Ultra Assay Research Articles

The case for expanding worldwide access to point of care molecular drug susceptibility testing for isoniazid

The Xpert MTB/XDR assay (Mycobacterium tuberculosis/extensively drug resistant; Cepheid, Sunnyvale, CA, USA) is a recent addition to the repertoire of tuberculosis (TB) drug susceptibility testing (DST) technologies and is set to bolster the early detection of drug-resistant TB [1]. Following on from the Xpert MTB/RIF (rifampin) Ultra assay, already used extensively to detect the presence of Mycobacterium tuberculosis complex and resistance to rifampicin, the Xpert MTB/XDR assay is designed to detect resistance to isoniazid, fluoroquinolones, ethionamide, and second-line injectables [1].

Detection of Mycobacterium tuberculosis complex DNA in oronasal swabs from infected African buffaloes (Syncerus caffer)

Mycobacterium bovis (M. bovis), a member of the Mycobacterium tuberculosis complex (MTBC), is the causative agent of bovine TB (bTB) in animals. Spread occurs through inhalation or ingestion of bacilli transmitted from infected individuals. Early and accurate detection of infected African buffaloes shedding M. bovis is essential for interrupting transmission. In this pilot study, we determined if MTBC DNA could be detected in M. bovis infected buffalo oronasal secretions using a molecular transport media (PrimeStore MTM) with oronasal swabs and a rapid qPCR assay (Xpert MTB/RIF Ultra). Bovine TB test-positive buffaloes were culled, then tissue samples and oronasal swabs collected post-mortem for mycobacterial culture and Ultra testing, respectively. The Ultra detected MTBC DNA in 5/12 swabs from M. bovis culture-confirmed buffaloes. Oronasal swabs from M. bovis negative buffaloes (n = 20) were negative on Ultra, indicating the high specificity of this test. This study showed that MTM can successfully preserve MTBC DNA in oronasal swabs. The proportion of MTBC positive oronasal swabs was higher than expected and suggests that the Ultra may be an additional method for identifying infected buffaloes. Further studies are needed to confirm the utility of the Ultra assay with oronasal swabs as an assay to evaluate possible MTBC shedding in buffaloes.

Diagnostic accuracy of Xpert ultra for childhood tuberculosis: A preliminary systematic review and meta-analysis.

Diagnosis of childhood tuberculosis (TB) is challenging. Xpert MTB/RIF and the new version Xpert MTB/RIF Ultra (Ultra) are molecular tests currently used to rapidly identify the infection. We reviewed the literature for the accuracy of Ultra assay in the diagnosis of tuberculosis and rifampicin resistance in children. We conducted a full search in PubMed, Web of Science (WOS), Embase, and Scopus, up to April 2021. A bivariate random‐effects model was used to determine the pooled sensitivity and specificity of Ultra, with a 95% confidence interval (CI), compared with culturing and the composite reference standard (CRS). In the ten included studies (2,427 participants), the pooled Ultra sensitivity and specificity, in diagnosing pulmonary tuberculosis (PTB), were 78% (95% CI, 73–82) and 92% (95% CI, 91–94), respectively, against culture. Since a high heterogeneity was found between studies, we created subgroups based on different samples and ages. Ultra‐pooled sensitivity was consistently lower against CRS (95% CI, 35%, 32–38). Compared to Xpert MTB/RIF, Ultra sensitivity tended toward higher values (Ultra: 73%, 67%–78% vs. Xpert MTB/RIF: 66%, 60%–72%), but specificity was lower (Ultra: 95%, 94%–96% vs. Xpert MTB/RIF: 99%, 98%–99%). Ultra has improved the definitive diagnosis of PTB, particularly in subjects with paucibacillary TB, including children. The lower specificity could be due to the fact that culture is an imperfect reference standard. Further studies are needed to evaluate the accuracy of Ultra in the diagnosis of childhood TB.

