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.

Culture filtrate supplementation can be used to improve Mycobacterium tuberculosis culture positivity for spinal tuberculosis diagnosis.

Culture remains the gold standard to diagnose spinal tuberculosis (STB) despite the paucibacillary nature of the disease. Current methods can take up to 42 days to yield a result, delaying the ability to rapidly detect drug resistance. Studies have demonstrated the use of supplementation with culture filtrate (CF) from an axenic culture of Mycobacterium tuberculosis (Mtb) as a source of growth factors to improve culture rates. Our objective was to test a modified culture assay, utilizing CF supplemented media (CFSM), to improve culture positivity rates for suspected STB. Twelve patients with suspected STB were assessed by conventional culture (BACTEC™ MGIT 960), GeneXpert™ and standard histopathological examination. Spinal biopsies were taken from areas of diseased vertebral tissue or abscess, predetermined from MRI. Additional biopsies were obtained to assess CFSM for improved detection and faster culture of Mtb. All cases were diagnosed as STB and treated empirically for tuberculosis based on either bacteriological evidence (GeneXpert™, MGIT and/or CFSM positive), or based on clinical presentation. 5 specimens (45.45%) were positive for Mtb DNA as detected by GeneXpert™ and 1 specimen (8.33%) was cultured using MGIT (time to detection; 18 days). CFSM was able to culture 7 specimens (58.3%), with all CFSM positive specimens yielding a culture within 14 days. Two samples were positive only using the CFSM assay pointing to additional yield for diagnostic workup. Modification of standard culture can improve detection of Mtb and reduce time to positivity in individuals with STB where culture material is a requirement.

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.

Investigating Non-sterilizing Cure in TB Patients at the End of Successful Anti-TB Therapy.

Mycobacterium tuberculosis (Mtb) is extremely recalcitrant to antimicrobial chemotherapy requiring 6 months to treat drug-sensitive tuberculosis (TB). Despite this, 4-10% of cured patients will develop recurrent disease within 12 months after completing therapy. Reasons for relapse in cured TB patients remains speculative, attributed to both pathogen and host factors. Populations of dormant bacilli are hypothesized to cause relapse in initially cured TB patients however, development of tests to convincingly demonstrate their presence at the end of anti-TB treatment has been challenging. Previous studies have indicated the utility of culture filtrate supplemented media (CFSM) to detect differentially culturable tubercle bacilli (DCTB). Here, we show that 3/22 of clinically cured patients retained DCTB in induced sputum and bronchoalveolar lavage fluid (BALF), with one DCTB positive patient relapsing within the first year of completing therapy. We also show a correlation of DCTB status with "unresolved" end of treatment FDG PET-CT imaging. Additionally, 19 end of treatment induced sputum samples from patients not undergoing bronchoscopy were assessed for DCTB, identifying a further relapse case with DCTB. We further show that induced sputum is a less reliable source for the DCTB assay at the end of treatment, limiting the utility of this assay in a clinical setting. We next investigated the host proteome at the site of disease (BALF) using multiplexed proteomic analysis and compared these to active TB cases to identify host-specific factors indicative of cure. Distinct signatures stratified active from cured TB patients into distinct groups, with a DCTB positive, subsequently relapsing, end of treatment patient showing a proteomic signature closer to active TB disease than cure. This exploratory study offers evidence of live Mtb, undetectable with conventional culture methods, at the end of clinically successful treatment and putative host protein biomarkers of active disease and cure. These findings have implications for the assessment of true sterilizing cure in TB patients and opens new avenues for targeted approaches to monitor treatment response.

Bacterial and host determinants of cough aerosol culture positivity in patients with drug-resistant versus drug-susceptible tuberculosis.

A burgeoning epidemic of drug-resistant tuberculosis (TB) threatens to derail global control efforts. Although the mechanisms remain poorly clarified, drug-resistant strains are widely believed to be less infectious than drug-susceptible strains. Consequently, we hypothesized that lower proportions of patients with drug-resistant TB would have culturable Mycobacterium tuberculosis from respirable, cough-generated aerosols compared to patients with drug-susceptible TB, and that multiple factors, including mycobacterial genomic variation, would predict culturable cough aerosol production. We enumerated the colony forming units in aerosols (≤10 µm) from 452 patients with TB (227 with drug resistance), compared clinical characteristics, and performed mycobacterial whole-genome sequencing, dormancy phenotyping and drug-susceptibility analyses on M. tuberculosis from sputum. After considering treatment duration, we found that almost half of the patients with drug-resistant TB were cough aerosol culture-positive. Surprisingly, neither mycobacterial genomic variants, lineage, nor dormancy status predicted cough aerosol culture positivity. However, mycobacterial sputum bacillary load and clinical characteristics, including a lower symptom score and stronger cough, were strongly predictive, thereby supporting targeted transmission-limiting interventions. Effective treatment largely abrogated cough aerosol culture positivity; however, this was not always rapid. These data question current paradigms, inform public health strategies and suggest the need to redirect TB transmission-associated research efforts toward host-pathogen interactions.

Extract from used Xpert MTB/RIF Ultra cartridges is useful for accurate second-line drug-resistant tuberculosis diagnosis with minimal rpoB-amplicon cross-contamination risk.

Xpert MTB/RIF Ultra (Ultra) detects Mycobacterium tuberculosis and rifampicin resistance. Follow-on drug susceptibility testing (DST) requires additional sputum. Extract from the diamond-shaped chamber of the cartridge (dCE) of Ultra's predecessor, Xpert MTB/RIF (Xpert), is useful for MTBDRsl-based DST but this is unexplored with Ultra. Furthermore, whether CE from non-diamond compartments is useful, the performance of FluoroType MTBDR (FT) on  CE, and rpoB cross-contamination risk associated with the extraction procedure are unknown. We tested MTBDRsl, MTBDRplus, and FT on CEs from chambers from cartridges (Ultra, Xpert) tested on bacilli dilution series. MTBDRsl on Ultra dCE on TB-positive sputa (n = 40) was also evaluated and, separately, rpoB amplicon cross-contamination risk . MTBDRsl on Ultra dCE from dilutions ≥103 CFU/ml (CTmin <25, >"low semi-quantitation") detected fluoroquinolone (FQ) and second-line injectable (SLID) susceptibility and resistance correctly (some SLIDs-indeterminate). At the same threshold (at which ~85% of Ultra-positives in our setting would be eligible), 35/35 (100%) FQ and 34/35 (97%) SLID results from Ultra dCE were concordant with sputa results. Tests on other chambers were unfeasible. No tubes open during 20 batched extractions had FT-detected rpoB cross-contamination. False-positive Ultra rpoB results was observed when dCE dilutions ≤10-3 were re-tested. MTBDRsl on Ultra dCE is concordant with isolate results. rpoB amplicon cross-contamination is unlikely. These data mitigate additional specimen collection for second-line DST and cross-contamination concerns.

Direct genotyping of Mycobacterium tuberculosis from Xpert® MTB/RIF remnants.

Genotyping of Mycobacterium tuberculosis (MTB) isolates has markedly improved our knowledge of its transmission dynamics. MIRU-VNTR is considered the reference molecular tool for MTB fingerprinting. However, the dependence of this technique on cultured isolates means that we lack molecular epidemiology data from many settings where culture facilities have not been implemented. Efforts have been made to adapt the MIRU-VNTR procedure to direct analysis of clinical specimens, although implementation of these efforts has not proven successful. The large-scale roll-out of Xpert MTB/RIF (Xpert) technology, which is now in almost every TB-endemic country, including many where MTB is not cultured, provides us with a new opportunity to explore whether MTB genotyping could be performed from the remnants of the Xpert cartridge. We ran a pilot study in Mozambique in which the remnants of 24 positive Xpert assays for detection of MTB were used as template material for the 15-locus or the more discriminatory 24-locus MIRU-VNTR technique. MTB fingerprinting was possible in specimens with a high bacterial burden, according to the Xpert load categories, and within the first week after Xpert was performed. Given the wide availability, simple processing, and rapid reporting of results with Xpert, our findings suggest that MIRU-VNTR-based fingerprinting from remnants of Xpert could play a major role in extending MTB molecular epidemiology studies to settings where information on the transmission dynamics of this pathogen is lacking.

False-Positive Xpert MTB/RIF Results in Retested Patients with Previous Tuberculosis: Frequency, Profile, and Prospective Clinical Outcomes.

Globally, Xpert MTB/RIF (Xpert) is the most widely used PCR test for the diagnosis of tuberculosis (TB). Positive results in previously treated patients, which are due to old DNA or active disease, are a diagnostic dilemma. We prospectively retested sputum from 238 patients, irrespective of current symptoms, who were previously diagnosed to be Xpert positive and treated successfully. Patients who retested as Xpert positive and culture negative were exhaustively investigated (repeat culture, chest radiography, bronchoscopy with bronchoalveolar lavage, long-term clinical follow-up). We evaluated whether the duration since previous treatment completion, mycobacterial burden (the Xpert cycle threshold [CT ] value), and reclassification of Xpert-positive results with a very low semiquantitation level to Xpert-negative results reduced the rate of false positivity. A total of 229/238 (96%) of patients were culture negative. Sixteen of 229 (7%) were Xpert positive a median of 11 months (interquartile range, 5 to 19 months) after treatment completion. The specificity was 93% (95% confidence interval [CI], 89 to 96%). Nine of 15 (40%) Xpert-positive, culture-negative patients reverted to Xpert negative after 2 to 3 months (1 patient declined further participation). Patients with false-positive Xpert results had a lower mycobacterial burden than patients with true-positive Xpert results (CT , 28.7 [95% CI, 27.2 to 30.4] versus 17.6 [95% CI, 16.9 to 18.2]; P < 0.001), an increased likelihood of a chest radiograph not compatible with active TB (5/15 patients versus 0/5 patients; P = 0.026), and less-viscous sputum (15/16 patients versus 2/5 patients whose sputum was graded as mucoid or less; P = 0.038). All patients who initially retested as Xpert positive and culture negative ("Xpert false positive") were clinically well without treatment after follow-up. The duration since the previous treatment poorly predicted false-positive results (a duration of ≤2 years identified only 66% of patients with false-positive results). Reclassifying Xpert-positive results with a very low semiquantitation level to Xpert negative improved the specificity (+3% [95% CI, +2 to +5%]) but reduced the sensitivity (-10% [95% CI, -4 to -15%]). Patients with previous TB retested with Xpert can have false-positive results and thus not require treatment. These data inform clinical practice by highlighting the challenges in interpreting Xpert-positive results, underscore the need for culture, and have implications for next-generation ultrasensitive tests.

Mycobacterial genomic DNA from used Xpert MTB/RIF cartridges can be utilised for accurate second-line genotypic drug susceptibility testing and spoligotyping.

Xpert MTB/RIF (Xpert) is a widely-used test for tuberculosis (TB) and rifampicin-resistance. Second-line drug susceptibility testing (DST), which is recommended by policymakers, typically requires additional specimen collection that delays effective treatment initiation. We examined whether cartridge extract (CE) from used Xpert TB-positive cartridges was, without downstream DNA extraction or purification, suitable for both genotypic DST (MTBDRplus, MTBDRsl), which may permit patients to rapidly receive a XDR-TB diagnosis from a single specimen, and spoligotyping, which could facilitate routine genotyping. To determine the limit-of-detection and diagnostic accuracy, CEs from dilution series of drug-susceptible and -resistant bacilli were tested (MTBDRplus, MTBDRsl). Xpert TB-positive patient sputa CEs (n = 85) were tested (56 Xpert-rifampicin-susceptible, MTBDRplus and MTBDRsl; 29 Xpert-rifampicin-resistant, MTBDRsl). Spoligotyping was done on CEs from dilution series and patient sputa (n = 10). MTBDRplus had high non-valid result rates. MTBDRsl on CEs from dilutions ≥103CFU/ml (CT ≤ 24, >"low" Xpert semiquantitation category) was accurate, had low indeterminate rates and, on CE from sputa, highly concordant with MTBDRsl isolate results. CE spoligotyping results from dilutions ≥103CFU/ml and sputa were correct. MTBDRsl and spoligotyping on CE are thus highly feasible. These findings reduce the need for additional specimen collection and culture, for which capacity is limited in high-burden countries, and have implications for diagnostic laboratories and TB molecular epidemiology.

Xpert MTB/RIF Results in Patients With Previous Tuberculosis: Can We Distinguish True From False Positive Results?

BACKGROUND: Patients with previous tuberculosis may have residual DNA in sputum that confounds nucleic acid amplification tests such as Xpert MTB/RIF. Little is known about the frequency of Xpert-positive, culture-negative ("false positive") results in retreatment patients, whether these are distinguishable from true positives, and whether Xpert's automated filter-based wash step reduces false positivity by removing residual DNA associated with nonintact cells. METHODS: Pretreatment patients (n = 2889) with symptoms of tuberculosis from Cape Town, South Africa, underwent a sputum-based liquid culture and Xpert. We also compared Xpert results from dilutions of intact or heat-lysed and mechanically lysed bacilli. RESULTS: Retreatment cases were more likely to be Xpert false-positive (45/321 Xpert-positive retreatment cases were false-positive) than new cases (40/461) (14% [95% confidence interval {CI}, 10%-18%] vs 8% [95% CI, 6%-12%];P= .018). Fewer years since treatment completion (adjusted odds ratio [aOR], 0.85 [95% CI, .73-.99]), less mycobacterial DNA (aOR, 1.14 [95% CI, 1.03-1.27] per cycle threshold [CT]), and a chest radiograph not suggestive of active tuberculosis (aOR, 0.22 [95% CI, .06-.82]) were associated with false positivity. CThad suboptimal accuracy for false positivity: 46% of Xpert-positives with CT> 30 would be false positive, although 70% of false positives would be missed. CT's predictive ability (area under the curve, 0.83 [95% CI, .76-.90]) was not improved by additional variables. Xpert detected nonviable, nonintact bacilli without a change in CTvs controls. CONCLUSIONS: One in 7 Xpert-positive retreatment patients were culture negative and potentially false positive. False positivity was associated with recent previous tuberculosis, high CT, and a chest radiograph not suggestive of active tuberculosis. Clinicians may consider awaiting confirmatory testing in retreatment patients with CT> 30; however, most false positives fall below this cut-point. Xpert can detect DNA from nonviable, nonintact bacilli.