Extended clinical sample incubation in the cepheid Xpert MTB/XDR test sample reagent : enhancing flexibility and workflows in high-volume laboratories

AVAILABILITY OF DATA AND MATERIALS : The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.Cepheid Xpert MTB/RIF ULTRA (ULTRA) and Xpert MTB/XDR are tests for detecting Mycobacterium tuberculosis (MTB) and drug resistance. Both tests involve a sample pre-processing step using the test's sample reagent (SR). The manufacturer recommends a four-hour limit for SR-treated samples prior to testing, posing challenges for high-volume laboratories conducting both tests. Implementing the XDR test as a follow-on to ULTRA positive specimen can be challenging in high-volume laboratories due to the time constraints imposed by the manufacturer's recommendations. To address this issue, this study investigated the impact of extended sample incubation in SR for durations longer than four hours at varying temperature conditions. Pre-characterized MTB isolates with diverse drug susceptibility profiles were incubated up to 36 hours at different temperatures including room temperature (RT), 2-8°C, and -20°C and tested using Xpert MTB/XDR. The study results indicate no adverse effects on sample stability or drug susceptibility detection. This suggests extended incubation could offer flexibility for conducting both tests on a single specimen, benefiting high-throughput laboratories.The National Health Laboratory Services (NHLS) Research Trust.https://www.elsevier.com/locate/diagmicrobiohj2024Medical MicrobiologySDG-03:Good heatlh and well-bein

New Horizons in the Diagnosis of Tuberculosis of the Spine: The Role of Whole Genome Sequencing

Study Design Prospective study. Purpose To evaluate the utility of whole genome sequencing (WGS) in drug resistance testing, lineage of the organisms, and organism-related factors responsible for bacilli settling in the spine. Overview of Literature The workstream for the diagnosis of tuberculosis (TB) involves isolation and culture of the organism and drug resistance testing using phenotypic methods. Xpert MTB/RIF Ultra is a genetic-based method that detects for Mycobacterium tuberculosis DNA in the rpoB gene. Meanwhile, WGS is a newer genetic-based method that assesses the whole genome of the bacterium. Very few studies have reported the use of WGS for extrapulmonary TB. Herein, we used WGS to diagnose spinal TB. Methods Tissues from 61 patients undergoing surgery for spinal TB underwent histologic examination, Xpert MTB/RIF Ultra, and culture and sensitivity testing. DNA from the cultured bacteria was sent for WGS. The test bacterial genome was compared to a reference strain of pulmonary TB. Results Acid-fast bacilli were observed in 9/58 specimens. Meanwhile, histology confirmed TB in all the patients. Bacilli were cultured in 28 patients (48.3%), and the average time to culture was 18.7 days. Xpert MTB/RIF Ultra was positive in 47 patients (85%). WGS was performed in 23 specimens. Overall, 45% of the strains belonged to lineage 2 (East Asian). There was one case of multidrug-resistant TB and two cases of non-tuberculous mycobacteria on WGS. We could not confirm any genomic difference between pulmonary and spinal TB strains. Conclusions Xpert MTB/RIF Ultra of tissues or pus is the investigation of choice when diagnosing spinal TB. Meanwhile, WGS can diagnose multidrug-resistant TB and non-tuberculous mycobacteria more accurately. No mutations were identified in spinal and pulmonary TB bacteria

Comparison of three commercial molecular assays for detection of rifampin and isoniazid resistance among Mycobacterium tuberculosis isolates in a high-HIV-prevalence setting

In a head to head comparison of the MTBDRplus Version 2.0 (Hain Lifescience), Xpert® MTB/RIF (Cepheid) and the Anyplex™ MTB/NTM (Seegene) assays we demonstrate equal sensitivity (59/61; 96.7%) and specificity (53/54; 98.1%) for detecting rifampicin resistance with further analysis of discordances. The Xpert does not detect Isoniazid resistance while Anyplex showed high false positivity.National Health Laboratory Serviceshttp://jcm.asm.org2016-03-31hb201