The value of lateral flow urine lipoarabinomannan assay and empirical treatment in Xpert MTB/RIF ultra negative patients with presumptive TB: a prospective cohort study

The value of Lateral Flow urine Lipoarabinomannan (LF-LAM) assay and the role of empiric tuberculosis (TB) treatment in the era of the highly sensitive Xpert MTB/RIF Ultra (Xpert Ultra) assay is unclear. This study aimed to assess the additional yield of LF-LAM assay when used in combination with Xpert Ultra, and the role of empiric TB treatment when Xpert Ultra used as the initial diagnostic in presumptive TB cases admitted to a tertiary hospital in Ethiopia. We performed a secondary analysis of a cohort of hospitalized Xpert MTB/RIF-negative patients. Sputum samples were examined for Mycobacterium tuberculosis by culture and Xpert Ultra. In HIV positive and severely ill patients, a urine sample was collected for the LF-LAM assay. Treatment outcome was assessed six months after enrollment. Logistic regression was used to identify factors predictive of deaths among Xpert Ultra negative patients. The Xpert Ultra assay diagnosed 31 of the 35 culture positive among the 250 hospitalized Xpert MTB/RIF-negative participants. The LF-LAM assay did not identify any case not detected by Xpert Ultra among the 52 (21.4%) participants living with HIV and the 16 patients with severe disease. Among Xpert Ultra negative patients, those who received empirical TB treatment had a similar odds of death (aOR 0.74, 95% CI: 0.1–2.7) as those not started on TB treatment. Low body mass index (≤ 18.5 kg/m2) was the only significant predictor of death in Xpert Ultra negative patients (aOR 4. 0, 95% CI: 1.08–14.6). In this prospective cohort, LF-LAM did not improve the diagnostic yield when used in combination with Xpert Ultra. Empiric TB treatment for Xpert Ultra negative presumptive TB cases was not associated with death at six months. Future studies in diverse settings should be to determine the optimal management of Xpert Ultra negative patients.

Accuracy and speed of molecular response reporting with Xpert BCR-ABL Ultra in vitro diagnostic test in CML patients

Purpose: The objective of this study was to introduce the GeneXpert Ultra cartridge-based BCR-ABL diagnostic test into routine molecular monitoring of minimal residual disease for chronic myeloid leukemia (CML) patients who are on tyrosine kinase inhibitors (TKIs).Methods: BCR-ABL1 transcript quantification was performed on 100 peripheral blood samples from 33 CMLpatients at different stages of the disease using automated GeneXpert BCR-ABL Ultra assay and following a standardized laboratory protocol (SP).Results: Analysis results were expressed as % on the International Scale (IS) for BCR-ABL1 real-time PCR quantification and estimated using molecular response (MR) according to the laboratory recommendations for scoring deep MRs following CML treatment. A comparison of the two methods showed that there was no statistically significant difference in the results expressed as % on the IS (p = 0.098). Samples were divided into six groups (MR1, MR2, MR3, MR4, MR4.5, and MR5) according to the estimated MR. A total of 74/100 samples had a completely identical MR with both methods, 12/100 samples had a higher MR obtained with GeneXpert, and 14/100 samples had a lower MR compared to the SP. In addition, 2/74 samples had a different MR for two classes obtained with GeneXpert compared to the SP, while the others 72/74 samples differed in one MR class. Analysis of the major molecular response resulted in 82% agreement. Minimal amounts of BCR-ABL1 copies were detected with GeneExpert in eight samples while the SP did not detect them. None of the GeneXpert results had any effect on the clinical decision for treatment with TKIsobtained using the SP.Conclusion: The Xpert BCR-ABL Ultra assay can be considered a useful and very fast clinical tool for molecular follow up of patients on TKIs.

Use of Xpert MTB/RIF Ultra assay on stool and gastric aspirate samples to diagnose pulmonary tuberculosis in children in a high-tuberculosis-burden but resource-limited area of China

