Utility of Second-Generation Line Probe Assay (Hain MTBDRplus) Directly on 2-Month Sputum Specimens for Monitoring Tuberculosis Treatment Response.

The utility of a line probe assay (Genotype MTBDRplus) performed directly on 2-month sputa to monitor tuberculosis treatment response is unknown. We assessed if direct testing of 2-month sputa with MTBDRplus can predict 2-month culture conversion and long-term treatment outcome. Xpert MTB/RIF-confirmed rifampin-susceptible tuberculosis cases were recruited at tuberculosis diagnosis and followed up at 2 and 5 to 6 months. MTBDRplus was performed directly on 2-month sputa and on all positive cultured isolates at 2 and 5 to 6 months. We also investigated the association of a positive direct MTBDRplus at 2 months with subsequent unsuccessful tuberculosis treatment outcome (failure/death during treatment or subsequent disease recurrence). A total of 279 patients (62% of whom were HIV-1 coinfected) were recruited. Direct MTBDRplus at 2 months had a sensitivity of 78% (95% confidence interval [CI], 65 to 87) and specificity of 80% (95% CI, 74 to 84) to predict culture positivity at 2 months with a high negative predictive value of 93% (95% CI, 89 to 96). Inconclusive genotypic susceptibility results for both rifampin and isoniazid were seen in 26% of MTBDRplus tests performed directly on sputum. Compared to a reference of MTBDRplus performed on positive cultures, the false-positive resistance rate for direct testing of MTBDRplus on sputa was 4% for rifampin and 2% for isoniazid. While a positive 2-month smear was not significantly associated with an unsuccessful treatment outcome (adjusted odds ratio [aOR], 2.69; 95% CI, 0.88 to 8.21), a positive direct MTBDRplus at 2 months was associated with an unsuccessful outcome (aOR 2.87; 95% CI, 1.11 to 7.42). There is moderate utility of direct 2-month MTBDRplus to predict culture conversion at 2 months and also to predict an unfavorable outcome.

Identification of nontuberculous mycobacteria using the GenoType CMdirect and GenoType Mycobacterium CM line probe assays in GeneXpert MTB/RIF Ultra negative sputum: A South African prevalence study.

The GenoType CMdirect Ver 1.0 and GenoType Mycobacterium Common Mycobacteria Ver 2.0-line probe assays were utilized to identify nontuberculous mycobacteria (NTM) from decontaminated sputum in a GeneXpert MTB/RIF Ultra negative cohort. A preliminary NTM prevalence of 1.7 % was observed, with a 100 % specificity. The high specificity advocates the CMdirect as a possible rule-in test for NTM disease.

Diagnostic accuracy of LiquidArray MTB-XDR VER1.0 for the detection of Mycobacterium tuberculosis complex, fluoroquinolone, amikacin, ethambutol, and linezolid susceptibility.

Drug susceptibility testing (DST) is essential for effectively starting people on effective tuberculosis (TB) regimens. No accuracy data exists for the new high-throughput LiquidArray MTB-XDR (LA-XDR) test, which detects Mycobacterium tuberculosis complex (MTBC) and susceptibility to the fluoroquinolones, amikacin, ethambutol, and linezolid (the latter two drugs have no rapid molecular DSTs available). We enrolled (n=720) people with presumptive TB who provided two sputa for Xpert MTB/RIF Ultra and culture (MTBC reference standard). Phenotypic DST and Sanger sequencing served as a composite reference standard. Manual FluoroLyse and automated GenoXtract-fleXT (fleXT) DNA extraction methods were compared. For MTBC, LA-XDR using fleXT-extracted or FluoroLyse-extracted DNA had similar sensitivities (85-87%; which improved upon eluate retesting) and specificities (99%). Drug susceptibility sensitivities varied: 94% (86, 98) for fluoroquinolones, 64% (45, 80) for amikacin, and 88% (79, 93) for ethambutol (specificities 97-100%). LA-XDR detected 86% (6/7) phenotypically resistant linezolid isolates. LA-XDR with fleXT had indeterminate proportions of 8% (21/251) for fluoroquinolones, 1% (2/251) for ethambutol, 25% (63/251) for amikacin, and 37% (93/251) for linezolid. In a hypothetical population of 100 smear-negative fluoroquinolones-resistant cases, 24% (24/100) could be missed due to an unsuccessful result (1 fleXT error and, for LA-XDR, 2 invalid results, 15 MTBC-negative, 6 fluoroquinolone-indeterminate, 1 false-susceptible). LA-XDR met the minimum WHO target product profile for a next-generation sputum-based moderate complexity DST with high sensitivity for fluoroquinolones and ethambutol resistance, moderate sensitivity for amikacin resistance, and promise for linezolid resistance, for which more data are needed. Improved MTBC detection would reduce missed resistance.

Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures.

Objectives: Nontuberculous mycobacteria (NTM) infections present a global health challenge. This study describes unidentified mycobacteria species from extrapulmonary sites, using advanced identification and sequencing techniques. Methods: Extrapulmonary mycobacteria growth indicator tube primary cultures collected retrospectively between 2019 and 2023, featuring unidentified mycobacteria species detected by GenoType Mycobacterium line probe assays, underwent multilocus targeted next-generation sequencing using Oxford Nanopore Technology, polymerase chain reaction amplicon Sanger sequencing, and Deeplex Myc-TB analysis. Previously collected clinical and laboratory data were reported. Results: A total of 28 cultures, collected from extrapulmonary sites, each from different patients, were included. Mycobacterial mixtures were identified in 19 of 28 (68%) cultures, with four of 28 (14%) showing unidentified species based on sequencing of rpoB and hsp65 targets. Mycobacterium monacense was present in 13 of 28 (46%) of the cultures. Culturable Mycobacterium tuberculosis complex (MTBC) was identified in five extrapulmonary specimens that previously tested negative for MTBC using Xpert MTB/RIF Ultra. The comparative analysis between Sanger and targeted next-generation sequencing using Oxford Nanopore Technology sequencing (for hsp65) demonstrated 27 of 28 (96%) agreement on the predominant strain. Deeplex Myc-TB could not identify NTM-MTBC co-infections in minor subpopulations. Conclusions: This study highlights the role of advanced sequencing in identifying NTM mixtures and mycobacterial co-infections. It calls for ongoing efforts to integrate next-generation sequencing into mycobacteria testing algorithms.

Intensive Care Unit Sluice Room Sinks as Reservoirs and Sources of Potential Transmission of Carbapenem-Resistant Bacteria in a South African Tertiary Care Hospital.

Purpose: Carbapenem-resistant bacteria (CRB) pose a major health risk to patients in intensive care units (ICU) across African hospitals. There are hardly any data about the role of hospital sinks as reservoirs of CRB in resource-poor African settings. Furthermore, the specific within-sink location of the highest concentration of pathogens and the role of splash back as a transmission mechanism remains poorly clarified. Methods: We swabbed ICU sluice room sinks in a tertiary hospital in Cape Town, South Africa. Swabs were taken from four different parts of the sluice room sinks (tap-opening, trap, below the trap, and u-bend). Dilutions were prepared and plated on carbapenem-infused agar. Colonies were identified and drug resistance profiles were determined using a biochemical analyser. To evaluate the potential transmission from the sink, similar plates were placed at fixed distances from the sink when the tap was turned on and off. Results: CRB were isolated from the trap, water interface below the trap, and the u-bend (the latter harboured the highest density of CRB species). Five CRB, resistant to at least 7 antibiotic classes, were isolated including Pseudomonas, Klebsiella, Citrobacter, Serratia, and Providencia. CRB could be cultured from droplets that fell on agar-containing plates placed at a varying distance from the trap. Conclusion: There is a higher density of CRB in the u-bend of ICU sluice room sinks which can act as a potential source of transmission. The data inform targeted CRB transmission-interruption strategies in resource-poor settings.

