Detection of Mycobacterium tuberculosis in Blood by Use of the Xpert MTB/RIF Assay

We have developed a novel blood lysis-centrifugation approach for highly sensitive Mycobacterium tuberculosis detection in large volumes of blood with the Xpert MTB/RIF assay. One through 20 ml of blood was spiked with 0.25 to 10 CFU/ml of the M. tuberculosis surrogate M. bovis BCG. Multiple replicates of each sample were processed by a new lysis-centrifugation method and tested with the Xpert MTB/RIF assay. The assay was very sensitive with increased blood volumes. In the 20-ml samples, BCG was detected in blood spiked with 10, 5, 1, and 0.25 CFU/ml 100, 100, 83, and 57% of the time, respectively, compared to 100, 66, 18, and 18%, of the time, respectively, in 1-ml blood samples. Assay sensitivity was influenced by the type of anticoagulant used, with acid-citrate-dextrose solution B (ACD-B) providing the best results. A limit of detection of 10 CFU/ml was established with BCG spiked into ACD-B-treated blood, and 92, 36, and 33% of the samples with 5, 1, and 0.5 CFU/ml, respectively, were assay positive. The lysis buffer was stable both at room temperature and at 4°C for 2 months. The assay was tested with blood stored for 8 days without a change in sensitivity as measured by cycle threshold. This new assay format extends the capability of the Xpert MTB/RIF test, enabling up to 20 ml of blood to be tested rapidly for the presence of M. tuberculosis. This approach may be a useful method to detect extrapulmonary tuberculosis and the risk of death in immunocompromised patients.     

Evaluation of Xpert MTB/RIF for Detection of Mycobacterium tuberculosis in Cerebrospinal Fluid

Studies investigating Xpert MTB/RIF diagnostic performance on cerebrospinal fluid (CSF) samples are lacking in resource-rich settings. Xpert MTB/RIF results for 740 CSF samples from 698 patients across England were retrospectively compared with the results of culture of the same and contemporary samples. The overall sensitivity was calculated at 55%.     

Comparison of the Xpert MTB/RIF and Cobas TaqMan MTB Assays for Detection of Mycobacterium tuberculosis in Respiratory Specimens

The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is a fully automated, cartridge-based real-time PCR assay designed to detect Mycobacterium tuberculosis and rifampin resistance within 2 h. The performance of the Xpert assay has been evaluated in various clinical settings. However, there are few data comparing the Xpert assay to the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), one of the most widely utilized molecular assays for M. tuberculosis detection. In this prospective study, 320 consecutive respiratory specimens were processed simultaneously using acid-fast bacillus (AFB) staining, mycobacterial cultures with both solid and liquid media, and the Cobas and Xpert assays. The Xpert assay was performed with direct respiratory specimens, while the Cobas assay was done with decontaminated concentrated specimens. Based on the culture as a reference method, the overall sensitivities of the Cobas and Xpert assays were 71.4% and 67.9%, respectively. When AFB smear results were taken into consideration, the sensitivities of the Cobas assay for smear-positive and -negative specimens were 87% and 54%, while those of the Xpert assay were 67% and 69%, respectively. The Cobas assay showed 100% specificity and 100% positive predictive value (PPV) regardless of smear results, while the Xpert assay showed 100% specificity and 100% PPV for smear-positive specimens but 98% specificity and 60% PPV for smear-negative specimens. In conclusion, the Xpert assay showed performance that was slightly inferior to that of the Cobas assay but seems useful for the rapid detection of M. tuberculosis, considering that it was performed without laborious and time-consuming decontamination and concentration procedures.     

Integrating the Xpert MTB/RIF Assay into a Diagnostic Workflow for Rapid Detection of Mycobacterium tuberculosis in a Low-Prevalence Area

The Xpert MTB/RIF assay is a rapid and fully automated real-time PCR assay. The performance of the Xpert MTB/RIF assay as a primary screening test for urgent clinical specimens was evaluated during a 2-year period. The results showed that replacing smear microscopy with the Xpert MTB/RIF assay facilitates laboratory handling and improves the sensitivity and specificity of Mycobacterium tuberculosis detection.     

Xpert MTB/RIF assay for diagnosis of pleural tuberculosis

We prospectively investigated the diagnostic utility of the Xpert MTB/RIF (Mycobacterium tuberculosis/rifampin [RIF] resistance) assay in 20 cases with confirmed tuberculous pleural effusion. The sensitivity and specificity of the Xpert assay in pleural fluid were 25% and 100%, respectively. All cases positive by the Xpert assay were also positive by pleural fluid culture.     