ObjectivesOur study analyzed the performance of Xpert MTB/RIF Ultra (Ultra) on stool and gastric aspirate (GA) samples for the diagnosis of pediatric pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis in a high-burden area of China. MethodsChildren with presumptive TB were enrolled in two hospitals in Sichuan Province (July 2019–Oct 2020). Because of the unavailability of sputum for etiological testing, gastric samples were aspirated and tested by bacterial culture, acid-fast bacillus microscopy, and Ultra. Stool samples were tested simultaneously using Ultra and Xpert. ResultsIn total, 141 children with active TB and 34 with non-TB respiratory tract infections were enrolled. Ultra-stool (60.3%, 85/141) and Ultra-GA (52.5%, 74/141) tests were similarly sensitive (p = 0.187). Among the subset of 48 children with confirmed TB, Ultra testing was equally sensitive on stool and GA samples (85.4%, 41/48). The agreement between Ultra-stool and Ultra-GA was moderate in children with active TB (kappa value = 0.527). After integrating Ultra-GA and Ultra-stool outcomes, 70.9% (100/141) of the children were considered to have confirmed TB. The specificities of Ultra-stool and Ultra-GA were 97.1% (33/34) and 100% (34/34), respectively (p = 0.314). ConclusionsIn children, stools can be used as alternative samples to GAs for Ultra tests. Stool- and GA-based Ultra tests are appropriate for bacteriological TB confirmation.

Xpert MTB/RIF Ultra assay for the detection of Mycobacterium tuberculosis in people with negative conventional Xpert MTB/RIF but chest imaging suggestive of tuberculosis in Dhaka, Bangladesh

BackgroundThe World Health Organization is considering substituting Xpert MTB/RIF (Xpert) with Xpert MTB/RIF Ultra (Ultra) for tuberculosis (TB) diagnosis, but supportive evidence is scarce, particularly among people more likely (presumptive) to have paucibacillary pulmonary TB (PTB). MethodsDuring January-July 2018, presumptive PTB patients visiting TB Screening and Treatment Centres of Dhaka for routine chest X-ray (CXR) and conventional Xpert were enrolled. Sputum specimens were additionally tested with microscopy, culture, and Ultra. Specimens with “Trace call” by Ultra (Ultra-trace) were retested. Yield and diagnostic accuracy using various approaches to Ultra-trace and concordance of Ultra with bacteriological-positive PTB were assessed. ResultsAltogether, 1,083 participants (104 ‘Xpert-positive’; 979 ‘Xpert-negative and CXR-suggestive’) were enrolled. All Xpert-positives and 900 (92%) Xpert-negatives were concordant with Ultra, however, seventy-nine (8.1%) Xpert-negative specimens tested positive with Ultra; 37 (46.8%) were categorically positives, and 42 (53.2%) were Ultra-trace. Sixteen of the 42 were retested, of whom eight (50.1%) Ultra-trace turned categorically positive, leading to 45 (4.6%) additionally detected by Ultra. Ultra sensitivity and specificity were 93.9% and 94.6%, and it additionally detected 5.4% more TB patients with a concordance of 94.6% (kappa, □=0.78) compared to any bacteriologically positive specimen (microscopy, culture, or Xpert). ConclusionUltra exhibited improved detection and accuracy among Xpert-negatives in a cohort with a high likelihood of PTB.

Detection of pulmonary tuberculosis in children using the Xpert MTB/RIF Ultra assay on sputum: a multicenter study.

Microbiological confirmation is rare in children with active tuberculosis; therefore, a more accurate test is needed to detect pulmonary tuberculosis in children. In this multicenter study, we evaluated the utility of the Xpert MTB/RIF Ultra (Ultra) on sputum, an assay recommended by the World Health Organization to test for childhood tuberculosis in high-burden settings. Children with symptoms suggestive of tuberculosis were enrolled at three hospitals in China and categorized as having active tuberculosis or nontuberculosis. The sensitivity and specificity of Ultra were 42.1% (48/114) and 99.0% (208/210), respectively. Using three MTB culture results as the reference, the sensitivity of Ultra in the subset of 38 children with culture-positive and 76 children with culture-negative was 68.4% (26/38) and 28.9% (22/76), respectively(p < 0.001). A single MTB culture combined with a single Ultra could detect 54 (54/114,47.4%) cases with active TB, while repeated MTB culture combined with a single Ultra detected 60 (60/114, 52.6%) cases with active TB(p = 0.427). Among 155 children (58 with TB and 97 with RTIs) simultaneously tested with the Ultra and Xpert MTB/RIF (Xpert), the sensitivity of the Xpert (24.1%, 14/58) was lower than that of the Ultra (41.4%, 24/58; p = 0.048). Eight children were found to have rifampin-resistant MTB strains. The Xpert MTB/RIF Ultra assay should be implemented to test for pulmonary tuberculosis in children to achieve higher confirmation rates.

