Evidence for Tuberculosis in Individuals With Xpert Ultra "Trace" Sputum During Screening of High-Burden Communities.

BACKGROUND: "Trace" results on Xpert MTB/RIF Ultra ("Ultra"; Cepheid) -a molecular diagnostic test for tuberculosis (TB)-are often interpreted as an indication for TB treatment, but may also represent detection of nonviable bacilli or analytical error. In community-screening settings where individual TB risk is low, there is limited guidance on how to interpret Ultra-trace results. METHODS: We conducted systematic Ultra TB screening of adults and adolescents (≥15 years) in Kampala, Uganda, through door-to-door and event-based sputum collection. We enrolled individuals with trace-positive sputum for detailed clinical, radiographic, and microbiological (including 2 sputum cultures, repeat Ultra, and for people with HIV, urine lipoarabinomannan) evaluation, and compared those findings with similar evaluations in controls with Ultra-negative and Ultra-positive (non-trace) sputum. RESULTS: Of 21 957 people screened with Ultra, 211 (1.0%) tested positive, including 96 (46% of positives) with trace results. Of 92 people enrolled with trace-positive sputum; 12% (11/92) were HIV-positive and 14% (13/92) had prior TB. The prevalence of TB among participants with trace-positive sputum results was 14% (13/92) by culture, 24% (22/92) using broader microbiological criteria, and 26% (24/92) after accounting for clinical diagnosis. The prevalence of cough and of abnormal chest computed tomography (CT) findings were 32% and 26%, respectively, if Ultra-negative; 34% and 54% if trace-positive/non-microbiologically confirmed; 72% and 95% if trace-positive/microbiologically confirmed; and 71% and 93% if Ultra-positive (more than trace). CONCLUSIONS: Most individuals with trace-positive sputum in Ugandan communities did not have microbiologically confirmed TB but had more symptoms and chest CT abnormalities than people with Ultra-negative sputum.

Comparative evaluation of the STANDARD M10 and Xpert C. difficile assays for detection of toxigenic Clostridioides difficile in stool specimens.

This study compared the performance of two commercial molecular assays, the STANDARD M10 Clostridioides difficile assay (M10) and the Xpert C. difficile assay (Xpert), for detecting toxigenic C. difficile in stool specimens. A total of 487 consecutive stool specimens submitted for routine C. difficile testing between June and November 2023 were included. Following routine testing using C. DIFF QUIK CHEK COMPLETE (QCC), M10 and Xpert were tested in parallel, alongside toxigenic culture (reference standard). Additionally, two-step algorithms, using QCC on the first step and either M10 or Xpert on the second step, were assessed. Both M10 and Xpert demonstrated a sensitivity and negative predictive value (NPV) of 100%. M10 exhibited significantly higher specificity and positive predictive value (PPV; 91.9% and 64.2%, respectively) than Xpert (90.3% and 59.8%, respectively). Both two-step algorithms showed a sensitivity and NPV of 98.4% and 99.8%, respectively. The specificity and PPV of the two-step algorithm using M10 (95.2% and 75.0%, respectively) were slightly higher than those of the one using Xpert (94.8% and 73.2%, respectively), without statistical significance. Receiver operating characteristic curve analysis, assessing the predictive ability of cycle threshold (Ct) values for the detection of free toxin, exhibited an area under the curve of 0.825 for M10 and 0.843 for Xpert. This indicates the utility of Ct values as predictors for the detection of free toxin in both assays. In conclusion, M10 proves to be an effective diagnostic tool with performance comparable to Xpert, whether utilized independently or as part of a two-step algorithm.

Performance comparison of BD Phoenix CPO detect panel with Cepheid Xpert Carba-R assay for the detection of carbapenemase-producing Klebsiella pneumoniae isolates.