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis

BACKGROUND : Modern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium(PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems. METHODS : Assessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28 days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation. RESULTS : Complete inactivation ofM. tuberculosis occurredwithin 30 min of exposure at a ratio of 1:3 for sputumto PS-MTM. Sputum specimen in PS-MTMshowed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170 CFU/ml). Furthermore, PS-MTM samples remained stable over 28 days at ambient temperature displaying no significant change over time in Ctvalues (b5% on a mean starting value of 22.47). Of the 256 clinical sputumspecimens, 10.2%were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples. CONCLUSIONS : Collecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.http://www.elsevier.com/locate/jmicmeth2016-10-31hb201

A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR + Ki67 + on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67 + HLA-DR + Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67 + HLA-DR + T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ + IL2 + TNFα + CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67 + HLA-DR + ) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment

Frequency of Circulating CD4+Ki67+HLA-DR− T Regulatory Cells Prior to Treatment for Multidrug Resistant Tuberculosis Can Differentiate the Severity of Disease and Predict Time to Culture Conversion

Identifying a blood circulating cellular biomarker that can be used to assess severity of disease and predict the time to culture conversion (TCC) in patients with multidrug resistant tuberculosis (MDR-TB) would facilitate monitoring response to treatment and may be of value in the design of future drug trials. We report on the frequency of blood Ki67+HLA-DR− CD4+ T regulatory (Treg) cells in predicting microbiological outcome before initiating second-line treatment for MDR-TB. Fifty-one patients with MDR-TB were enrolled and followed over 18 months; a subset of patients was sputum culture (SC) negative at baseline (n = 9). SC positive patients were divided into two groups, based on median TCC: rapid responders (≤71 days TCC; n = 21) and slow responders (>71 days TCC; n = 21). Whole blood at baseline, months 2 and 6 was stimulated with M tuberculosis (Mtb) antigens and Treg cells were then identified as CD3+CD4+CD25hiFoxP3+CD127−CD69− and further delineated as Ki67+HLA-DR− Treg. The frequency of these cells was significantly enlarged at baseline in SC positive relative to SC negative and smear positive relative to smear negative patients and in those with lung cavitation. This difference was further supported by unsupervised hierarchical clustering showing a significant grouping at baseline of total and early differentiated memory Treg cells in slow responders. Conversely, there was a clustering of a lower proportion of Treg cells and activated IFNγ-expressing T cells at baseline in the rapid responders. Examining changes over time revealed a more gradual reduction of Treg cells in slow responders relative to rapid responders to treatment. Receiver operating curve analysis showed that baseline Mtb-stimulated Ki67+HLA-DR− Treg cells could predict the TCC of MDR-TB treatment response with 81.2% sensitivity and 85% specificity (AUC of 0.87, p < 0.0001), but this was not the case after 2 months of treatment. In conclusion, our data show that the frequency of a highly defined Mtb-stimulated blood Treg cell population at baseline can discriminate MDR-TB disease severity and predict time to culture clearance

Comparison between the BACTEC MGIT 960 system and the agar proportion method for susceptibility testing of multidrug resistant tuberculosis strains in a high burden setting of South Africa

BACKGROUND: The increasing problem of multi-drug-resistant (MDR) tuberculosis (TB) [ie resistant to at least isoniazid (INH) and rifampicin (RIF)] is becoming a global problem. Successful treatment outcome for MDR-TB depends on reliable and accurate drug susceptibility testing of first-line and second-line anti-TB drugs. METHOD: Consecutive M. tuberculosis isolates identified as MDR-TB during August 2007 to January 2008 using the BACTEC MGIT 960 systems and the agar proportion method were included in this study. Susceptibility testing of MDR-TB isolates against ethambutol (EMB) and streptomycin (STR) as well as two second-line anti-TB drugs, kanamycin (KAN) and ofloxacin (OFX) was performed using the BACTEC MGIT 960 systems at a routine diagnostic laboratory. The results were compared to those obtained by the agar proportion method. RESULT: The agreement between the BACTEC MGIT 960 system and the agar proportion method was 44% for EMB, 61% for STR and 89% for both KAN and OFX. The sensitivity and specificity of the BACTEC MGIT 960 system using the agar proportion method as a gold standard was 92% and 37% for EMB, 95% and 37% for STR, 27% and 97% for KAN and 84% and 90% for OFX, respectively. CONCLUSIONS: The BACTEC MGIT 960 system showed acceptable sensitivity for EMB, STR, and OFX; however, the BACTEC MGIT 960 system was less specific for EMB and STR and demonstrated a low sensitivity for KAN. The lower agreement found between the two methods suggests the unreliability of the BACTEC MGIT 960 system for the drugs tested. The reasons for the lower agreement between the two methods need to be investigated and further studies are needed in this setting to confirm the study finding.The project was supported by a grant from the NHLS.http://www.biomedcentral.com/1471-2334/12/369am2013ay201