Retrospective diagnosis of lymphatic tuberculosis in frozen samples using two genetic amplification methods, Xpert® MTB/RIF ULTRA and Abbott RealTime MTB Assay.

ObjectivesThe main objective of the present study is to assess the sensitivity and specificity of a retrospective diagnostic of lymphatic tuberculosis (LTB), testing frozen samples using gene amplification PCR methods. The secondary objective was to compare the results of two different commercial tuberculosis gene amplification methods for this purpose.Material and methodsWe retrospectively studied 38 frozen samples, previously processed for mycobacterial culture between January 2014 and August 2019. The results of the previous cultures were: 21 samples positive for Mycobacterium tuberculosis complex (MTB) (5 being smear positive), 7 samples culture positive for Mycobacterium avium-intracellulare complex and 10 samples which were mycobacterial culture negative and discarded for LTB diagnosis, used as controls. The samples were processed using two gene amplification methods: Xpert® MTB/RIF Ultra (Cepheid) and Abbott RealTime MTB Assay (Abbott).ResultsCompared to initial culture results the sensitivity and specificity of Xpert® MTB/RIF Ultra were 57.1% and 100% and 52.3 % and 92.5%, respectively for the Abbott RealTime MTB assay. The differences were not statistically significant. In addition, there were no differences according to the period of freezing.ConclusionsGene amplification of frozen samples confirmed the diagnosis of lymphatic TB in almost 60% of cases, allowing retrospective diagnosis in initially non suspected cases. Both gene amplification techniques tested were equally useful.

Detectable troponin below the 99th percentile predicts survival in patients with cardiovascular disease

Abstract Background Patients with cardiovascular disease and elevated troponin above the 99th percentile of the upper reference limit are at increased risk for major adverse events, and usually require urgent treatment, including coronary angiography. Meanwhile, patients with detectable troponin levels below the 99th percentile represent a more heterogeneous collective at need for further risk stratification. Purpose This study aims to determine the prognostic implications of detectable troponin below the 99th percentile of the upper reference limit compared to troponin lower than the detectable range in patients with cardiovascular disease. Methods The ECAD registry was screened for patients without detectable troponin and with detectable troponin below the 99th percentile upon admission. Patients with ST-segment elevation myocardial infarction and patients with admission troponin above the 99th percentile were excluded. Troponin was determined by Siemens Dimension Troponin I (detectable limit: 40 ng/L, 99th percentile: 70 ng/L) and contemporary Centaur high-sensitive Troponin I Ultra (detectable limit: 6 ng/L, 99th percentile: 40 ng/L) assays. Overall survival was defined as the primary endpoint. Cox regression analysis was used to determine the association of troponin groups with incident mortality, adjusting for age, sex, systolic blood pressure, low-density lipoprotein (LDL) cholesterol, smoking status, and family history of premature cardiovascular disease. Results 14,776 consecutive patients (mean age was 65.35±12.74 years, with 71.3% male) with hospital admissions between 2004 and 2019 were included to the analysis. 11,965 patients had troponin levels below the detectable limits, while 2,811 patients had detectable troponin below the 99th percentile. During a mean follow-up of 4.25±3.76 years, 2379 (16.1%) deaths of any cause occurred. The overall mortality was higher in patients with detectable troponin below the 99th percentile compared to patients without detectable troponin (20.8% vs. 15.0%, p&amp;lt;0.001). In multivariable regression analysis, detectable troponin below the 99th percentile was significantly associated with all-cause mortality (HR 1.71; 95% CI 1.46–2.01; p&amp;lt;0.001). At 30 months, there was a significant stepwise relationship with increasing overall mortality between the tertiles of troponin levels (tertile 1, HR 1.62 (1.39–1.90); tertile 2, HR 1.88 (1.63–2.16); tertile 3, HR 2.02 (1.74–2.35)). Conclusions Detectable troponin below the 99th percentile is an independent predictor of overall mortality in patients with cardiovascular disease, and shows a gradually higher risk with increasing troponin levels. Every finding of detectable troponin in patients with cardiovascular disease should therefore prompt further diagnostic work-up. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): UMEA Young Clinical Scientist Grant, Medical faculty, University Duisburg-Essen (Hendricks)

