Search

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.

BackgroundSerological assays have been used in seroprevalence studies to inform the dynamics of COVID-19. Lateral flow immunoassay (LFIA) tests are a very practical technology to use for this objective; however, one of their challenges may be variable diagnostic performance. Given the numerous available LFIA tests, evaluation of their accuracy is critical before real-world implementation.MethodsWe performed a retrospective diagnostic evaluation study to independently determine the diagnostic accuracy of 4 different antibody-detection LFIA tests. The sample panel was comprised of specimens collected and stored in biobanks; specifically, specimens that were RT-PCR positive for SARS-CoV-2 collected at various times throughout the COVID-19 disease course and those that were collected before the pandemic, during 2018 or earlier, from individuals with upper respiratory symptoms but were negative for tuberculosis. Clinical performance (sensitivity and specificity) was analyzed overall, and subset across individual antibody isotypes, and days from symptoms onset.ResultsA very high specificity (98% - 100%) was found for all four tests. Overall sensitivity was variable, ranging from 29% [95% CI: 21%-39%] to 64% [95% CI: 54%-73%]. When considering detection of IgM only, the highest sensitivity was 42% [95% CI: 32%-52%], compared to 57% [95% CI: 47%-66%] for IgG only. When the analysis was restricted to at least 15 days since symptom onset, across any isotype, the sensitivity reached 90% for all four brands.ConclusionAll four LFIA tests proved effective for identifying COVID-19 antibodies when two conditions were met: 1) at least 15 days have elapsed since symptom onset and 2) a sample is considered positive when either IgM or IgG is present. With these considerations, the use of this assays could help in seroprevalence studies or further exploration of its potential uses.

IntroductionThe surge of the COVID-19 pandemic challenged health services globally, and in Lesotho, the HIV and tuberculosis (TB) services were similarly affected. Integrated, multi-disease diagnostic services were proposed solutions to mitigate these disruptions. We describe and evaluate the effect of an integrated, hospital-based COVID-19, TB and HIV screening and diagnostic model in two rural districts in Lesotho, during the period between December 2020 and August 2022.MethodsAdults and children above 5 years attending two hospitals were screened for COVID-19 and TB symptoms. After a positive screening, participants were offered to enroll in a service model that included clinical evaluation, chest radiography, SARS-CoV-2, Xpert MTB/RIF Ultra and HIV testing. Participants diagnosed with COVID-19, TB, or HIV were contacted after 28 days evaluate their health status, and linkage to HIV or TB services.ResultsOf the 179160 participants screened, 6623(37%) screened positive, and 4371(66%) were enrolled in this service model, yielding a total of 458 diagnoses. One positive rapid antigen test for SARS-CoV-2 was found per 11 participants screened, one Xpert-positive TB case was diagnosed per 85 people screened, and 1 new HIV diagnosis was done per 182 people screened. Of the 321(82.9%) participants contacted after 28 days of diagnosis, 304(94.7%) reported to be healthy. Of the individuals that were newly diagnosed with HIV or TB, 18/24(75.0%) and 46/51(90.1%) started treatment. This service showed no difference in the detection of new HIV and TB cases when compared to other hospitals, where no such integrated service model was provided.ConclusionThis screening and diagnostic model successfully maintained same-day, integrated COVID-19, TB, and HIV testing services through different COVID-19 incidence periods in a resource-limited context. There were positive effects in avoiding diagnostic delays and ensuring linkage to services, however, efficiencies were contingent on the successful adaptation to the changing environment.

Purpose Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤ 85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Methods This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results The study was conducted between November 2nd 2020 and 4th of December 2020. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI 75.2–87.5%). Specificity was 99.3% (CI 98.3–99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI 86.2–97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2–56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling. Trial registration number and registration date DRKS00021220 and 01.04.2020

We evaluated the performance of nasal and nasopharyngeal Standard Q COVID-19 [coronavirus disease 2019] Ag tests (SD Biosensor) and the Panbio COVID-19 Ag Rapid Test Device (nasal; Abbott) against the Abbott RealTime severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assay during the Omicron (clades 21M, 21K, and 21L) wave in South Africa. Overall, all evaluated tests performed well, with high sensitivity (range, 77.78%–81.42%) and excellent specificity values (>99%). The sensitivity of rapid antigen tests increased above 90% in samples with cycle threshold