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

BACKGROUND: Xpert MTB/RIF Ultra (Ultra) is a widely used rapid front-line tuberculosis and rifampicin-susceptibility testing. Mycobacterium Growth Indicator Tube (MGIT) 960 liquid culture is used as an adjunct but is vulnerable to contamination. We aimed to assess whether Ultra can be used on to-be-discarded contaminated cultures. METHODS: We stored contaminated MGIT960 tubes (growth-positive, acid-fast bacilli [AFB]-negative) originally inoculated at a high-volume laboratory in Cape Town, South Africa, to diagnose patients with presumptive pulmonary tuberculosis. Patients who had no positive tuberculosis results (smear, Ultra, or culture) at contamination detection and had another, later specimen submitted within 3 months of the contaminated specimen were selected. We evaluated the sensitivity and specificity of Ultra on contaminated growth from the first culture for tuberculosis (next-available non-contaminated culture result reference standard) and rifampicin resistance (vs MTBDRplus on a later isolate). We calculated potential time-to-diagnosis improvements and also evaluated the immunochromatographic MPT64 TBc assay. FINDINGS: Between June 1 and Aug 31, 2019, 36 684 specimens from 26 929 patients were processed for diagnostic culture. 2402 (7%) cultures from 2186 patients were contaminated. 1068 (49%) of 2186 patients had no other specimen submitted. After 319 exclusions, there were 799 people with at least one repeat specimen submitted; of these, we included in our study 246 patients (31%) with a culture-positive repeat specimen and 429 patients (54%) with a culture-negative repeat specimen. 124 patients (16%) with a culture-contaminated repeat specimen were excluded. When Ultra was done on the initial contaminated growth, sensitivity was 89% (95% CI 84-94) for tuberculosis and 95% (75-100) for rifampicin-resistance detection, and specificity was 95% (90-98) for tuberculosis and 98% (93-100) for rifampicin-resistance detection. If our approach were used the day after contamination detection, the time to tuberculosis detection would improve by a median of 23 days (IQR 13-45) and provide a result in many patients who had none. MPT64 TBc had a sensitivity of 5% (95% CI 0-25). INTERPRETATION: Ultra on AFB-negative growth from contaminated MGIT960 tubes had high sensitivity and specificity, approximating WHO criteria for sputum test target product performance and exceeding drug susceptibility testing. Our approach could mitigate negative effects of culture contamination, especially when repeat specimens are not submitted. FUNDING: The European & Developing Countries Clinical Trials Partnership, National Institutes of Health.

Characteristics and early outcomes of patients with Xpert MTB/RIF-negative pulmonary tuberculosis diagnosed during screening before antiretroviral therapy.

BACKGROUND: A proportion of patients with tuberculosis diagnosed by sputum culture during screening before antiretroviral therapy (ART) have false-negative Xpert MTB/RIF assay results (Xpert-negative tuberculosis). We determined the characteristics and early ART program outcomes of such patients. METHODS: Adult patients who enrolled in a South African township ART service were systematically screened for pulmonary tuberculosis regardless of symptoms by testing paired sputum samples with Xpert MTB/RIF and liquid culture. The ART service provided follow-up for all patients, and early (90-day) programmatic outcomes were determined. RESULTS: Among 602 patients screened, 523 had ≥1 Xpert and culture result, yielding 89 culture-positive tuberculosis diagnoses. Of these, 37 (42%) of the patients with tuberculosis were Xpert-negative when a single sputum sample was tested, compared with 25 (28%) when 2 samples were tested. Compared with patients with Xpert-positive tuberculosis, those with Xpert-negative tuberculosis (using either definition) had substantially higher CD4 cell counts, lower plasma viral loads, higher hemoglobin concentrations, and higher body mass index. Their tuberculosis was also less advanced, with a lower frequency of prolonged cough (≥2 weeks), less extensive radiographic abnormalities, and a lower frequency of detectable lipoarabinomannan antigenuria and mycobacteriuria. Xpert-negative cases were all sputum smear negative with prolonged time to culture positivity (median, 21 days). Despite greater delays in starting tuberculosis treatment, Xpert-negative patients were less likely to die during follow-up. CONCLUSIONS: Compared to patients with Xpert-positive tuberculosis diagnosed during pre-ART screening, Xpert-negative cases had less advanced immunosuppression and less advanced tuberculosis and did not have adverse outcomes despite substantial delays in starting tuberculosis treatment.

Frequent Suboptimal Thermocycler Ramp Rate Usage Negatively Impacts GenoType MTBDRsl VER 2.0 Performance for Second-Line Drug-Resistant Tuberculosis Diagnosis.

