chain reaction (RT-PCR) chip-based assay that can detect Mycobacterium tuberculosis (Mtb) and rifampicin (RIF) drug resistance using portable, battery-operated devices. The National TB Elimination Program (NTEP) in India introduced this novel tool at the district and subdistrict level in 2020. This study aimed to assess the level and causes of inconclusive results (invalid results, errors, and indeterminate results) in MTB and RIF testing at NTEP sites and the root causes of these in the programmatic setting. Truenat testing data from 1,690 functional Truenat sites under the NTEP from April to June 2021 were analyzed to assess the rates of errors, invalid MTB results, and indeterminate RIF results. Following this analysis, 12 Truenat sites were selected based on site performance in Truenat testing, diversity of climatic conditions, and geographical terrain. These sites were visited to assess the root causes of their high and low rates of inconclusive results using a structured checklist. A total of 327,649 Truenat tests performed for MTB and RIF testing were analyzed. The rate of invalid MTB results was 5.2% [95% confidence interval (CI): 5.11-5.26; n = 16,998] and the rate of errors was 2.5% (95% CI: 2.46-2.57; n = 8,240) in Truenat MTB chip testing. For Mtb-positive samples tested using the Truenat RIF chip for detection of RIF resistance (n = 40,926), the rate of indeterminate results was 15.3% (95% CI: 14.97-15.67; n = 6,267) and the rate of errors was 1.6% (95% CI: 1.53-1.78; n = 675). There was a 40.1% retesting gap for Mtb testing and a 78.2% gap for inconclusive RR results. Among the inconclusive results retested, 27.9% (95% CI: 27.23-28.66; n = 4,222) were Mtb-positive, and 9.2% (95% CI: 7.84-10.76; n = 139) were detected as RR. The main causes affecting Truenat testing performance include suboptimal adherence to standard operating procedures (SOPs), inadequate training, improper storage of testing kits, inadequate sputum quality, lack of quality control, and delays in the rectification of machine issues. Root cause analysis identified that strengthening of training, external quality control, and supervision could improve the rate of inconclusive results. Ensuring hands-on training of technicians for Truenat testing and retesting of samples with inconclusive results are major recommendations while planning for Truenat scale-up. The recommendations from the study were consolidated into technical guidance documents and videos and disseminated to laboratory staff working at the tiered network of TB laboratories under the NTEP in order to improve Truenat MTB-RIF testing performance.

Value of ultrasound guided biopsy combined with Xpert Mycobacterium tuberculosis/resistance to rifampin assay in the diagnosis of chest wall tuberculosis.

The thoracic wall lesions, particularly chest wall tuberculosis, and chest wall tumors and other pyogenic wall and actinomycetes infections, almost always present as a diagnostic challenge. To explore the value of ultrasound-guided biopsy combined with the Xpert Mycobacterium tuberculosis/resistance to rifampin (MTB/RIF) assay to diagnose chest wall tuberculosis. We performed a retrospective study of patients with chest wall lesions from March 2018 to March 2021. All patients received the ultrasound-guided biopsy for pathology examination, acid-fast Bacillus staining, mycobacterial culture, and Xpert MTB/RIF analysis. The sensitivity, specificity, and area under the curve (AUC) were calculated for these diagnostic tests, either individually or combined. Rifampicin resistance results were compared between the mycobacterial culture and the Xpert MTB/RIF assay. In 31 patients with the chest wall lesion biopsy, 22 patients were diagnosed with chest wall tuberculosis. Of them, 3, 6, and 21 patients tested positive for mycobacterial culture, acid-fast stain, and Xpert MTB/RIF assay, respectively. The rifampicin resistance results of the 3 culture-positive patients were consistent with their Xpert MTB/RIF assay results. When considering the sensitivity, specificity, and AUC value, the Xpert MTB/RIF assay (95.5%, 88.9%, and 0.92, respectively) was a better choice than the acid-fast Bacillus stain (27.3%, 100.0%, and 0.64, respectively) and mycobacterial culture (13.6%, 100.0%, 0.57, respectively). No complications were reported during the procedure. Ultrasound guided biopsy combined with Xpert MTB/RIF has high value in the diagnosis of chest wall tuberculosis, and can also detect rifampicin resistance.