BACKGROUND: We aimed to compare the performance of carbapenemase classification in carbapenem-resistant Klebsiella pneumoniae (CRKP) obtained using the BD Phoenix CPO Detect panel (CPO panel) and Cepheid Xpert Carba-R assays. We analyzed 55 CRKP strains from clinical specimens collected between November 2020 and November 2022. The CPO panel was used to detect both antibiotic susceptibility and phenotypic carbapenemase classes, while Xpert Carba-R was employed to identify KPC, NDM, VIM, OXA-48, and IMP genes. Due to the limited availability of molecular kits, we arbitrarily selected 55 isolates, identified as carbapenemase-producing according to the CPO panel and with meropenem minimum inhibitory concentration values > 8 mg/L. RESULTS: According to the Xpert Carba-R assay, 16 of the 55 isolates (29.1%) were categorised as Ambler Class A (11 of which matched CPO panel Class A identification); three isolates (5.5%) were identified as Class B and 27 isolates (49.1%) as Class D (in both cases consistent with CPO panel B and D classifications). A further eight isolates (14.5%) exhibited multiple carbapenemase enzymes and were designated as dual-carbapenemase producers, while one isolate (1.8%) was identified as a non-carbapenemase-producer. The CPO panel demonstrated positive and negative percent agreements of 100% and 85.7% for Ambler Class A, 100% and 100% for Class B, and 96.4% and 100% for Class D carbapenemase detection, respectively. CONCLUSION: While the CPO panel's phenotypic performance was satisfactory in detecting Class B and D carbapenemases, additional confirmatory testing may be necessary for Class A carbapenemases as part of routine laboratory procedures.

Improving detection of carcinoma in situ in bladder cancer: urinary cytology vs the Xpert® BC Monitor.

OBJECTIVE: To investigate and compare the performance of urinary cytology and the Xpert BC Monitor test in the detection of bladder cancer in various clinically significant patient cohorts, including patients with carcinoma in situ (CIS), in a prospective multicentre setting, aiming to identify potential applications in clinical practice. PATIENTS AND METHODS: A total of 756 patients scheduled for transurethral resection of bladder tumour (TURBT) were prospectively screened between July 2018 and December 2020 at six German University Centres. Central urinary cytology and Xpert BC Monitor tests were performed prior to TURBT. The diagnostic performance of urinary cytology and the Xpert BC Monitor was evaluated according to sensitivity (SN), specificity (SC), negative predictive value (NPV) and positive predictive value (PPV). Statistical comparison of urinary cytology and the Xpert BC Monitor was conducted using the McNemar test. RESULTS: Of 756 screened patients, 733 (568 male [78%]; median [interquartile range] age 72 [62-79] years) were included. Bladder cancer was present in 482 patients (65.8%) with 258 (53.5%) high-grade tumours. Overall SN, SC, NPV and PPV were 39%, 93%, 44% and 92% for urinary cytology, and 75%, 69%, 59% and 82% for the Xpert BC Monitor. In patients with CIS (concomitant or solitary), SN, SC, NPV and PPV were 59%, 93%, 87% and 50% for urinary cytology, and 90%, 69%, 95% and 50% for the Xpert BC Monitor. The Xpert BC Monitor missed four tumours (NPV = 98%) in patients with solitary CIS, while potentially avoiding 63.3% of TURBTs in inconclusive or negative cystoscopy and a negative Xpert result. CONCLUSION: Positive urinary cytology may indicate bladder cancer and should be taken seriously. The Xpert BC Monitor may represent a useful diagnostic tool for correctly identifying patients with solitary CIS and unsuspicious or inconclusive cystoscopy.

Performance of fine needle aspiration cytology and Ziehl-Neelsen staining technique in the diagnosis of tuberculosis lymphadenitis.

INTRODUCTION: Proper diagnosis of tuberculosis (TB) lymphadenitis is critical for its treatment and prevention. Fine needle aspirate cytology (FNAC) is the mainstay method for the diagnosis of TB lymphadenitis in Ethiopia; however, the performance of FNAC has not been evaluated in the Eastern Region of Ethiopia. This study aimed to evaluate the performance of FNAC and Ziehl-Neelsen (ZN) staining compared with that of GeneXpert for the diagnosis of TB lymphadenitis. METHODS: Fine needle aspiration (FNA) specimens collected from 291 patients suspected of having TB lymphadenitis were examined using FNAC, ZN, and GeneXpert to diagnose TB lymphadenitis. Gene-Xpert was considered the reference standard method for comparison. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa coefficient were determined using SPSS version 25. RESULTS: The sensitivity, specificity, PPV, and NPV of ZN for diagnosing TB lymphadenitis were 73.2%, 97.4%, 96.2%, and 80.1% respectively. There was poor agreement between ZN and GeneXpert (Kappa=-0.253). The sensitivity, specificity, PPV, and NPV of FNAC were 83.3%, 94.8%, 93.5%, and 86.3% respectively. There was moderate agreement between the FNAC and GeneXpert (Kappa = 0.785). CONCLUSION: The fine needle aspiration cytology (FNAC) is a more sensitive test for the diagnosis of TB lymphadenitis than ZN. The FNAC showed a moderate agreement with the GeneXpert assay. This study recommends the FNA GeneXpert MTB/RIF test in preference to FNAC for the diagnosis of TB lymphadenitis to avoid a missed diagnosis of smear-negative TB lymphadenitis.

Pooling sputum samples for the Xpert MTB/RIF assay: a practical screening strategy for highly infectious tuberculosis cases.

The Xpert MTB/RIF test (Xpert) can help in the accurate screening of tuberculosis, however, its widespread use is limited by its high cost and lack of accessibility. Pooling of sputum samples for testing is a strategy to cut expenses and enhance population coverage but may result in a decrease in detection sensitivity due to the dilution of Mycobacterium tuberculosis (Mtb) by sample mixing. We investigated how the mixing ratio affected the detection performance of Xpert. We used frozen sputum samples that had been kept after individual Xpert assays of the sputa from Mtb-confirmed TB patients and non-TB patients. Our results showed that the overall sensitivity of the Xpert pooling assay remained higher than 80% when the mixing ratio was between 1/2 and 1/8. When the mixing ratio was raised to 1/16, the positive detection rate fell to 69.0%. For patients with either a high sputum Mtb smear score ≥ 2+, a time-to-positive culture ≤ 10 days, or an Xpert test indicating a high or medium abundance of bacteria, the pooling assay positivity rates were 93.3%, 96.8%, and 100% respectively, even at a 1/16 mixing ratio. For participants with cavities and cough, the pooling assay positivity rates were 86.2% and 90.0% at a 1/8 ratio, higher than for those without these signs. Our results show that the Xpert pooled assay has a high overall sensitivity, especially for highly infectious patients. This pooling strategy with lower reagent and labor costs could support TB screening in communities with limited resources, thereby facilitating reductions in the community transmission and incidence of TB worldwide.

Diagnostic accuracy of the Xpert® MTB/XDR assay for detection of Isoniazid and second-line antituberculosis drugs resistance at central TB reference laboratory in Tanzania.

INTRODUCTION: Early diagnosis of tuberculosis (TB) and universal access to drug-susceptibility testing (DST) are critical elements of the WHO End TB Strategy. Current rapid tests (e.g., Xpert® MTB/RIF and Ultra-assays) can detect rifampicin resistance-conferring mutations, but cannot detect resistance to Isoniazid and second-line anti-TB agents. Although Line Probe Assay is capable of detecting resistance to second-line anti-TB agents, it requires sophisticated laboratory infrastructure and advanced skills which are often not readily available in settings replete with TB. A rapid test capable of detecting Isoniazid and second-line anti-TB drug resistance is highly needed. METHODS: We conducted a diagnostic accuracy study to evaluate a new automated Xpert MTB/XDR 10-colour assay for rapid detection of Isoniazid and second-line drugs, including ethionamide, fluoroquinolones, and injectable drugs (Amikacin, Kanamycin, and Capreomycin). Positive Xpert MTB/RIF respiratory specimens were prospectively collected through routine diagnosis and surveillance of drug resistance at the Central TB Reference Laboratory in Tanzania. Specimens were tested by both Xpert XDR assay and LPA against culture-based phenotypic DST as the reference standard. FINDINGS: We analysed specimens from 151 TB patients with a mean age (SD) of 36.2 (12.7) years. The majority (n = 109, 72.2%) were males. The sensitivity for Xpert MTB/XDR was 93.5% (95% CI, 87.4-96.7); for Isoniazid, 96.6 (95% CI, 92.1-98.6); for Fluoroquinolone, 98.7% (95% Cl 94.8-99.7); for Amikacin, 96.6%; and (95% CI 92.1-98.6) for Ethionamide. Ethionamide had the lowest specificity of 50% and the highest was 100% for Fluoroquinolone. The diagnostic performance was generally comparable to that of LPA with slight variations between the two assays. The non-determinate rate (i.e., invalid M. tuberculosis complex detection) of Xpert MTB/XDR was 2·96%. CONCLUSION: The Xpert MTB/XDR demonstrated high sensitivity and specificity for detecting resistance to Isoniazid, Fluoroquinolones, and injectable agents. This assay can be used in clinical settings to facilitate rapid diagnosis of mono-isoniazid and extensively drug-resistant TB.

Diagnostic efficacy of endobronchial ultrasound-guided transbronchoscopic lung biopsy for identifying tuberculous nodules.

BACKGROUND: Microbiological diagnosis of pulmonary tuberculosis (PTB) is hampered by a low pathogen burden, low compliance and unreliable sputum sampling. Although endobronchial ultrasound-guided transbronchoscopic lung biopsy (EBUS-TBLB) has been found to be useful for the assessment of intrapulmonary nodules in adults, few data are available for the clinical diagnosis of pulmonary tuberculosis. Here, we evaluated EBUS-TBLB as a diagnostic procedure in adult patients with radiologically suspected intrapulmonary tuberculous nodules. METHODS: This was a retrospective analysis of patients admitted with pulmonary nodules between January 2022 and January 2023 at Hangzhou Red Cross Hospital. All patients underwent EBUS-TBLB, and lung biopsy samples were obtained during hospitalization. All samples were tested for Mycobacterium tuberculosis using acid‒fast smears, Bactec MGIT 960, Xpert MTB/RIF, next-generation sequencing (NGS), and DNA (TB‒DNA) and RNA (TB‒RNA). The concordance between different diagnostic methods and clinical diagnosis was analysed via kappa concordance analysis. The diagnostic efficacy of different diagnostic methods for PTB was analysed via ROC curve. RESULTS: A total of 107 patients were included in this study. Among them, 86 patients were diagnosed by EBUS-TBLB, and the overall diagnostic rate was 80.37%. In addition, 102 enrolled patients had benign lesions, and only 5 were diagnosed with lung tumours. Univariate analysis revealed that the diagnostic rate of EBUS-TBLB in pulmonary nodules was related to the location of the probe. The consistency analysis and ROC curve analysis revealed that NGS had the highest concordance with the clinical diagnosis results (agreement = 78.50%, κ = 0.558) and had the highest diagnostic efficacy for PTB (AUC = 0.778). In addition, Xpert MTB/RIF + NGS had the highest concordance with the clinical diagnosis results (agreement = 84.11%, κ = 0.667) and had the highest efficacy in the diagnosis of PTB (AUC = 0.826). CONCLUSION: EBUS-TBLB is a sensitive and safe method for the diagnosis of pathological pulmonary nodules. Xpert MTB/RIF combined with NGS had the highest diagnostic efficacy and can be used in the initial diagnosis of PTB.

PneumoniaCheck, a novel aerosol collection device, permits capture of airborne Mycobacterium tuberculosis and characterisation of the cough aeromicrobiome in people with tuberculosis.

BACKGROUND: Tuberculosis (TB), a major cause of disease and antimicrobial resistance, is spread via aerosols. Aerosols have diagnostic potential and airborne-microbes other than Mycobacterium tuberculosis complex (MTBC) may influence transmission. We evaluated whether PneumoniaCheck (PMC), a commercial aerosol collection device, captures MTBC and the aeromicrobiome of people with TB. METHODS: PMC was done in sputum culture-positive people (≥ 30 forced coughs each, n = 16) pre-treatment and PMC air reservoir (bag, corresponding to upper airways) and filter (lower airways) washes underwent Xpert MTB/RIF Ultra (Ultra) and 16S rRNA gene sequencing (sequencing also done on sputum). In a subset (n = 6), PMC microbiota (bag, filter) was compared to oral washes and bronchoalveolar lavage fluid (BALF). FINDINGS: 54% (7/13) bags and 46% (6/14) filters were Ultra-positive. Sequencing read counts and microbial diversity did not differ across bags, filters, and sputum. However, microbial composition in bags (Sphingobium-, Corynebacterium-, Novosphingobium-enriched) and filters (Mycobacterium-, Sphingobium-, Corynebacterium-enriched) each differed vs. sputum. Furthermore, sequencing only detected Mycobacterium in bags and filters but not sputum. In the subset, bag and filter microbial diversity did not differ vs. oral washes or BALF but microbial composition differed. Bags vs. BALF were Sphingobium-enriched and Mycobacterium-, Streptococcus-, and Anaerosinus-depleted (Anaerosinus also depleted in filters vs. BALF). Compared to BALF, none of the aerosol-enriched taxa were enriched in oral washes or sputum. INTERPRETATION: PMC captures aerosols with Ultra-detectable MTBC and MTBC is more detectable in aerosols than sputum by sequencing. The aeromicrobiome is distinct from sputum, oral washes and BALF and contains differentially-enriched lower respiratory tract microbes.

Evaluation of targeted sequencing for pathogen identification in bone and joint infections: a cohort study from China.

PURPOSE: Bone and joint tuberculosis (BJTB) is a distinct variant of tuberculosis in which clinical diagnosis often leads to relative misdiagnosis and missed diagnoses. This study aimed to evaluate the diagnostic accuracy of the targeted nanopore sequencing (TNPseq) assay for BJTB patients in China. METHOD: The study enrolled a cohort of 163 patients with suspected BJTB. Diagnostic testing was performed using the TNPseq assay on samples including punctured tissue, pus, and blood. The diagnostic accuracy of the TNPseq assay was then compared with that of the T-SPOT and Xpert MTB/RIF assays. RESULT: TNPseq exhibited superior performance in terms of accuracy, demonstrating a sensitivity of 76.3% (95% CI: 71.0-81.6%) and a specificity of 98.8% (95% CI: 93.5-100%) in clinical diagnosis. When evaluated against a composite reference standard, TNPseq demonstrated a sensitivity of 74.4% (95% CI: 69.3-79.5%) and a specificity of 98.8% (95% CI: 93.7-100%). These results exceed the performance of both the T-SPOT and Xpert MTB/RIF tests. Notably, TNPseq demonstrated high specificity and accuracy in puncture specimens, with a sensitivity of 75.0% (95% CI: 70.2-79.8%) and a specificity of 98.3% (95% CI: 92.7-100%), as well as in pus samples, with a sensitivity of 83.3% (95% CI: 78.6-88.1%) and a specificity of 100% (95% CI: 100-100%). Additionally, TNPseq facilitated the detection of mixed infection scenarios, identifying 20 cases of bacterial-fungal co-infection, 17 cases of bacterial-viral co-infection, and two cases of simultaneous bacterial-fungal-viral co-infection. CONCLUSION: TNPseq demonstrated great potential in the diagnosis of BJTB due to its high sensitivity and specificity. The ability of TNPseq to diagnose pathogens and detect drug resistance genes can also guide subsequent treatment. Expanding the application scenarios and scope of TNPseq will enable it to benefit more clinical treatments.

Establishment and evaluation of a new fluorescent probe method based on loop-mediated isothermal amplification for the detection of Mycobacterium tuberculosis complex.

We aimed to develop a novel diagnostic method called multiplex fluorescence of loop primer upon self-dequenching loop-mediated isothermal amplification (mFLOS-LAMP) for the rapid detection of Mycobacterium tuberculosis complex (MTBC). A set of specific primers was designed to target the detection of IS1081 and IS6110 genes, which are insertion sequences within the MTBC. The 110 sputum specimens collected were assessed using the established mFLOS-LAMP method, multiplex polymerase chain reaction, Xpert MTB/RIF, and smear microscopy. The optimal reaction temperature and duration for mFLOS-LAMP were determined to be 65°C and 30 min, respectively, by optimizing the entire system. The detection sensitivity of mFLOS-LAMP was 6.0 × 101 CFU/mL, by Bacillus Calmette-Guerin, and the mFLOS-LAMP sensitivity of M. tuberculosis H37Rv genomic DNA was 500 fg, and the specificity was 100%. The sensitivity of mFLOS-LAMP was 94.2% and the specificity was 96.6%, when Xpert MTB/RIF was used as the reference method. There was no statistically significant difference in their detection rate (χ2 = 0, P = 1.000), and the consistency was good (kappa = 0.909, P < 0.001). The receiver operating characteristic analysis yielded the maximum area under the curve of 0.954. The mFLOS-LAMP method demonstrated high sensitivity and specificity, allowing for swift and accurate detection of MTBC.

Yield of Facility-based Targeted Universal Testing for Tuberculosis With Xpert and Mycobacterial Culture in High-Risk Groups Attending Primary Care Facilities in South Africa.

BACKGROUND: We report the yield of targeted universal tuberculosis (TB) testing of clinic attendees in high-risk groups. METHODS: Clinic attendees in primary healthcare facilities in South Africa with one of the following risk factors underwent sputum testing for TB: human immunodeficiency virus (HIV), contact with a TB patient in the past year, and having had TB in the past 2 years. A single sample was collected for Xpert-Ultra (Xpert) and culture. We report the proportion positive for Mycobacterium tuberculosis. Data were analyzed descriptively. The unadjusted clinical and demographic factors' relative risk of TB detected by culture or Xpert were calculated and concordance between Xpert and culture is described. RESULTS: A total of 30 513 participants had a TB test result. Median age was 39 years, and 11 553 (38%) were men. The majority (n = 21734, 71%) had HIV, 12 492 (41%) reported close contact with a TB patient, and 1573 (5%) reported prior TB. Overall, 8.3% were positive for M. tuberculosis by culture and/or Xpert compared with 6.0% with trace-positive results excluded. In asymptomatic participants, the yield was 6.7% and 10.1% in symptomatic participants (with trace-positives excluded). Only 10% of trace-positive results were culture-positive. We found that 55% of clinic attendees with a sputum result positive for M. tuberculosis did not have a positive TB symptom screen. CONCLUSIONS: A high proportion of clinic attendees with specific risk factors (HIV, close TB contact, history of TB) test positive for M. tuberculosis when universal testing is implemented.

Point-of-Care Urine Lipoarabinomannan Testing to Guide Tuberculosis Treatment Among Severely Ill Inpatients With Human Immunodeficiency Virus in Real-World Practice: A Multicenter Stepped Wedge Cluster-Randomized Trial From Ghana.

BACKGROUND: The lateral flow urine lipoarabinomannan assay, Determine TB LAM (Determine LAM), offers the potential for timely tuberculosis (TB) treatment among people with human immunodeficiency virus (PWH). METHODS: In this cluster-randomized trial, Determine LAM was made available with staff training with performance feedback at 3 hospitals in Ghana. Newly admitted PWH with a positive World Health Organization four-symptom screening for TB, severe illness, or advanced HIV were enrolled. The primary outcome was days from enrollment to TB treatment initiation. We also reported the proportion of patients with a TB diagnosis, initiating TB treatment, all-cause mortality, and Determine LAM uptake at 8 weeks. RESULTS: We enrolled 422 patients including 174 (41.2%) in the intervention group. The median CD4 count was 87 (interquartile range [IQR], 25-205) cells/µL, and 32.7% were on antiretroviral therapy. More patients were diagnosed with TB in the intervention compared with the control group: 59 (34.1%) versus 46 (18.7%) (P < .001). Time to TB treatment remained constant, but patients were more likely to initiate TB treatment (adjusted hazard ratio, 2.19 [95% CI, 1.60-3.00]) during the intervention. Of patients with a Determine LAM test available, 41 (25.3%) tested positive. Of those, 19 (46.3%) initiated TB treatment. Overall, 118 patients had died (28.2%) at 8 weeks of follow-up. CONCLUSIONS: The Determine LAM intervention in real-world practice increased TB diagnosis and the probability of TB treatment but did not reduce time to treatment initiation. Despite high uptake, only half of the LAM-positive patients initiated TB treatment.

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

BACKGROUND: The 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). METHODS: A 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. RESULTS: For 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. CONCLUSION: The 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.

To Test or Not? Xpert MTB/RIF as an Alternative to Smear Microscopy to Guide Line Probe Assay Testing for Drug-Resistant Tuberculosis.

Xpert MTB/RIF (Xpert) revolutionized tuberculosis (TB) diagnosis. Laboratory decision making on whether widely-used reflex drug susceptibility assays (MTBDRplus, first-line resistance; MTBDRsl, second-line) are conducted is based on smear status, with smear-negative specimens often excluded. We performed receiver operator characteristic (ROC) curve analyses using bacterial load information (smear microscopy grade, Xpert-generated semi-quantitation categories and minimum cycle threshold [CTmin] values) from Xpert rifampicin-resistant sputum for the prediction of downstream line probe assay results as "likely non-actionable" (no resistance or susceptible results generated). We evaluated actionable-to-non-actionable result ratios and pay-offs with missed resistance versus LPAs done universally. Smear-negatives were more likely than smear-positive specimens to generate a non-actionable MTBDRplus (23% [133/559] versus 4% [15/381]) or MTBDRsl (39% [220/559] versus 12% [47/381]) result. However, excluding smear-negatives would result in missed rapid diagnoses (e.g., only 49% [264/537] of LPA-diagnosable isoniazid resistance would be detected if smear-negatives were omitted). Testing smear-negatives with a semi-quantitation category ≥ "medium" had a high ratio of actionable-to-non-actionable results (12.8 or a 4-fold improvement versus testing all using MTBDRplus, 4.5 or 3-fold improvement for MTBDRsl), which would still capture 64% (168/264) and 77% (34/44) of LPA-detectable smear-negative resistance, respectively. Use of CTmins permitted optimization of this ratio with higher specificity for non-actionable results but decreased resistance detected. Xpert quantitative information permits identification of a smear-negative subset in whom the payoffs of the ratio of actionable-to-non-actionable LPA results with missed resistance may prove acceptable to laboratories, depending on context. Our findings permit the rational expansion of direct DST to certain smear-negative sputum specimens.

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

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

Evaluation of NG-Test CARBA 5 version 2, Cepheid Xpert Carba-R, and carbapenem inactivation methods in comparison to whole-genome sequencing for the identification of carbapenemases in non-fermenting Gram-negative bacilli.

NG-Test CARBA 5 (NG-Biotech) is a rapid in vitro multiplex immunoassay for the phenotypic detection and differentiation of the "big five" carbapenemase families (KPC, OXA-48-like, VIM, IMP, and NDM). Version 2 of this assay was evaluated alongside the Xpert Carba-R assay (Cepheid, Inc.), the modified carbapenem inactivation method (mCIM), and the CIMTris assay, with a collection of carbapenem-resistant non-fermenting Gram-negative bacilli comprising 138 Pseudomonas aeruginosa and 97 Acinetobacter baumannii isolates. Whole-genome sequencing (WGS) was used as the reference standard. For P. aeruginosa, NG-Test CARBA 5 produced an overall percentage agreement (OPA) with WGS of 97.1%, compared with 92.8% forXpert Carba-R and 90.6% for mCIM. For A. baumannii, as OXA-type carbapenemases (non-OXA-48) are not included, both the NG-Test CARBA 5 and Xpert Carba-R only had an OPA of 6.2%, while the CIMTris performed well with an OPA of 99.0%. The majority of A. baumannii isolates (95.9%) tested falsely positive for IMP on NG-Test CARBA 5; no IMP genes were found on WGS. No clear cause was found for this phenomenon; a cross-reacting protein antigen unique to A. baumannii is a possible culprit. NG-Test CARBA 5 performed well for carbapenemase detection in P. aeruginosa. However, results from A. baumannii isolates should be interpreted with caution.

A mutation responsible for impaired detection by the Xpert SARS-CoV-2 assay independently emerged in different lineages during the SARS-CoV-2 pandemic.

BACKGROUND: COVID-19 diagnosis lies on the detection of SARS-CoV-2 on nasopharyngeal specimens by RT-PCR. The Xpert-Xpress SARS-CoV-2 assay provides results in less than one hour from specimen reception, which makes it suitable for clinical/epidemiological circumstances that require faster responses. The analysis of a COVID-19 outbreak suspected in the neonatology ward from our institution showed that the Ct values obtained for the targeted genes in the Xpert assay were markedly different within each specimen (N Ct value > 20 cycles above the E Ct value). RESULTS: We identified the mutation C29200T in the N gene as responsible for an impairment in the N gene amplification by performing whole genome sequencing of the specimens involved in the outbreak (Omicron variant). Subsequently, a retrospective analysis of all specimens sequenced in our institution allowed us to identify the same SNP as responsible for similar impairments in another 12 cases (42% of the total cases reported in the literature). Finally, we found that the same SNP emerged in five different lineages independently, throughout almost all the COVID-19 pandemic. CONCLUSIONS: We demonstrated for the first time the impact of this SNP on the Xpert assay, when harbored by new Omicron variants. We extend our observation period throughout almost all the COVID-19 pandemic, offering the most updated observations of this phenomenon, including sequences from the seventh pandemic wave, until now absent in the reports related to this issue. Continuous monitoring of emerging SNPs that could affect the performance of the most commonly used diagnostic tests, is required to redesign the tests to restore their correct performance.

Differentiating gastrointestinal tuberculosis and Crohn's disease- a comprehensive review.

Gastrointestinal Tuberculosis (GITB) and Crohn's disease (CD) are both chronic granulomatous diseases with a predilection to involve primarily the terminal ileum. GITB is often considered a disease of the developing world, while CD and inflammatory bowel disease are considered a disease of the developed world. But in recent times, the epidemiology of both diseases has changed. Differentiating GITB from CD is of immense clinical importance as the management of both diseases differs. While GITB needs anti-tubercular therapy (ATT), CD needs immunosuppressive therapy. Misdiagnosis or a delay in diagnosis can lead to catastrophic consequences. Most of the clinical features, endoscopic findings, and imaging features are not pathognomonic for either of these two conditions. The definitive diagnosis of GITB can be clinched only in a fraction of cases with microbiological positivity (acid-fast bacilli, mycobacterial culture, or PCR-based tests). In most cases, the diagnosis is often based on consistent clinical, endoscopic, imaging, and histological findings. Similarly, no single finding can conclusively diagnose CD. Multiparametric-based predictive models incorporating clinical, endoscopy findings, histology, radiology, and serology have been used to differentiate GITB from CD with varied results. However, it is limited by the lack of validation studies for most such models. Many patients, especially in TB endemic regions, are initiated on a trial of ATT to see for an objective response to therapy. Early mucosal response assessed at two months is an objective marker of response to ATT. Prolonged ATT in CD is recognized to have a fibrotic effect. Therefore, early discrimination may be vital in preventing the delay in the diagnosis of CD and avoiding a complicated course.

Challenges with the use of Xpert HPV as a screening tool for oral HPV among people living with HIV (PLHIV): experiences from Pune, India.

BACKGROUND: People living with HIV (PLHIV) are at higher risk for human papillomavirus (HPV)-related oropharyngeal cancers compared to the general population. Xpert HPV test is a polymerase chain reaction (PCR) assay capable of rapid HPV detection. Performing the assay requires minimal intervention by laboratory personnel. Its use could improve oropharyngeal cancer screening among PLHIV living in low-and middle-income countries (LMICs) with limited diagnostic capacities. However, Xpert HPV performance for oral samples has not been evaluated. Here, we describe our experience with Xpert HPV and compare its results with traditional PCR, for oral samples. METHODS: Oral samples from 429 PLHIV receiving care at a tertiary care hospital affiliated antiretroviral therapy center in Pune, India were used. Samples were collected either after a 30s oral rinse and gargle (n = 335) or in combination with cytobrush scraping of the oral mucosa (n = 91). Unsuccessful tests were those that generated an invalid or error result on Xpert HPV. Successful tests were those that generated a positive or negative result. Kappa statistic was used to compare concordance between Xpert HPV and traditional real-time PCR results. RESULTS: There were 29.8% (n = 127) unsuccessful tests, of which 78.7% (n = 100) were invalid and 21.3% (n = 27) were error results. Adding cytobrush scraping to oral rinse as a collection procedure did not significantly reduce the proportion of unsuccessful tests (p = 0.9). For successful tests, HPV positivity on Xpert was 0.3% (n = 1/299). Kappa statistic was 0.11, indicating poor agreement between Xpert HPV and traditional PCR results. CONCLUSIONS: Presently, Xpert HPV appears to have limited use for oral HPV detection among PLHIV using oral samples. More research to improve the diagnostic capabilities of Xpert HPV for oral samples among PLHIV is needed.