Xpert MTB/XDR for rapid detection of drug-resistant tuberculosis beyond rifampicin

Effective control of drug-resistant tuberculosis (DR-TB) requires rapid diagnosis and prompt treatment. 1 WHOWHO operational handbook on tuberculosis. Module 3: diagnosis—rapid diagnostics for tuberculosis detention, 2021 update. World Health Organization, Geneva2021 Google Scholar However, access to diagnostic assays for DR-TB remains a challenge, especially in highly endemic countries. The Xpert MTB/RIF assay and its successor the Xpert MTB/RIF Ultra (Cepheid, Sunnyvale, CA, USA) are used extensively worldwide to detect Mycobacterium tuberculosis and rifampicin resistance but cannot assess resistance to isoniazid and second-line drugs. Tests for second-line-drug resistance are technically demanding and require advanced laboratory infrastructure. Therefore, their use is restricted to central or national tuberculosis laboratories. Almost a decade since the introduction of the GeneXpert technology, the Xpert MTB/XDR assay has been approved for reflex testing of sputum samples after a positive result for M tuberculosis complex on the Xpert MTB/RIF or Ultra assays. Detection of isoniazid, fluoroquinolone, ethionamide, amikacin, kanamycin, and capreomycin resistance by the Xpert MTB/XDR assay: a cross-sectional multicentre diagnostic accuracy studyThe Xpert MTB/XDR assay showed high diagnostic accuracy and met WHO's minimum target product profile criteria for a next-generation drug susceptibility test. The assay has the potential to diagnose drug-resistant tuberculosis rapidly and accurately and enable optimum treatment. Full-Text PDF

Molecular detection of Mycobacterium tuberculosis sensu stricto in the soil of Niger

Mycobacterium tuberculosis (MTB) complex is comprising of pathogenic mycobacteria responsible for human and animal tuberculosis, a major public health problem in Niger. Although infected individuals are paramount sources of contamination, nevertheless alternative, neglected sources may play some role in minority forms of the infection. Accordingly, we investigated the presence of Mycobacterium tuberculosis complex in soil samples in Niger. A total of 103 soil samples were collected in six different areas in Niger in October and November 2018 and April and May 2020 from residential areas of tuberculosis patients. Screening PCR targeting M. tuberculosis complex CRISPR-Csm4 and Xpert MTB/RIF Ultra assay were applied to detect the M. tuberculosis complex. M. tuberculosis DNA was positively detected in five of 103 (5/103; 4.8%) soil samples (Dosso: one sample, Zinder: one sample and Niamey: three samples) using the CRISPR-Csm4 system. CRISPR-Csm4 gene sequence identified four M. tuberculosis sensu stricto (may be lineages 1, 3 or 4) and one M. tuberculosis L2 lineage (Beijing). Moreover, the five positive samples were confirmed by Xpert MTB/RIF Ultra assay as rifampicin-susceptible M. tuberculosis complex strains. However, culture remained negative after 42 days. In this study, we announced for the first time the presence of M. tuberculosis sensu stricto in the soil of Niger. Moreover, these detected lineages were identical to the dominant M. tuberculosis lineages in patients. The presence of common lineages of M. tuberculosis between the soil and human highlight the risk of transmission from the soil to human.

False detection of rifampicin resistance using Xpert® MTB/RIF Ultra assay due to an A451V mutation in Mycobacterium tuberculosis.