Xpert MTB/RIF技术对结核分枝杆菌的诊断价值

目的 对Xpert MTB/RIF技术检测结核分枝杆菌（MTB）的结果进行分析并评估其价值。方法 采集2017年10月~2018年10月本院就诊疑为结核分枝杆菌感染的患者共580例,利用Xpert MTB/RIF技术对其痰液（或体液）标本进行DNA检测,同时将标本进行固体培养及涂片抗酸染色并对检测结果进行比较。结果 Xpert MTB/RIF技术检测MTB的阳性率为18.97%（110/580）,和固体培养法的阳性率18.62%（108/580）相比差异无统计学意义（P＞0.05）;和抗酸染色法的阳性率15.00%（87/580）相比则更为敏感,差异有统计学意义（P＜0.05）。 结论 Xpert MTB/RIF检测技术和固体培养法不相上下,且由于抗酸染色法,可以用于MTB的快速筛选。     

Diagnostic Accuracy of Xpert MTB/RIF Assay in Extrapulmonary Tuberculosis

Introduction: The WHO endorsed Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay, has been evaluated for pulmonary TB in a number of studies but very few have investigated it for extrapulmonary specimens. The present study evaluates the performance of Xpert MTB/RIF assay in the diagnosis of extrapulmonary TB (EPTB). Aim and Objectives: The aim of the study is to determine sensitivity and specificity of Xpert MTB/RIF assay for diagnosis of EPTB and RIF resistance in comparison to culture on Lowenstein–Jensen (LJ) medium and proportion method (PM), respectively. Materials and Methods: A total of 738 specimens from clinically suspected cases of EPTB were subjected to Ziehl–Neelsen staining, Xpert MTB/RIF assay and culture on LJ medium. PM was done on MTB isolates. Results: The sensitivity, specificity of Xpert MTB/RIF assay for diagnosis of EPTB were 84.91% (95% confidence interval [CI] 72.41%–93.25%) and 86.72% (95% CI 83.94%–89.17%) and for RIF resistance detection were 60.00% (95% CI 32.29%–83.66%) and 94.74% (95% CI 73.97%–99.87%), respectively. Among culture-positive cases, the sensitivity of Xpert MTB/RIF assay was 94.12% in smear positive and 80.56% in smear-negative cases. Xpert MTB/RIF showed maximum sensitivity of MTB detection from lymph node specimens (100% [95% CI 54.07%–100.00%]) and other body fluids (100% [95% CI 15.81%–100.00%]). Conclusion: The present study establishes Xpert MTB/RIF assay as a promising tool in the rapid diagnosis of EPTB and detection of RIF resistance.     

Evaluation of the Analytical Performance of the Xpert MTB/RIF Assay

We performed the first studies of analytic sensitivity, analytic specificity, and dynamic range for the new Xpert MTB/RIF assay, a nucleic acid amplification-based diagnostic system that detects Mycobacterium tuberculosis and rifampin (RIF) resistance in under 2 h. The sensitivity of the assay was tested with 79 phylogenetically and geographically diverse M. tuberculosis isolates, including 42 drug-susceptible isolates and 37 RIF-resistant isolates containing 13 different rpoB mutations or mutation combinations. The specificity of the assay was tested with 89 nontuberculosis bacteria, fungi, and viruses. The Xpert MTB/RIF assay correctly identified all 79 M. tuberculosis isolates and correctly excluded all 89 nontuberculosis isolates. RIF resistance was correctly identified in all 37 resistant isolates and in none of the 42 susceptible isolates. Dynamic range was assessed by adding 10² to 10⁷ CFU of M. tuberculosis into M. tuberculosis-negative sputum samples. The assay showed a log-linear relationship between cycle threshold and input CFU over the entire concentration range. Resistance detection in the presence of different mixtures of RIF-resistant and RIF-susceptible DNA was assessed. Resistance detection was dependent on the particular mutation and required between 65% and 100% mutant DNA to be present in the sample for 95% certainty of resistance detection. Finally, we studied whether assay specificity could be affected by cross-contaminating amplicons generated by the GenoType MTBDRplus assay. M. tuberculosis was not detected until at least 10⁸ copies of an MTBDRplus amplicon were spiked into 1 ml of sputum, suggesting that false-positive results would be unlikely to occur.Conventional diagnostic methods for Mycobacterium tuberculosis are slow and/or lack sensitivity. A number of new diagnostic approaches have brought incremental improvements to detection and drug susceptibility testing; however, the technical complexity of these assays and their dependence on dedicated laboratory infrastructure have limited their adoption, especially in low-resource, high-burden settings (1, 11, 12, 21). The recently introduced Xpert MTB/RIF (manufactured and marketed by Cepheid, Sunnyvale, CA) assay simultaneously detects the presence of M. tuberculosis and its susceptibility to the important first-line drug rifampin (RIF) (7). A sample processing system and an automated heminested real-time PCR assay are integrated into a single disposable cartridge. The assay can be performed directly from a clinical sputum sample or from a decontaminated sputum pellet and can generally be completed in less than 2 h (7).The Xpert MTB/RIF assay detects M. tuberculosis and RIF resistance by PCR amplification of the rifampin resistance-determining region (RRDR) of the M. tuberculosis rpoB gene and subsequent probing of this region for mutations that are associated with RIF resistance. Approximately 95% of RIF-resistant tuberculosis cases contain mutations in this 81-bp region (16). Our previous work has established that the Xpert MTB/RIF assay has a limit of detection (LOD), defined as the minimum number of bacilli that can be detected with 95% confidence) of 131 CFU per ml of clinical sputum (7). The assay was also able to identify RIF resistance in samples containing 23 common clinically occurring rpoB mutations. None of the 20 nontuberculosis mycobacteria (NTM) species tested, including the NTM species commonly described as causing human disease were falsely identified as M. tuberculosis (7), suggesting high specificity. Several small studies using clinical samples demonstrated 98% to 100% sensitivity overall, 72% sensitivity in smear-negative patients, and a specificity of 100% (7).In the present study, we expand upon our previous work and report on several critical analytical assay performance characteristics, including dynamic range, sensitivity, specificity, RIF resistance detection in heterogeneous samples, and resiliency against cross-contamination by other nucleic acid amplification techniques (NAATs).     

The Diagnostic Performance of the GenoType MTBDRplus Version 2 Line Probe Assay Is Equivalent to That of the Xpert MTB/RIF Assay

Molecular diagnostics for Mycobacterium tuberculosis have recently been endorsed by the World Health Organization. The Xpert MTB/RIF assay was endorsed for use on patient material, regardless of smear gradation, while the GenoType MTBDRplus (version 1) has been limited for use on smear-positive patient material. In this study, we evaluated the diagnostic performance of the Xpert MTB/RIF and GenoType MTBDRplus (version 2) assays on smear-positive and smear-negative patient specimens submitted to a high-throughput diagnostic laboratory. A total of 282 consecutive specimens were subjected to the two new molecular assays, and their performance characteristics were assessed relative to the routine diagnostic standard. Both assays showed similar diagnostic performance characteristics. The sensitivities of the GenoType MTBDRplus (v2.0) and Xpert MTB/RIF assays for the detection of culture-positive M. tuberculosis were 73.1% and 71.2%, respectively, while the specificities of both assays were 100%. Both assays were able to diagnose the presence of M. tuberculosis in 57 to 58% of smear-negative cases, suggesting that the performance characteristics were dependent on bacillary load. The detection of M. tuberculosis in culture-negative specimens confirmed that molecular assays should not be used for treatment monitoring. The sensitivity and specificity for rifampin resistance detection were 100% in both assays; however, the GenoType MTBDRplus (v2.0) assay provided additional information on isoniazid susceptibility. The GenoType MTBDRplus (v2.0) assay will complement the Xpert MTB/RIF screening assay by validating rifampin susceptibility and providing information on isoniazid susceptibility. In addition, the GenoType MTBDRplus (v2.0) assay will provide pharmacogenetic information that may be critical in guiding appropriate treatment.     

Xpert MTB/RIF系统在盆腔结核诊断中的应用价值分析

目的探讨Xpert MTB/RIF系统检测盆腔积液中结核分枝杆菌对于盆腔结核的诊断价值。方法采集30例临床确诊为盆腔结核患者及30例盆腔恶性肿瘤患者(对照组)的盆腔积液,对积液同时采用离心涂片法、荧光定量PCR法和Xpert MTB/RIF系统进行结核分枝杆菌检测,比较三种方法的敏感性及特异性。结果离心涂片法、荧光定量PCR法和Xpert MTB/RIF系统检测结核分枝杆菌的敏感性分别为6.67%(2/30)、53.33%(16/30)和73.33%(22/30),三种方法间差异均有统计学意义(P均<0.05)。三种方法特异度分别为100.0%,96.7%,100.0%,差异无统计学意义。结论采用Xpert MTB/RIF系统对盆腔积液进行结核分枝杆菌检测敏感性高,特异度好,具有较高的临床应用价值。     

Evaluation of the Cepheid Xpert MTB/RIF assay for direct detection of Mycobacterium tuberculosis complex in respiratory specimens

A total of 217 specimens submitted for routine smear and culture from three different sites within the western United States were used to evaluate the GeneXpert MTB/RIF assay (for research use only) (Cepheid, Sunnyvale, CA). Overall agreement compared to culture was 89% (98% for smear positives and 72% for smear negatives) for detection of Mycobacterium tuberculosis.     

Direct Comparison of Xpert MTB/RIF Assay with Liquid and Solid Mycobacterial Culture for Quantification of Early Bactericidal Activity

The early bactericidal activity of antituberculosis agents is usually determined by measuring the reduction of the sputum mycobacterial load over time on solid agar medium or in liquid culture. This study investigated the value of a quantitative PCR assay for early bactericidal activity determination. Groups of 15 patients were treated with 6 different antituberculosis agents or regimens. Patients collected sputum for 16 h overnight at baseline and at days 7 and 14 after treatment initiation. We determined the sputum bacterial load by CFU counting (log CFU/ml sputum, reported as mean ± standard deviation [SD]), time to culture positivity (TTP, in hours [mean ± SD]) in liquid culture, and Xpert MTB/RIF cycle thresholds (C_T, n [mean ± SD]). The ability to discriminate treatment effects between groups was analyzed with one-way analysis of variance (ANOVA). All measurements showed a decrease in bacterial load from mean baseline (log CFU, 5.72 ± 1.00; TTP, 116.0 ± 47.6; C_T, 19.3 ± 3.88) to day 7 (log CFU, −0.26 ± 1.23, P = 0.2112; TTP, 35.5 ± 59.3, P = 0.0002; C_T, 0.55 ± 3.07, P = 0.6030) and day 14 (log CFU, −0.55 ± 1.24, P = 0.0006; TTP, 54.8 ± 86.8, P < 0.0001; C_T, 2.06 ± 4.37, P = 0.0020). The best discrimination between group effects was found with TTP at day 7 and day 14 (F = 9.012, P < 0.0001, and F = 11.580, P < 0.0001), followed by log CFU (F = 4.135, P = 0.0024, and F = 7.277, P < 0.0001). C_T was not significantly discriminative (F = 1.995, P = 0.091, and F = 1.203, P = 0.316, respectively). Culture-based methods are superior to PCR for the quantification of early antituberculosis treatment effects in sputum.     

DNA Sequencing for Confirmation of Rifampin Resistance Detected by Cepheid Xpert MTB/RIF Assay

DNA sequencing of rpoB and culture-based drug susceptibility results were evaluated for samples referred for confirmation of rifampin resistance detected by the Cepheid Xpert MTB/RIF assay. Silent mutations and mutations associated with low-level resistance were found in the study population. These data support CDC recommendations to confirm Xpert rifampin resistance results.     

Performance of Xpert MTB/RIF RUO assay and IS6110 real-time PCR for Mycobacterium tuberculosis detection in clinical samples

The Cepheid Xpert MTB/RIF research-use-only (RUO) assay and a laboratory-developed test (LDT) targeting IS6110 were evaluated and compared to mycobacterial culture as the gold standard. The performance characteristics of both molecular assays were determined by using 112 specimens from 90 patients, including 89 pulmonary specimens and 23 extrapulmonary specimens. Of the specimens tested, 37 (33%) were culture positive for the Mycobacterium tuberculosis complex; 29 were pulmonary, and 8 were extrapulmonary. Of these culture-positive specimens, 83% of the pulmonary specimens and 50% of the extrapulmonary specimens were smear positive. There was complete concordance between the smear-positive culture-positive specimens, independent of the anatomical site (100% sensitivity). The sensitivity of the MTB/RIF RUO assay for smear-negative specimens was 60% for pulmonary and 75% for extrapulmonary specimens, while the IS6110 LDT sensitivities were 40% and 0%, respectively. There was also complete concordance among the culture-negative specimens tested. Both assays showed 95% specificity, with four culture-negative specimens testing as positive. A review of patient records indicated that there was a high likelihood of the presence of M. tuberculosis complex DNA in the false-positive specimens. Biosafety analysis was performed and showed an acceptable reduction in organism viability using the processing methods described above. Both molecular assays are suitable for the detection of M. tuberculosis isolates in smear-positive pulmonary and extrapulmonary specimens, while the sensitivity of the detection of M. tuberculosis isolates in smear-negative specimens was variable.     

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

Among 452 samples that were positive by the Xpert MTB/RIF (Xpert) assay and MGIT 960 system (MGIT), 440 and 10 Mycobacterium tuberculosis samples 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. rpoB sequencing 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 the rpoB gene.     

Evaluation of the Xpert MTB/RIF test accuracy for diagnosis of tuberculosis in areas with a moderate tuberculosis burden

The Xpert MTB/RIF assay (Xpert) is a molecular assay used for direct detection of Mycobacterium tuberculosis (MTB) in clinical specimens. In this study, we aimed to assess the accuracy of the Xpert assay for the diagnosis of tuberculosis (TB) in TB suspected patients from the northern region of Iran. The obtained results were compared with the culture method. The sputum specimens were examined using the Xpert assay, smear microscopy, and solid culture media as a reference diagnostic tool. Among 293 presumptive TB cases, 92 (31.4%) were positive according to the culture method. The Xpert method detected 88 (95.7%) cases that were positive according to the culture method, compared with 78 (84.8%) positive cases according to smear microscopy. The overall sensitivity and specificity of the Xpert method for TB diagnosis were 95.7% and 99%, respectively. Also, the sensitivity and specificity for smear microscopy were 84.8% and 97.5%, respectively. The Xpert assay showed high overall sensitivity and specificity; thus, it can be effectively used for the early and accurate diagnosis of MTB in TB endemic areas. In addition, the agreement between semi‐quantitative results of Xpert and smear microscopy assays could be helpful in evaluating transmission potential in TB patients.     

Comparison of the Genedia MTB Detection Kit and the Cobas TaqMan MTB Assay for Detection of Mycobacterium tuberculosis in Respiratory Specimens

The performance of the Genedia MTB detection kit was compared with that of the Cobas TaqMan MTB test using respiratory specimens. The Genedia and Cobas assays showed comparable sensitivities (81.8% and 78.8%, respectively) and specificities (99.8% and 99.5%, respectively), while the Genedia assay produced fewer invalid results and required less turnaround time and labor.     

Xpert MTB/RIF assay for the diagnosis of extrapulmonary tuberculosis: a diagnostic evaluation study

ObjectivesThe diagnosis of extrapulmonary tuberculosis (EPTB) is often made on clinical suspicion alone, resulting in both under- and overdiagnosis and relatively poor outcomes. In this study, we evaluated the clinical utility of the Xpert MTB/RIF on routinely collected extrapulmonary specimens in Ethiopia.MethodsThis study was carried out at Jimma University Specialized Hospital, Southwest Ethiopia. Extrapulmonary specimens were collected from 572 patients clinically suspected of suffering from EPTB. All specimens were tested for TB by smear microscopy, culture, and Xpert MTB/RIF. The diagnostic accuracy of Xpert MTB/RIF was calculated and compared to a composite reference standard (CRS), comprising clinical and laboratory results.ResultsIn total, 572 extrapulmonary specimens (279 lymph node, 159 pleural, 80 peritoneal, 45 cerebrospinal, and nine pericardial fluids) were tested. The pooled sensitivity and specificity of Xpert MTB/RIF were calculated to be 75% (95% CI 70–80) and 98% (95% CI 97–100) respectively when compared to the CRS. The highest sensitivity was documented for lymph node specimens (90%; 95% CI 86–94), moderate sensitivity for cerebrospinal fluid (53%; 95% CI 28–79), while the sensitivity was lowest for pleural (30%; 95% CI 17–44) and peritoneal (32%; 95% CI 12–51) fluids. Xpert MTB/RIF in addition detected rifampicin resistance in 13 patients, in perfect agreement with results from the line probe assay.ConclusionsXpert MTB/RIF may be used as initial diagnostic tool for testing of lymph node specimens from patients suspected of having TB lymphadenitis. The added value of Xpert MTB/RIF to diagnose pleural or peritoneal TB is limited by its poor sensitivity.     

Comparison of the Xpert MTB/RIF test with an IS6110-TaqMan real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens

The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P = 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P = 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P = 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.