Strengthening second-line drug-resistant tuberculosis (TB) detection is a priority. GenoType MTBDRplus VER 2.0 performance is reduced with non-recommended ramp rate usage (temperature change speed between PCR cycles); however, ramp rate's effect on GenoType MTBDRsl VER 2.0 (MTBDRsl) performance, is unknown. Fifty-two Xpert MTB/RIF Ultra-positive rifampicin-resistant smear-negative sputa and a Mycobacterium tuberculosis dilution series were tested at a manufacturer-recommended (2.2°C/second) or suboptimal (4.0°C/second) ramp rate. M. tuberculosis-complex-DNA positivity, indeterminates, fluoroquinolone- and second-line injectable-resistance accuracy, banding differences, and, separately, inter-reader variability were assessed. Five (39%) of 13 re-surveyed laboratories did not use the manufacturer-recommended ramp rate. On sputum, 2.2°C/second improved indeterminates versus 4.0°C/second (0 of 52 versus 7 of 51; P = 0.006), incorrect drug-class diagnostic calls (0 of 104 versus 6 of 102; P = 0.013), and incorrect banding calls (0 of 1300 versus 54 of 1275; P < 0.001). Similarly, 2.2°C/second improved valid results [(52 of 52 versus 41 of 51; +21% (P = 0.001)] and banding call inter-reader variability [34 of 1300 (3%) versus 52 of 1300 (4%); P = 0.030]. At the suboptimal ramp rate, false-resistance and false-susceptible calls resulted from wild-type band absence rather than mutant band appearance, resulting in misclassification of moxifloxacin resistance level from high-to-low. Suboptimal ramp rate contributes to poor MTBDRsl performance. Laboratories must ensure that the manufacturer-recommended ramp rate is used.

Microbiological diagnosis of pulmonary tuberculosis in children by oral swab polymerase chain reaction.

Microbiological diagnosis of pediatric pulmonary tuberculosis (TB) is challenging due to the difficulty of collecting and testing sputum from children. We investigated whether easily-obtained oral swab samples are useful alternatives or supplements to sputum. Oral swabs and induced sputum (IS) were collected from 201 South African children with suspected pulmonary TB. IS samples were tested by mycobacterial culture and Xpert MTB/RIF. Oral swabs were tested by PCR targeting IS6110. Children were categorized as Confirmed TB (microbiologic confirmation on IS), Unconfirmed TB (clinical diagnosis only), or Unlikely TB (recovery without TB treatment). Relative to Confirmed TB, PCR on two oral swabs per child was 43% sensitive and 93% specific. This sensitivity fell below that of sputum Xpert (64%). Among children with either Confirmed or Unconfirmed TB, PCR on two oral swabs per child was 31% sensitive and 93% specific, which was more sensitive than sputum testing among this group (21%). Although oral swab analysis had low sensitivity in sputum-positive children, it detected TB in a significant proportion of sputum-negative children who were clinically diagnosed with TB. Specificity at 93% was suboptimal but may improve with the use of automated methods. With further development, oral swabs may become useful supplements to sputum as samples for diagnosis of pulmonary TB in children.

Accuracy of Xpert Mtb/Rif Ultra for the Diagnosis of Pulmonary Tuberculosis in Children.

We determined the accuracy of the new Xpert MTB/RIF Ultra assay for diagnosis of pulmonary tuberculosis in children, using banked-induced sputum. Sensitivity and specificity were 75.3% and 96.9%, respectively, compared with a culture result from the same specimen. Of those children with microbiologically confirmed tuberculosis (positive Xpert, Ultra or culture), Xpert detected 63.2%, Ultra 73.7% and culture 82.9%.

Xpert MTB/RIF Ultra for detection of Mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre diagnostic accuracy study.