BackgroundIn a 12 month period, three Irish-born adult cases with pulmonary TB were initially diagnosed by Xpert® MTB/RIF Ultra assay, which detected a rifampicin resistance-conferring mutation prompting treatment as potential MDR cases.MethodsFurther laboratory investigations on the cultured isolates included GenoType MTBDRplus assay, phenotypic drug susceptibility tests using the BD BACTEC MGIT culture system and MIC broth microdilution tests. Sequencing of the rpoB gene was performed using Sanger sequencing and WGS.ResultsPhenotypic drug susceptibility tests determined the isolates to be rifampicin susceptible. Molecular investigations identified an A451V (codon 532) mutation in the Mycobacterium tuberculosis rpoB gene that has not previously been found to cause rifampicin resistance. Genome sequencing revealed that the three isolates’ genomes differed by ≤5 SNPs, indicating a high likelihood of recent transmission events. Furthermore, a cluster of six related M. tuberculosis isolates from our in-house typing database showed four were highly related; all were rifampicin susceptible and lacked this mutation.ConclusionsFalse detection of rifampicin resistance, albeit rare, should be considered possible with Xpert® MTB/RIF Ultra assay, particularly in low TB incidence settings. Confirmatory sequencing methods should be performed to prevent the unnecessary use of second-line anti-tuberculous drugs.

Selective detection of glutathione by flower-like NiV2O6 with only peroxidase-like activity at neutral pH

In view of the broad application prospect of peroxidase-like nanozymes in biomedical analysis, it is of great significance to eliminate the interference of their oxidase-like activity and enable them to work under neutral conditions. Herein, flower-like NiV2O6 was synthesized and their enzyme-mimicking activity was investigated. Through the regulation of pH, NiV2O6 nanozyme showed only peroxidase-like activity but not oxidase-like activity under neutral conditions, which could catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine into its blue product in the presence of H2O2. Furthermore, based on the competitive effect of glutathione (GSH) on the catalytic activity of nanozymes, a semi-quantitative/quantitative colorimetric assay was established for GSH detection by using peroxidase-like NiV2O6. The assay exhibited a good linear relationship in GSH concentration ranging from 3–100 μmol L−1, with a detection limit of 0.89 μmol L−1. Moreover, in the presence of formaldehyde as masking agent, this method showed satisfactory specificity for GSH under the interference of a variety of interfering substances and even biothiols. Concerning the practical application, the system was applied to monitor GSH level in fetal bovine serum, human serum and SiHa cells. Satisfyingly, the obtained results were consistent well with those of Ultra performance liquid chromatography (UPLC) and assay kit, indicating the constructed assay has great potential in clinical application.

Xpert® BCR-ABL Ultra, a High Sensitivity Assay, Demonstrates a Limit of Detection Reaching MR4.5 and below