Next-generation sequencing for identifying pyrazinamide resistance in Mycobacterium tuberculosis

No abstract available.http://cid.oxfordjournals.orgam201

Field evaluation of a novel preservation medium to transport sputum specimens for molecular detection of Mycobacterium tuberculosis in a rural African setting

Molecular tests are revolutionizing diagnosis of tuberculosis (TB). Disease burden is concentrated in resource-poor countries with inadequate infrastructure and capacity resulting in delays for specimens to reach the laboratory. We assessed the performance of an innovative method using a swab to inoculate sputum in a transport medium, PrimeStore® - Molecular Transport Medium (PS-MTM) for subsequent molecular detection of Mycobacterium tuberculosis at a centralized facility. A sputum specimen was obtained from suspected TB patients at rural healthcare facilities in South Africa and a swab taken and placed into PS-MTM from this specimen, prior to it being processed by either liquid culture or Xpert MTB/Rif assay (Xpert). A subset from a larger cohort study of a 141 patients was included for analysis, which included 47 laboratory-confirmed TB patients. M. tuberculosis was detected at 29% by culture, 29% by Xpert and 31% and 36% by real-time PCR of PS-MTM for the culture and Xpert specimen respectively. Concordance between the method under evaluation with culture was 82% (McNemar, p=0.55) and 84% (McNemar, p=0.05) for Xpert. Stratified by culture result, detection rate by real-time PCR of PS-MTM was similar to Xpert for patients with positive culture (p=0.32), but significantly higher if culture was negative (p=0.008). These results suggest that swab collection of sputum into PS-MTM provides a promising application for diagnosis of TB in rural healthcare settings thereby potentially improving the options available for the diagnosis of TB in countries incapable of applying decentralized high-tech molecular testing.Department of Medical Microbiology, University of Pretoria and Anova Health Institute.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-31562017-06-30hb2016Medical Microbiolog

Molecular detection of Mycobacterium tuberculosis from sputum transported in PrimeStore(®) from rural settings

SETTING : Mopani District, South Africa. OBJECTIVE : To explore remote, molecular detection of Mycobacterium tuberculosis from sputum transported using PrimeStorew Molecular Transport Medium (PSMTM) compared to settings where microscopy or Xpertw MTB/RIF is used as the baseline test. DESIGN : Two sputum specimens were collected from patients with cough of72 weeks at clinics in rural South Africa. Shortly after expectoration and before processing using Xpert, microscopy and liquid culture, a flocked swab was swirled in each of these specimens and placed in PS-MTM. Swabs were stored and transported to the United States at ambient temperature for real-time PrimeMixw polymerase chain reaction (PM-PCR). RESULTS : Of 132 patients, 23 (17%) were positive on microscopy, 39 (30%) on Xpert and 44 (33%) by PSMTM/PSMTM/ PM-PCR. Concordance of PS-MTM/PM-PCR with positive microscopy and Xpert was respectively 96% and 85%. Of 107 microscopy-negative samples, 22 (21%) were positive using PS-MTM/PM-PCR, while 11/91 (12%) Xpert-negative samples were PS-MTM/ PM-PCR-positive. PS-MTM/PM-PCR positivity was significantly higher than smear microscopy positivity (P , 0.001), but similar to Xpert (P ¼ 0.33). CONCLUSION: PCR testing of specimens transported in PS-MTM would enhance TB diagnosis in settings where smear microscopy is the baseline diagnostic test, and could provide an alternative in settings where Xpert testing is not available.http://www.ingentaconnect.comcontent/iuatld/ijtld2015-11-30hb201