BACKGROUND: The Xpert MTB/RIF assay is an automated molecular test that has improved the detection of tuberculosis and rifampicin resistance, but its sensitivity is inadequate in patients with paucibacillary disease or HIV. Xpert MTB/RIF Ultra (Xpert Ultra) was developed to overcome this limitation. We compared the diagnostic performance of Xpert Ultra with that of Xpert for detection of tuberculosis and rifampicin resistance. METHODS: In this prospective, multicentre, diagnostic accuracy study, we recruited adults with pulmonary tuberculosis symptoms presenting at primary health-care centres and hospitals in eight countries (South Africa, Uganda, Kenya, India, China, Georgia, Belarus, and Brazil). Participants were allocated to the case detection group if no drugs had been taken for tuberculosis in the past 6 months or to the multidrug-resistance risk group if drugs for tuberculosis had been taken in the past 6 months, but drug resistance was suspected. Demographic information, medical history, chest imaging results, and HIV test results were recorded at enrolment, and each participant gave at least three sputum specimen on 2 separate days. Xpert and Xpert Ultra diagnostic performance in the same sputum specimen was compared with culture tests and drug susceptibility testing as reference standards. The primary objectives were to estimate and compare the sensitivity of Xpert Ultra test with that of Xpert for detection of smear-negative tuberculosis and rifampicin resistance and to estimate and compare Xpert Ultra and Xpert specificities for detection of rifampicin resistance. Study participants in the case detection group were included in all analyses, whereas participants in the multidrug-resistance risk group were only included in analyses of rifampicin-resistance detection. FINDINGS: Between Feb 18, and Dec 24, 2016, we enrolled 2368 participants for sputum sampling. 248 participants were excluded from the analysis, and 1753 participants were distributed to the case detection group (n=1439) and the multidrug-resistance risk group (n=314). Sensitivities of Xpert Ultra and Xpert were 63% and 46%, respectively, for the 137 participants with smear-negative and culture-positive sputum (difference of 17%, 95% CI 10 to 24); 90% and 77%, respectively, for the 115 HIV-positive participants with culture-positive sputum (13%, 6·4 to 21); and 88% and 83%, respectively, across all 462 participants with culture-positive sputum (5·4%, 3·3 to 8·0). Specificities of Xpert Ultra and Xpert for case detection were 96% and 98% (-2·7%, -3·9 to -1·7) overall, and 93% and 98% for patients with a history of tuberculosis. Xpert Ultra and Xpert performed similarly in detecting rifampicin resistance. INTERPRETATION: For tuberculosis case detection, sensitivity of Xpert Ultra was superior to that of Xpert in patients with paucibacillary disease and in patients with HIV. However, this increase in sensitivity came at the expense of a decrease in specificity. FUNDING: Government of Netherlands, Government of Australia, Bill & Melinda Gates Foundation, Government of the UK, and the National Institute of Allergy and Infectious Diseases.

Urine lipoarabinomannan testing for diagnosis of pulmonary tuberculosis in children: a prospective study.

BACKGROUND: Urine tests for mycobacterial lipoarabinomannan might be useful for point-of-care diagnosis of tuberculosis in adults with advanced HIV infection, but have not been assessed in children. We assessed the accuracy of urine lipoarabinomannan testing for the diagnosis of pulmonary tuberculosis in HIV-positive and HIV-negative children. METHODS: We prospectively recruited children (aged ≤ 15 years) who presented with suspected tuberculosis at a primary health-care clinic and paediatric referral hospital in South Africa, between March 1, 2009, and April 30, 2012. We assessed the diagnostic accuracy of urine lipoarabinomannan testing with lateral fl ow assay and ELISA, with mycobacterial culture of two induced sputum samples as the reference standard. Positive cultures were identified by acid-fast staining and tested to confirm Mycobacterium tuberculosis and establish susceptibility to rifampicin and isoniazid. FINDINGS: 535 children (median age 42.5 months, IQR 19.1 ­ 66.3) had urine and two induced specimens available for testing. 89 (17%) had culture-confirmed tuberculosis and 106 (20%) had HIV. The lateral fl ow lipoarabinomannan test showed poor accuracy against the reference standard, with sensitivity of 48.3% (95% CI 37.6 ­ 59.2), specificity of 60.8% (56.1 ­ 65.3), and an area under the receiver operating characteristic curve of 0.53 (0.46 ­ 0.60) for children without HIV and 0.64 (0.51 ­ 0.76) for children with HIV. ELISA had poor sensitivity in children without HIV (sensitivity 3.0%, 95% CI 0.4 ­ 10.5) and children with HIV (0%, 0.0 ­ 14.3); overall specificity was 95.7% (93.4 ­ 97.4). INTERPRETATION: Urine lipoarabinomannan tests have insufficient sensitivity and specificity to diagnose HIV-positive and HIV-negative children with tuberculosis and should not be used in this patient population.