The ability to consistently detect low levels of BCR-ABL transcripts in patients with chronic myeloid leukemia (CML) is important in the assessment of treatment outcomes in patients on tyrosine kinase inhibitor (TKI) therapy. Particularly, BCR-ABL assays that are sensitive in the measurement of deep level response may aid in the identification of potential candidates for treatment discontinuation. Xpert® BCR-ABL Ultra detects the most common BCR-ABL transcripts below MR4.5 (Molecular Response at 4.5-log reduction) or 0.0032%, which is widely accepted as the clinical threshold that defines candidates who can safely discontinue TKI therapy. The present studies were designed to verify the limit of detection (LoD) for the Xpert® BCR-ABL Ultra assay below MR4.5 on the international Scale (IS) in clinical samples for both the b3a2 and b2a2 transcripts.To overcome the challenge of testing numerous replicates requiring large volumes of patient samples, serial dilutions ranging from BCR-ABL/ABL levels of 10% to <0.001% (IS) were prepared as contrived samples using CML patient blood with initial BCR-ABL level >10% (IS) and pooled blood from CML negative patients, ranged from 10% to <0.001% (IS). Twenty-one replicates of each dilution were measured for %BCR-ABL/ABL (IS). Determination of the LoD was performed by the statistical analysis to identify the lowest concentration of %BCR-ABL/ABL (IS) per test that can be reproducibly distinguished from negative samples with 95% confidence. The acceptable precision for %BCR-ABL/ABL (IS) is defined as the ability to detect at least a 3-fold difference for all concentrations tested. In addition, analytical LoD studies were performed using spike-in CML cell lines and cell-line derived RNAs, carrying either b3a2 or b2a2 transcripts. Furthermore, the clinical sensitivity study was conducted using blood from twelve low BCR-ABL transcripts level CML patients on TKI therapy, who had achieved and maintained MMR (Major Molecular Response) [0.1% (IS)] with reporting below 0.05% (IS).Consistent results were observed in the both the diluted CML patient blood and spike-in CML cell lines or cell-line derived RNA studies for both the b3a2 and b2a2 transcripts, demonstrating an assay LoD of MR4.5 and below with a less than 2-fold difference at the LoD levels. With the clinical sensitivity study, eleven out of twelve low CML subjects were detected in at least 19 out of 20 replicates tested per subject over a range of 0.038% (IS) (SD=~0.17 Log) to 0.0011% (IS) (SD=~0.4 Log). The overall ABL copy number present in clinical samples in each study was at least 5-10 times the required minimal ABL copy number of ≥32,000 to support a claim of MR4.5 and ≥100,000 for MR5.0.These LoD evaluations demonstrate that the Xpert® BCR-ABL Ultra assay complies with the international guidelines for assay sensitivity achieving MR4.5 with 5-10 times more than the required ABL copies to confidently identify candidate patients that may benefit from the discontinuation of TKI therapy. DisclosuresLevitas:Cepheid: Employment. Uy:Novartis: Consultancy, Other: Travel Suppport; Boehringer Ingelheim: Consultancy; GlycoMimetics: Consultancy. Mims:Novartis: Honoraria. Shridhar:Cepheid: Employment. Wu:Cepheid: Employment. Huang:Cepheid: Employment. Wei:Cepheid: Employment. Lykke:Cepheid: Employment. Bates:Cepheid: Employment. Wong:Cepheid: Employment. Day:Cepheid: Employment.

Laboratory comparison of stool processing methods for Xpert® Ultra.

TB disease diagnosis in children is difficult due to non-specific symptoms, paucibacillary disease and the need for invasive procedures to obtain diagnostic specimens. In many settings, these specimens are simply not collected and therefore stool, easily obtained, has emerged as a promising specimen for the diagnosis of child TB. In this study, stool from a healthy adult was spiked with known concentrations of bacille Calmette-Guérin vaccine and tested using the Xpert® Ultra assay to determine the relative detection and error rate associated with four different published stool processing methods.

The Simple One-Step (SOS) Stool Processing Method for Use with the Xpert MTB/RIF Assay for a Child-Friendly Diagnosis of Tuberculosis Closer to the Point of Care.

ABSTRACTYoung children cannot easily produce sputum for diagnosis of pulmonary tuberculosis (TB). Alternatively, Mycobacterium tuberculosis complex bacilli can be detected in stool by using the Xpert MTB/RIF (Ultra) assay (Xpert). Published stool processing methods contain somewhat complex procedures and require additional supplies. The aim of this study was to develop a simple one-step (SOS) stool processing method based on gravity sedimentation only, similar to Xpert testing of sputum samples, for the detection of M. tuberculosis in stool samples. We first assessed whether the SOS stool method could provide valid Xpert results without the need for bead-beating, dilution, and filtration steps. We concluded that this was the case, and we then validated the SOS stool method by testing spiked stool samples. By using the SOS stool method, 27 of the 29 spiked samples gave valid Xpert results, and M. tuberculosis was recovered from all 27 samples. The proof of principle of the SOS stool method was demonstrated in routine settings in Addis Ababa, Ethiopia. Nine of 123 children with presumptive TB had M. tuberculosis-positive results for nasogastric aspiration (NGA) samples, and 7 (77.8%) of those children also had M. tuberculosis-positive Xpert results for stool samples. Additionally, M. tuberculosis was detected in the stool samples but not the NGA samples from 2 children. The SOS stool processing method makes use of the standard Xpert assay kit, without the need for additional supplies or equipment. The method can potentially be rolled out to any Xpert site, bringing a bacteriologically confirmed diagnosis of TB in children closer to the point of care.