Background\ud The World Health Organization (WHO) End TB Strategy stresses universal access to drug susceptibility testing (DST). DST determines whether Mycobacterium tuberculosis bacteria are susceptible or resistant to drugs. Xpert MTB/XDR is a rapid nucleic acid amplification test for detection of tuberculosis and drug resistance in one test suitable for use in peripheral and intermediate level laboratories. In specimens where tuberculosis is detected by Xpert MTB/XDR, Xpert MTB/XDR can also detect resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin.\ud \ud Objectives\ud To assess the diagnostic accuracy of Xpert MTB/XDR for pulmonary tuberculosis in people with presumptive pulmonary tuberculosis (having signs and symptoms suggestive of tuberculosis, including cough, fever, weight loss, night sweats).\ud \ud To assess the diagnostic accuracy of Xpert MTB/XDR for resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin in people with tuberculosis detected by Xpert MTB/XDR, irrespective of rifampicin resistance (whether or not rifampicin resistance status was known) and with known rifampicin resistance.\ud \ud Search methods\ud We searched multiple databases to 23 September 2021. We limited searches to 2015 onwards as Xpert MTB/XDR was launched in 2020.\ud \ud Selection criteria\ud Diagnostic accuracy studies using sputum in adults with presumptive or confirmed pulmonary tuberculosis. Reference standards were culture (pulmonary tuberculosis detection); phenotypic DST (pDST), genotypic DST (gDST),composite (pDST and gDST) (drug resistance detection).\ud \ud Data collection and analysis\ud Two review authors independently reviewed reports for eligibility and extracted data using a standardized form. For multicentre studies, we anticipated variability in the type and frequency of mutations associated with resistance to a given drug at the different centres and considered each centre as an independent study cohort for quality assessment and analysis. We assessed methodological quality with QUADAS‐2, judging risk of bias separately for each target condition and reference standard. For pulmonary tuberculosis detection, owing to heterogeneity in participant characteristics and observed specificity estimates, we reported a range of sensitivity and specificity estimates and did not perform a meta‐analysis. For drug resistance detection, we performed meta‐analyses by reference standard using bivariate random‐effects models. Using GRADE, we assessed certainty of evidence of Xpert MTB/XDR accuracy for detection of resistance to isoniazid and fluoroquinolones in people irrespective of rifampicin resistance and to ethionamide and amikacin in people with known rifampicin resistance, reflecting real‐world situations. We used pDST, except for ethionamide resistance where we considered gDST a better reference standard.\ud \ud Main results\ud We included two multicentre studies from high multidrug‐resistant/rifampicin‐resistant tuberculosis burden countries, reporting on six independent study cohorts, involving 1228 participants for pulmonary tuberculosis detection and 1141 participants for drug resistance detection. The proportion of participants with rifampicin resistance in the two studies was 47.9% and 80.9%. For tuberculosis detection, we judged high risk of bias for patient selection owing to selective recruitment. For ethionamide resistance detection, we judged high risk of bias for the reference standard, both pDST and gDST, though we considered gDST a better reference standard.\ud \ud Pulmonary tuberculosis detection\ud \ud ‐ Xpert MTB/XDR sensitivity range, 98.3% (96.1 to 99.5) to 98.9% (96.2 to 99.9) and specificity range, 22.5% (14.3 to 32.6) to 100.0% (86.3 to 100.0); median prevalence of pulmonary tuberculosis 91.3%, (interquartile range, 89.3% to 91.8%), (2 studies; 1 study reported on 2 cohorts, 1228 participants; very low‐certainty evidence, sensitivity and specificity).\ud \ud Drug resistance detection\ud \ud People irrespective of rifampicin resistance\ud \ud ‐ Isoniazid resistance: Xpert MTB/XDR summary sensitivity and specificity (95% confidence interval (CI)) were 94.2% (87.5 to 97.4) and 98.5% (92.6 to 99.7) against pDST, (6 cohorts, 1083 participants, moderate‐certainty evidence, sensitivity and specificity).\ud \ud ‐ Fluoroquinolone resistance: Xpert MTB/XDR summary sensitivity and specificity were 93.2% (88.1 to 96.2) and 98.0% (90.8 to 99.6) against pDST, (6 cohorts, 1021 participants; high‐certainty evidence, sensitivity; moderate‐certainty evidence, specificity).\ud \ud People with known rifampicin resistance\ud \ud ‐ Ethionamide resistance: Xpert MTB/XDR summary sensitivity and specificity were 98.0% (74.2 to 99.9) and 99.7% (83.5 to 100.0) against gDST, (4 cohorts, 434 participants; very low‐certainty evidence, sensitivity and specificity).\ud \ud ‐ Amikacin resistance: Xpert MTB/XDR summary sensitivity and specificity were 86.1% (75.0 to 92.7) and 98.9% (93.0 to 99.8) against pDST, (4 cohorts, 490 participants; low‐certainty evidence, sensitivity; high‐certainty evidence, specificity).\ud \ud Of 1000 people with pulmonary tuberculosis, detected as tuberculosis by Xpert MTB/XDR:\ud \ud ‐ where 50 have isoniazid resistance, 61 would have an Xpert MTB/XDR result indicating isoniazid resistance: of these, 14/61 (23%) would not have isoniazid resistance (FP); 939 (of 1000 people) would have a result indicating the absence of isoniazid resistance: of these, 3/939 (0%) would have isoniazid resistance (FN).\ud \ud ‐ where 50 have fluoroquinolone resistance, 66 would have an Xpert MTB/XDR result indicating fluoroquinolone resistance: of these, 19/66 (29%) would not have fluoroquinolone resistance (FP); 934 would have a result indicating the absence of fluoroquinolone resistance: of these, 3/934 (0%) would have fluoroquinolone resistance (FN).\ud \ud ‐ where 300 have ethionamide resistance, 296 would have an Xpert MTB/XDR result indicating ethionamide resistance: of these, 2/296 (1%) would not have ethionamide resistance (FP); 704 would have a result indicating the absence of ethionamide resistance: of these, 6/704 (1%) would have ethionamide resistance (FN).\ud \ud ‐ where 135 have amikacin resistance, 126 would have an Xpert MTB/XDR result indicating amikacin resistance: of these, 10/126 (8%) would not have amikacin resistance (FP); 874 would have a result indicating the absence of amikacin resistance: of these, 19/874 (2%) would have amikacin resistance (FN).\ud \ud Authors' conclusions\ud Review findings suggest that, in people determined by Xpert MTB/XDR to be tuberculosis‐positive, Xpert MTB/XDR provides accurate results for detection of isoniazid and fluoroquinolone resistance and can assist with selection of an optimised treatment regimen. Given that Xpert MTB/XDR targets a limited number of resistance variants in specific genes, the test may perform differently in different settings. Findings in this review should be interpreted with caution. Sensitivity for detection of ethionamide resistance was based only on Xpert MTB/XDR detection of mutations in the inhA promoter region, a known limitation. High risk of bias limits our confidence in Xpert MTB/XDR accuracy for pulmonary tuberculosis.\ud \ud Xpert MTB/XDR's impact will depend on its ability to detect tuberculosis (required for DST), prevalence of resistance to a given drug, health care infrastructure, and access to other tests.

Background Rapid tuberculosis (TB) drug susceptibility testing (DST) is crucial. Genotype MTBDRsl is a widely deployed World Health Organization (WHO)–endorsed assay. Programmatic performance data, including non-actionable results from smear-negative sputum, are scarce. Methods Sputa from Xpert MTB/RIF individuals (n = 951) were routinely-tested using Genotype MTBDRplus and MTBDRsl (both version 2). Phenotypic DST was the second-line drug reference standard. Discrepant results underwent Sanger sequencing. Findings 89% (849 of 951) of individuals were culture-positive (56%, 476 of 849 smear-negative). MTBDRplus had at least 1 nonactionable result (control and/or TB-detection bands absent or invalid, precluding resistance reporting) in 19% (92 of 476) of smear-negatives; for MTBDRsl, 40% (171 of 427) were nonactionable (28%, 120 of 427 false-negative TB; 17%, 51 of 427 indeterminate). In smear-negatives, MTBDRsl sensitivity for fluoroquinolones was 84% (95% confidence interval, 67%–93), 81% (54%–95%) for second-line injectable drugs, and 57% (28%–82%) for both. Specificities were 93% (89%–98%), 88% (81%–93%), and 97% (91%–99%), respectively. Twenty-three percent (172 of 746) of Xpert rifampicin-resistant specimens were MTBDRplus isoniazid-susceptible. Days-to-second-line-susceptibility reporting with the programmatic advent of MTBDRsl improved (6 [5–7] vs 37 [35–46]; P < .001). Conclusions MTBDRsl did not generate a result in 4 of 10 smear-negatives, resulting in substantial missed resistance. However, if MTBDRsl generates an actionable result, that is accurate in ruling-in resistance. Isoniazid DST remains crucial. This study provides real-world, direct, second-line susceptibility testing performance data on non-actionable results (that, if unaccounted for, cause an overestimation of test utility), accuracy, and care cascade impact.

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 “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.

Four moderate-complexity automated nucleic acid amplification tests for the diagnosis of tuberculosis are reported as having laboratory analytical and clinical performance similar to that of the Cepheid Xpert MTB/RIF assay. These assays are the Abbott RealTime MTB and RealTime MTB RIF/INH Resistance, Becton Dickinson MAX MDR-TB, the Hain Lifescience/Bruker FluoroType MTBDR, and the Roche cobas MTB and MTB RIF/INH assays. The study compared feasibility, ease of use, and operational characteristics of these assays/platforms. Manufacturer input was obtained for technical characteristics. Laboratory operators were requested to complete a questionnaire on the assays' ease of use. A time-in-motion analysis was also undertaken for each platform. For ease-of-use and operational requirements, the BD MAX MDR-TB assay achieved the highest scores (86% and 90%) based on information provided by the user and manufacturer, respectively, followed by the cobas MTB and MTB-RIF/INH assay (68% and 86%), the FluoroType MTBDR assay (67% and 80%), and the Abbott RT-MTB and RT MTB RIF/INH assays (64% and 76%). The time-in-motion analysis revealed that for 94 specimens, the RealTime MTB assay required the longest processing time, followed by the cobas MTB assay and the FluoroType MTBDR assay. The BD MAX MDR-TB assay required 4.6 hours for 22 specimens. These diagnostic assays exhibited different strengths and weaknesses that should be taken into account, in addition to affordability, when considering placement of a new platform.

Background: \ud There are an abundance of commercially available lateral flow assays (LFAs) that detect antibodies to SARS-CoV-2. Whilst these are usually evaluated by the manufacturer, externally performed diagnostic accuracy studies to assess performance are essential. Herein we present an evaluation of 12 LFAs. \ud Methods: \ud Sera from 100 SARS-CoV-2 reverse-transcriptase polymerase chain reaction (RT-PCR) positive participants were recruited through the FASTER study. A total of 105 pre-pandemic sera from participants with other infections were included as negative samples. \ud Results: \ud At presentation sensitivity against RT-PCR ranged from 37.4-79% for IgM/IgG, 30.3-74% for IgG, and 21.2-67% for IgM. Sensitivity for IgM/IgG improved ≥21 days post symptom onset for 10/12 tests. Specificity ranged from 74.3-99.1% for IgM/IgG, 82.9-100% for IgG, and 75.2-98% for IgM. Compared to the EuroImmun IgG enzyme-linked immunosorbent assay (ELISA), sensitivity and specificity ranged from 44.6-95.4% and 85.4-100%, respectively. \ud Conclusion: \ud There are many LFAs available with varied sensitivity and specificity. Understanding the diagnostic accuracy of these tests will be vital as we come to rely more on the antibody status of a person moving forward, and as such manufacturer-independent evaluations are crucial.

Bioaerosol capture and analysis is emerging as a non-invasive diagnostic method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this proof-of-concept study conducted in Lesotho, we evaluated the novel and simple AL2 bioaerosol detection device in comparison to conventional nasopharyngeal sampling methods. We demonstrated for the first time that SARS-CoV-2 can be detected using the AL2 bioaerosol capture device. However, studies with a larger sample size are needed to further evaluate this bioaerosol capture device for the detection of SARS-CoV-2.

The study of genetic minority variants is fundamental to the understanding of complex processes such as evolution, fitness, transmission, virulence, heteroresistance and drug tolerance in Mycobacterium tuberculosis (Mtb). We evaluated the performance of the variant calling tool LoFreq to detect de novo as well as drug resistance conferring minor variants in both in silico and clinical Mtb next generation sequencing (NGS) data. The in silico simulations demonstrated that LoFreq is a conservative variant caller with very high precision (≥96.7%) over the entire range of depth of coverage tested (30x to1000x), independent of the type and frequency of the minor variant. Sensitivity increased with increasing depth of coverage and increasing frequency of the variant, and was higher for calling insertion and deletion (indel) variants than for single nucleotide polymorphisms (SNP). The variant frequency limit of detection was 0.5% and 3% for indel and SNP minor variants, respectively. For serial isolates from a patient with DR-TB; LoFreq successfully identified all minor Mtb variants in the Rv0678 gene (allele frequency as low as 3.22% according to targeted deep sequencing) in whole genome sequencing data (median coverage of 62X). In conclusion, LoFreq can successfully detect minor variant populations in Mtb NGS data, thus limiting the need for filtering of possible false positive variants due to sequencing error. The observed performance statistics can be used to determine the limit of detection in existing whole genome sequencing Mtb data and guide the required depth of future studies that aim to investigate the presence of minor variants.

The limit of detection (LOD) of thirty-two antigen lateral flow tests (Ag-RDT) were evaluated with the SARS-CoV-2 Gamma variant. Twenty-eight of thirty-two Ag-RDTs exceeded the World Health Organization criteria of an LOD of 1.0×106 genome copy numbers/ml and performance was equivalent as with the 2020 B.1 lineage and Alpha variant.

During the last year, mass screening campaigns have been carried out to identify immunological response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and establish a possible seroprevalence. The obtained results gained new importance with the beginning of the SARS-CoV-2 vaccination campaign, as the lack of doses has persuaded several countries to introduce different policies for individuals who had a history of COVID-19. Lateral flow assays (LFAs) may represent an affordable tool to support population screening in low-middle-income countries, where diagnostic tests are lacking and epidemiology is still widely unknown. However, LFAs have demonstrated a wide range of performance, and the question of which one could be more valuable in these settings still remains. We evaluated the performance of 11 LFAs in detecting SARS-CoV-2 infection, analyzing samples collected from 350 subjects. In addition, samples from 57 health care workers collected at 21 to 24 days after the first dose of the Pfizer-BioNTech vaccine were also evaluated. LFAs demonstrated a wide range of specificity (92.31% to 100%) and sensitivity (50% to 100%). The analysis of postvaccination samples was used to describe the most suitable tests to detect IgG response against S protein receptor binding domain (RBD). Tuberculosis (TB) therapy was identified as a potential factor affecting the specificity of LFAs. This analysis identified which LFAs represent a valuable tool not only for the detection of prior SARS-CoV-2 infection but also for the detection of IgG elicited in response to vaccination. These results demonstrated that different LFAs may have different applications and the possible risks of their use in high-TB-burden settings. IMPORTANCE Our study provides a fresh perspective on the possible employment of SARS-CoV-2 LFA antibody tests. We developed an in-depth, large-scale analysis comparing LFA performance to enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence immunoassay (ECLIA) and evaluating their sensitivity and specificity in identifying COVID-19 patients at different time points from symptom onset. Moreover, for the first time, we analyzed samples of patients undergoing treatment for endemic poverty-related diseases, especially tuberculosis, and we evaluated the impact of this therapy on test specificity in order to assess possible performance in TB high-burden countries.

Eis promoter mutations can confer reduced Mycobacterium tuberculosis kanamycin susceptibility. GenoType MTBDRsl, a widely used assay evaluating this region, wrongly classified 17/410 isolates as eis promoter wild type. Six out of seventeen isolates harbored mutations known to confer kanamycin resistance, and the remainder harbored either novel eis promoter mutations (7/11) or disputed mutations (4/11). GenoType MTBDRsl can miss established and new variants that cause reduced susceptibility. These data highlight the importance of reflex phenotypic kanamycin testing.

BackgroundDuring the last year, mass screening campaigns have been carried out to identify immunological response to SARS-CoV-2 and establish a possible seroprevalence. The obtained results gained new importance with the beginning of SARS-CoV-2 vaccination campaign, as the lack of doses has persuaded several countries to introduce different policies for individuals who had a history of COVID 19.LFAs may represent an affordable tool to support population screening in LMICs, where diagnostic tests are lacking, and epidemiology is still widely unknown. However, LFAs have demonstrated a wide range of performance and the question of which one could be more valuable in these settings still remains.MethodsWe evaluated the performance of 11 LFAs in detecting SARS-CoV-2 infection, analysing samples collected from 350 subjects. In addition, samples from 57 health care workers collected at 21-24 days after the first dose of Pfizer-BioNTech vaccine were also evaluated.FindingsLFAs demonstrated a wide range of specificity (92.31% to 100%) and sensitivity (50 to 100%). The analysis of serum samples post vaccination was used to describe the most suitable tests to detect IgG response against S protein RBD. History of TB therapy was identified as a potential factor affecting the specificity of LFAs.ConclusionsThis analysis identified which LFAs represent a valuable tool not only for the detection of prior SARS-CoV-2 infection, but also to detect IgG elicited in response to vaccination. These results demonstrated that different LFAs may have different applications and the possible risks of their use in high TB burden settings.

Eis promoter mutations can confer reduced Mycobacterium tuberculosis kanamycin susceptibility. GenoType MTBDRsl, a widely used assay evaluating this region, wrongly classified 17/410 isolates as eis promoter wild type. Six out of seventeen isolates harbored mutations known to confer kanamycin resistance, and the remainder harbored either novel eis promoter mutations (7/11) or disputed mutations (4/11). GenoType MTBDRsl can miss established and new variants that cause reduced susceptibility. These data highlight the importance of reflex phenotypic kanamycin testing.

BackgroundIn 2020, the World Health Organization (WHO) recommended two SARS-CoV-2 lateral flow antigen detecting rapid diagnostics tests (Ag-RDTs), both initially with nasopharyngeal (NP) sample collection. Independent head-to-head studies demonstrated for SARS-CoV-2 Ag-RDTs nasal sampling to be a comparable and reliable alternative for nasopharyngeal (NP) sampling.MethodsWe conducted a head-to-head comparison study of a supervised, self-collected nasal mid-turbinate (NMT) swab and a professional-collected NP swab, using the Panbio Ag-RDT (the second WHO-listed SARS-CoV-2 Ag-RDT, distributed by Abbott). We calculated positive and negative percent agreement and, compared to the reference standard reverse transcription polymerase chain reaction (RT-PCR), sensitivity and specificity for both sampling techniques.ResultsA SARS-CoV-2 infection could be diagnosed by RT-PCR in 45 of 290 participants (15.5%). Comparing the NMT and NP sampling the positive percent agreement of the Ag-RDT was 88.1% (37/42 PCR positives detected; CI 75.0% - 94.8%). The negative percent agreement was 98.8% (245/248; CI 96.5% - 99.6%). The overall sensitivity of Panbio with NMT sampling was 84.4% (38/45; CI 71.2% - 92.3%) and 88.9% (40/45; CI 76.5% - 95.5%) with NP sampling. Specificity was 99.2% (243/245; CI 97.1% - 99.8%) for both, NP and NMT sampling. The sensitivity of the Panbio test in participants with high viral load (> 7 log10 SARS-CoV-2 RNA copies/mL) was 96.3% (CI 81.7% - 99.8%) for both, NMT and NP sampling.ConclusionFor the Panbio Ag-RDT supervised NMT self-sampling yields to results comparable to NP sampling. This suggests that nasal self-sampling could be used for scale-up population testing.

In 2020, the World Health Organization (WHO) recommended two SARS-CoV-2 lateral flow antigen-detecting rapid diagnostics tests (Ag-RDTs), both initially with nasopharyngeal (NP) sample collection. Independent head-to-head studies are necessary for SARS-CoV-2 Ag-RDT nasal sampling to demonstrate comparability of performance with nasopharyngeal (NP) sampling. We conducted a head-to-head comparison study of a supervised, self-collected nasal mid-turbinate (NMT) swab and a professional-collected NP swab, using the Panbio™ Ag-RDT (distributed by Abbott). We calculated positive and negative percent agreement between the sampling methods as well as sensitivity and specificity for both sampling techniques compared to the reference standard reverse transcription polymerase chain reaction (RT-PCR). A SARS-CoV-2 infection could be diagnosed by RT-PCR in 45 of 290 participants (15.5%). Comparing the NMT and NP sampling the positive percent agreement of the Ag-RDT was 88.1% (37/42 PCR positives detected; CI 75.0–94.8%). The negative percent agreement was 98.8% (245/248; CI 96.5–99.6%). The overall sensitivity of Panbio with NMT sampling was 84.4% (38/45; CI 71.2–92.3%) and 88.9% (40/45; CI 76.5–95.5%) with NP sampling. Specificity was 99.2% (243/245; CI 97.1–99.8%) for both, NP and NMT sampling. The sensitivity of the Panbio test in participants with high viral load (> 7 log10 SARS-CoV-2 RNA copies/mL) was 96.3% (CI 81.7–99.8%) for both, NMT and NP sampling. For the Panbio supervised NMT self-sampling yields comparable results to NP sampling. This suggests that nasal self-sampling could be used for to enable scaled-up population testing. Clinical Trial DRKS00021220. Supplementary Information The online version contains supplementary material available at 10.1007/s00430-021-00710-9.

BackgroundRapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection.Materials and MethodsThis multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use.ResultsStudy conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings.ConclusionThe LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

Failure to rapidly identify drug-resistant tuberculosis (TB) increases the risk of patient mismanagement, the amplification of drug resistance, and ongoing transmission. We generated comparative analytical data for four automated assays for the detection of TB and multidrug-resistant TB (MDR-TB): Abbott RealTime MTB and MTB RIF/INH (Abbott), Hain Lifescience FluoroType MTBDR (Hain), BD Max MDR-TB (BD), and Roche cobas MTB and MTB-RIF/INH (Roche)., Failure to rapidly identify drug-resistant tuberculosis (TB) increases the risk of patient mismanagement, the amplification of drug resistance, and ongoing transmission. We generated comparative analytical data for four automated assays for the detection of TB and multidrug-resistant TB (MDR-TB): Abbott RealTime MTB and MTB RIF/INH (Abbott), Hain Lifescience FluoroType MTBDR (Hain), BD Max MDR-TB (BD), and Roche cobas MTB and MTB-RIF/INH (Roche). We included Xpert MTB/RIF (Xpert) and GenoType MTBDRplus as comparators for TB and drug resistance detection, respectively. We assessed analytical sensitivity for the detection of the Mycobacterium tuberculosis complex using inactivated strains (M. tuberculosis H37Rv and M. bovis) spiked into TB-negative sputa and computed the 95% limits of detection (LOD95). We assessed the accuracy of rifampicin and isoniazid resistance detection using well-characterized M. tuberculosis strains with high-confidence mutations accounting for >85% of first-line resistance mechanisms globally. For H37Rv and M. bovis, we measured LOD95 values of 3,781 and 2,926 (Xpert), 322 and 2,182 (Abbott), 826 and 4,301 (BD), 10,398 and 23,139 (Hain), and 2,416 and 2,136 (Roche) genomes/ml, respectively. Assays targeting multicopy genes or targets (Abbott, BD, and Roche) showed increased analytical sensitivity compared to Xpert. Quantification of the panel by quantitative real-time PCR prevents the determination of absolute values, and results reported here can be interpreted for comparison purposes only. All assays showed accuracy comparable to that of Genotype MTBDRplus for the detection of rifampicin and isoniazid resistance. The data from this analytical study suggest that the assays may have clinical performances similar to those of WHO-recommended molecular TB and MDR-TB assays.

In a manufacturer-independent laboratory validation study, the Xpert MTB/XDR® assay demonstrated equivalent limit of detection to Xpert MTB/RIF®, detected 100% of tested resistance mutations and showed some utility for resistance detection in strain mixtures. The Xpert MTB/XDR assay is a reliable, sensitive assay for tuberculosis and expanded resistance detection.

Mycobacterium tuberculosis has developed extensive resistance to numerous antimycobacterial agents used in the treatment of tuberculosis. Insufficient intracellular accumulation of active moieties allows for selective survival of mycobacteria with drug resistance mutations and accordingly promotes the development of microbial drug resistance. Discovery of compounds with new mechanisms of action and physicochemical properties that promote intracellular accumulation, or compounds that act synergistically with other antimycobacterial drugs, has the potential to reduce and prevent further drug resistance. To this end, antimycobacterial activity, mechanism of action, and synergism in combination therapy were investigated for a series of polycyclic amine derivatives. Compound selection was based on the presence of moieties with possible antimycobacterial activity, the inclusion of bulky lipophilic carriers to promote intracellular accumulation, and previously demonstrated bioactivity that potentially support inhibition of efflux pump activity. The most potent antimycobacterial demonstrated a minimum inhibitory concentration (MIC99) of 9.6 μM against Mycobacterium tuberculosis H37Rv. Genotoxicity and inhibition of the cytochrome bc1 respiratory complex were excluded as mechanisms of action for all compounds. Inhibition of cell wall synthesis was identified as a likely mechanism of action for the two most active compounds (14 and 15). Compounds 5 and 6 demonstrated synergistic activity with the known Rv1258c efflux pump substrate, spectinomycin, pointing to possible efflux pump inhibition. For this series, the nature of the side chain, rather than the type of polycyclic carrier, seems to play a determining role in the antimycobacterial activity and cytotoxicity of the compounds. Contrariwise, the nature of the polycyclic carrier, particularly the azapentacycloundecane cage, appears to promote synergistic activity. Results point to the possibility of combining an azapentacycloundecane carrier with a side chain that promotes antimycobacterial activity to develop dual acting molecules for the treatment of Mycobacterium tuberculosis.

BackgroundAntigen-detecting rapid diagnostic tests for SARS-CoV-2 offer new opportunities for the quick and laboratory-independent identification of infected individuals for control of the SARS-CoV-2 pandemic.MethodsWe performed a prospective, single-center, point of care validation of two antigen-detecting rapid diagnostic tests (Ag-RDT) in comparison to RT-PCR on nasopharyngeal swabs.FindingsBetween October 9th and 23rd, 2020, 1064 participants were enrolled. The Panbio™Covid-19 Ag Rapid Test device (Abbott) was validated in 535 participants, with 106 positive Ag-RDT results out of 124 positive RT-PCR individuals, yielding a sensitivity of 85.5% (95% CI: 78.0–91.2). Specificity was 100.0% (95% CI: 99.1–100) in 411 RT-PCR negative individuals. The Standard Q Ag-RDT (SD Biosensor, Roche) was validated in 529 participants, with 170 positive Ag-RDT results out of 191 positive RT-PCR individuals, yielding a sensitivity of 89.0% (95%CI: 83.7–93.1). One false positive result was obtained in 338 RT-PCR negative individuals, yielding a specificity of 99.7% (95%CI: 98.4–100). For individuals presenting with fever 1-5 days post symptom onset, combined Ag-RDT sensitivity was above 95%.InterpretationWe provide an independent validation of two widely available commercial Ag-RDTs, both meeting WHO criteria of ≥80% sensitivity and ≥97% specificity. Although less sensitive than RT-PCR, these assays could be beneficial due to their rapid results, ease of use, and independence from existing laboratory structures. Testing criteria focusing on patients with typical symptoms in their early symptomatic period onset could further increase diagnostic value.FundingFoundation of Innovative Diagnostics (FIND), Fondation privée des HUG, Pictet Charitable Foundation.

Background Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. Methods We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. Results Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. Conclusion The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.

Whole genome sequencing (WGS) can investigate the entire Mycobacterium tuberculosis (Mtb) genome but currently requires large amounts of mycobacterial DNA, necessitating culture. Culture-free Mtb WGS could revolutionize the clinical use of WGS but is hampered by the high viscosity, low mycobacterial load, and high contamination with bacterial and human DNA in sputum samples. To improve the sputum liquefaction and decontamination step prior to DNA extraction, we assessed the efficiency of Myco-TB, MycoPrep, and Sputolysin with/without TiKa-Kic in liquefying and decontaminating sputum and aimed to evaluate the effect of these approaches on mycobacterial viability, and Mtb DNA quality and quantity. Experiments using spiked sputum samples showed that Myco-TB and BD MycoPrep with standard (15 min) or increased (30 min) incubation time, but not reduced (7,5 min) incubation time performed well in liquefying and decontaminating sputum. No difference in DNA quality or quantity, contamination, or the amount of human DNA present was observed. In comparison, Sputolysin with/without TiKa-Kic was less effective for liquefaction and decontamination of sputum. PCR amplification of the human GAPDH gene after sputum treatment, showed the presence of human DNA in all samples, regardless of sputum treatment. Focused efforts are needed to deplete contaminating DNA for culture-free Mtb WGS

Objectives: To evaluate the accuracy of two new molecular diagnostic tests for the detection of drug-resistant tuberculosis, the FluoroType MTB and MTBDR VER 2.0 assays, in combination with manual and automated DNA extraction methods. Methods: Sputa from 360 Xpert Ultra Mycobacterium tuberculosis complex (MTBC)-positive patients and 250 Xpert Ultra MTBC-negative patients were tested. GenoType MTBDRplus served as reference for MTBC and drug resistance detection. Sanger sequencing was used to resolve discrepancies. Results: FluoroType MTB VER 2.0 showed similar MTBC sensitivity compared with FluoroType MTBDR VER 2.0 (manual DNA extraction: 91.6% (294/321) versus 89.8% (291/324); p 0.4); automated DNA extraction: 92.1% (305/331) versus 87.7% (291/332); p 0.05)). FluoroType MTBDR VER2.0 showed comparable diagnostic accuracy to FluoroType MTBDR VER1.0 as previously reported for the detection of MTBC and rifampicin and isoniazid resistance. Conclusions: The FluoroType MTB and MTBDR VER 2.0 assays together with an automated DNA extraction and PCR set-up platform may improve laboratory operational efficiency for the diagnosis of MTBC and resistance to rifampicin and isoniazid and show promise for the implementation in a centralized molecular drug susceptibility testing model. (C) 2021 The Author(s). Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.

International audience; Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.