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

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. 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. 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). 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. The European & Developing Countries Clinical Trials Partnership, National Institutes of Health.

Validity of the GenXpert MTB/RIF in Diagnosis of Pulmonary Tuberculosis &amp; Description of Pulmonary TB Resistance Profile in Abbasia Chest Hospital

Background Tuberculosis (TB) is a global public health problem; early diagnosis will enhance the treatment and decrease the resistance to the anti TB drug which improve the overall treatment outcome Objectives Comparing the validity of GeneXpert Mycobacterium tuberculosis /RIF in diagnosing pulmonary TB and detecting Rifampicin resistance with Direct smear microscopy. Subjects&amp; methods Cross sectional study was conducted to collect a sample of 393 clinically suspected cases (January,2016 to February 2018). All cases underwent Direct smear microscopy, GeneXpert (GX) MTB/RIF and Lowenstein– Jensen Cultures Golden standard test for diagnosis of TB and comparing the validity of the two tests. Comparison between GX and the golden standard culture results was done by sensitivity and specificity calculation as well as the Kappa test for agreement between results of tests. Results For 393 clinically suspected TB cases, TB culture confirmed 196 (49.9%) TB positive cases and 197 (50.1%) TB negative cases and 32 Rifampicin resistant cases (16.3%) of the positive TB cases. GX detected 206 (52.4%) TB positive cases and 187(47.6%) TB negative cases and 34 Rifampicin resistant cases. For TB diagnosis, GX sensitivity was 91.8%, specificity was 86.8%, PVP= 87.4%, PVN=91.4Accuracy= 89.3, and substantial agreement between GX and culture as level of agreement =78.6%. As for Rifampicin resistant cases the GX sensitivity =100%, specificity=98.9%, PVP= 94.1%, PVN=98.9%, Accuracy= 99%, and GOOD agreement between GX and culture as kappa = 0.964 (level of agreement is 96.4%). Direct smear microscopy detected 166 (42.2%) TB positive cases and 227(57.8%) TB negative cases. Sensitivity of direct microscopy was 53.6% while specificity was 69%, PVP= 63.3%, PVN=59.9%, Accuracy= 61.3%, and fair agreement between Direct smear microscopy and culture with level of agreement =22.6%. Conclusion GX has the advantage of pulmonary TB rapid testing of high sensitivity and specificity that allows early treatment and decrease of the disease transmission in comparison to the currently used Direct smear microscopy for TB diagnosis

Comparison of Xpert MTB/RIF Ultra with Xpert MTB/RIF for the detection of Mycobacterium tuberculosis and rifampicin resistance in a primary-level clinic in rural China

The Xpert MTB/RIF Ultra (Ultra) is not yet used for the diagnosis of tuberculosis (TB) in China. We compared the performance of the Xpert and Ultra for detecting Mycobacterium tuberculosis and rifampicin resistance in a primary-level clinic in rural China. Sputum samples from suspected pulmonary TB patients were collected and subjected to smear microscopy, liquid culture, Xpert and Ultra tests. We then compared the sensitivity and specificity of Xpert and Ultra for diagnosing TB against liquid culture. Whole-genome sequencing was performed to predict rifampicin resistance and the results were compared with the Xpert and Ultra tests. The sensitivities of Xpert and Ultra were 88.1% and 95.1%, and the specificities were 91.9% and 84.4%, respectively. Among the 61 smear-negative culture-positive patients, the sensitivities of Xpert and Ultra were 80.3% and 91.8%. All Xpert-positive patients were Ultra-positive. Among culture-negative Xpert or Ultra-positive patients, 69.6% were taking anti-TB drugs or had a previous history of TB. Of the samples that Ultra classified as trace, nearly 25% were probably false-positives. Both Xpert and Ultra accurately detected all rifampicin-resistant patients. In conclusion, Ultra was more sensitive than Xpert, especially for smear-negative patients but had decreased specificity with more false-positives, especially with Ultra trace results.

Determining the diagnostic potential of Truenat MTB Plus for Tubercular lymphadenitis and detection of drug resistance and a comparison with GeneXpert Ultra

SettingTubercular lymphadenitis (TBLA), the most common form of extrapulmonary tuberculosis, is a diagnostic challenge. ObjectiveTruenat MTB Plus (TruPlus) along with Truenat Rif assay (TruRif) was evaluated for detection of TBLA and rifampicin resistance and compared with GeneXpert Ultra (Xpert Ultra). Design100 fine-needle aspirated specimens [50 confirmed by culture/smear/cytology, 20 clinically suspected, and 30 controls], processed in the mycobacteriology division of department of microbiology were subjected to TruPlus and TruRif, Xpert Ultra and multiplex PCR. The results of TBLA detection were compared against composite reference standard (CRS) and those of rifampicin resistance were compared against phenotypic drug susceptibility testing and rpoB gene sequencing. ResultsIn comparison to CRS, the diagnostic yield of TruPlus, Xpert Ultra and MPCR was 77.14%, 59.18% and 84.28%, respectively; with substantial agreement for TruPlus (k = 0.66) and MPCR (k = 0.76) and moderate for Xpert Ultra (k = 0.60). TruRif reported four cases as RifR and Xpert Ultra reported two. On comparing with phenotypic DST and gene sequencing, only two cases of RifR were confirmed, hence TruRif reported false-RifR in two cases. ConclusionTruPlus could be used as a reliable tool for diagnosing TBLA. The reporting of RifR by TruRif should be confirmed by phenotypic DST or gene sequencing.

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

ObjectiveTo 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.ResultsThey 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 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.

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).

Epidemiology of Molecular Probes in Xpert MTB/RIF Assay in AJK, Pakistan

Objective: This study aimed to detect rifampicin-resistant tuberculosis cases and assess the frequency of missing probes in different study populations in Azad Jammu and Kashmir (AJK), Pakistan. Methodology: The study was conducted at the State TB Reference Laboratory, District Headquarters Teaching Hospital, Mirpur, AJK. A total of 2,790 specimens collected between March 2016 to August 2019 were analyzed. Pulmonary TB (PTB) accounted for 94% of the cases, while 6% were classified as extra-pulmonary cases. All respiratory and non-respiratory samples underwent fluorescence smear microscopy (AFB) and a real-time PCR test (Xpert MTB/RIF assay) to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance. Results: Among the 2,790 suspected MTB patients, 734 (26%) were confirmed to have MTB using the Xpert MTB/RIF assay, while 564 (20%) tested positive by fluorescence microscopy. Of the MTB-positive patients, 720 (98%) were diagnosed with pulmonary TB, and 14 (2%) had extra-pulmonary TB. Rifampicin resistance (RR) was detected in 66 (9%) cases, with 97% of the resistant cases being pulmonary and 3% extra-pulmonary. The most frequently missing probe was E (Codon 529-533), accounting for 34% of the cases, followed by probe D (Codon 523-529) at 26%. The least frequently missing probe was C (Codon 523-529), observed in 3% of the cases. Probe B (Codon 512-518) was missing in 15.4% of cases, while probe A (Codon 518-523) was missing in 9.4% of cases. Conclusion: The utilization of molecular diagnostic techniques, such as the Xpert MTB/RIF assay, enables rapid identification of MTB and detection of rifampicin resistance. This study provides valuable baseline data on the prevalence of 81 bp mutations in the rpoB gene, highlighting the need for further evaluation of mutation patterns in AJK.