Performance of Xpert MTB/RIF Ultra for diagnosis of pulmonary and extra-pulmonary tuberculosis, one year of use in a multi-centric hospital laboratory in Brussels, Belgium.

Among the challenges in controlling tuberculosis, a rapid and accurate diagnostic test for the detection of Mycobacterium tuberculosis complex (MTBc) and its resistance to first line therapies is crucial. We evaluated the performance of the Xpert MTB/RIF Ultra assay (Xpert Ultra) for the rapid detection of MTBc and rifampicin resistance (RR) in 1120 pulmonary and 461 extra-pulmonary clinical specimens and compared it with conventional phenotypic techniques. The Xpert Ultra assay detected MTBc in 223 (14.1%) samples with an overall sensitivity and specificity, using culture as the “gold standard”, of 91.1% (95% CI, 85.6–95.1) and 94.5% (95% CI, 93.1–95.6), respectively. The sensitivity of the Xpert Ultra test for smear-negative extra-pulmonary specimens was high (87.1%), even higher than with smear-negative pulmonary specimens (81.8%). But this enhanced sensitivity came with a low overall specificity of smear-negative extra-pulmonary specimens (66.7%). For 73 patients, 79/1423 (3.4%) negative mycobacterial culture samples were found to be positive with Xpert Ultra. Clinical data was necessary to correctly interpret potential false-positive results, especially trace-positive results. Sensitivity of the Xpert Ultra to detect RR compared to drug susceptibility testing was 100% (95% CI, 29.2–100) and specificity was 99.2% (95% CI, 95.8–100). We concluded that the Xpert Ultra test is able to provide a reliable TB diagnosis within a significantly shorter turnaround time than culture. This is especially true for paucibacillary samples such as smear-negative pulmonary specimens and extra-pulmonary specimens.

Discordance of the Repeat GeneXpert MTB/RIF Test for Rifampicin Resistance Detection Among Patients Initiating MDR-TB Treatment in Uganda.

BackgroundThe Global Laboratory Initiative (GLI) guidelines recommend repeat for GeneXpertMTB/RIF (XpertMTB/RIF) in patients with a low pretest probability of rifampicin resistance (RR).MethodsThis was a cross-sectional study using results of sputum specimens collected from participants screened for the STREAM 2 trial. We recruited all patients with XpertMTB/RIF RR-TB detected who were referred for RR/multidrug-resistant (MDR) TB treatment initiation at Mulago National Referral Hospital, Kampala, between September 2017 and October 2019. At baseline, smear microscopy, repeat XpertMTB/RIF, Xpert Ultra, and MTBDRplus assays were done on sputum specimens. Culture-based drug susceptibility testing (DST) was performed on discordant specimens. We analyzed the prevalence and factors associated with discordance between initial and repeat XpertMTB/RIF RR and false XpertMTB/RIF RR. False XpertMTB/RIF RR was defined as no RR detected by any of Xpert Ultra, LPA, or culture DST (reference comparator).ResultsA total of 126/130 patients had repeat XpertMTB/RIF results, of whom 97 (77.0%) had M. tuberculosis detected, 81 (83.5%) had RR detected, and 1 (1.0%) had RR indeterminate. The prevalence of discordant XpertMTB/RIF RR was 15/96 (15.6%), whereas false XpertMTB/RIF RR prevalence was 10/96 (10.4%).Low–bacillary load sputum specimens were more likely to have discordant XpertMTB/RIF RR and false XpertMTB/RIF RR results (adjusted odds ratio [aOR], 0.04; 95% CI, 0.00–0.37; P = .01; aOR, 0.02; 95% CI, 0.01–0.35; P = .01, respectively).ConclusionsOur findings show a high false-positive rifampicin resistance rate in low–TB burden patients, which calls for repeat testing in order to prevent unnecessary prescription of anti-MDR-TB therapy.

Novel molecular transport medium used in combination with Xpert MTB/RIF ultra provides rapid detection of Mycobacterium bovis in African buffaloes

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore MTM at ambient temperature until Xpert MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis-unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis-positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis-unexposed animals, showing the potentially high specificity of Ultra with PrimeStore swabs. PrimeStore MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes.