Diagnostic accuracy of tongue swab testing on two automated tuberculosis diagnostic platforms, Cepheid Xpert MTB/RIF Ultra and Molbio Truenat MTB Ultima.

Tongue dorsum swabbing is a potential alternative to sputum collection for tuberculosis (TB) testing. Previous studies showed that Cepheid Xpert MTB/RIF Ultra (Xpert Ultra) can detect Mycobacterium tuberculosis DNA on tongue swabs stored in buffer, with 72% sensitivity and 100% specificity relative to a sputum microbiological reference standard (sputum MRS). The present study evaluated a more convenient sample collection protocol (dry swab storage), combined with streamlined sample processing protocols, for evaluating two commercial TB diagnostic tests: Xpert Ultra and Molbio Truenat MTB Ultima (MTB Ultima). Copan FLOQSwabs were self-collected or collected by study workers from 321 participants in Western Cape, South Africa. All participants had symptoms suggestive of TB, and 245 of them had sputum MRS-confirmed TB (by sputum MGIT culture and/or Xpert Ultra). One tongue swab per participant was tested on Xpert Ultra, and another tongue swab was tested with MTB Ultima. Xpert Ultra was 75.5% sensitive and 100% specific relative to sputum MRS, similar to previous methods that used swabs stored in buffer. MTB Ultima was 71.6% sensitive and 96.9% specific relative to sputum MRS. When sample lysates that were false-negative or invalid by MTB Ultima were frozen, thawed, and re-tested, MTB Ultima sensitivity rose to 79.1%. Both tests were more sensitive with swabs from participants with higher sputum Xpert Ultra semi-quantitative results. Although additional development could improve diagnostic accuracy, these results further support tongue swabs as easy-to-collect samples for TB testing. IMPORTANCE: Tongue dorsum swabbing is a promising alternative to sputum collection for tuberculosis (TB) testing. Our results lend further support for tongue swabs as exceptionally easy-to-collect samples for high-throughput TB testing.

Detection of minor variants in Mycobacterium tuberculosis whole genome sequencing data.

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.

Non-actionable Results, Accuracy, and Effect of First- and Second-line Line Probe Assays for Diagnosing Drug-Resistant Tuberculosis, Including on Smear-Negative Specimens, in a High-Volume Laboratory.

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.

Clinical Evaluation of Severe Acute Respiratory Syndrome Coronavirus 2 Rapid Antigen Tests During the Omicron Wave in South Africa.

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 <20, and all 3 tests performed best within the first week after symptom onset.

Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™: An antigen-detecting point-of-care device for SARS-CoV-2.

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.

Xpert MTB/XDR for detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin.

BACKGROUND: 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. OBJECTIVES: 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). 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. SEARCH METHODS: We searched multiple databases to 23 September 2021. We limited searches to 2015 onwards as Xpert MTB/XDR was launched in 2020. SELECTION CRITERIA: 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). DATA COLLECTION AND ANALYSIS: 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. MAIN RESULTS: 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. Pulmonary tuberculosis detection - 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). Drug resistance detection People irrespective of rifampicin resistance - 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). - 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). People with known rifampicin resistance - 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). - 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). Of 1000 people with pulmonary tuberculosis, detected as tuberculosis by Xpert MTB/XDR: - 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). - 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). - 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). - 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). AUTHORS' CONCLUSIONS: 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. 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.

Melting the eis: Nondetection of Kanamycin Resistance Markers by Routine Diagnostic Tests and Identification of New eis Promoter Variants.

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.

Comparative Analytical Evaluation of Four Centralized Platforms for the Detection of Mycobacterium tuberculosis Complex and Resistance to Rifampicin and Isoniazid.

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.

Head-to-head performance comparison of self-collected nasal versus professional-collected nasopharyngeal swab for a WHO-listed SARS-CoV-2 antigen-detecting rapid diagnostic test.

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.

A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic.

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.

Diagnostic Accuracy and Utility of FluoroType MTBDR, a New Molecular Assay for Multidrug-Resistant Tuberculosis.

Most cases of multidrug-resistant (MDR) tuberculosis (TB) are never diagnosed (328,300 of the ∼490,000 cases in 2016 were missed). The Xpert MTB/RIF assay detects resistance only to rifampin, despite ∼20% of rifampin-resistant cases being susceptible to isoniazid (a critical first-line drug). Consequently, many countries require further testing with the GenoType MTBDRplus assay. However, MTBDRplus is not recommended for use on smear-negative specimens, and thus, many specimens require culture-based drug susceptibility testing. Furthermore, MTBDRplus requires specialized expertise, lengthy hands-on time, and significant laboratory infrastructure and interpretation is not automated. To address these gaps, we evaluated the accuracy of the FluoroType MTBDR (FluoroType) assay. Sputa from 244 smear-positive and 204 smear-negative patients with presumptive TB (Xpert MTB positive, n = 343) were tested. Culture and MTBDRplus on isolates served as reference standards (for active TB and MDR-TB, respectively). Sanger sequencing and MTBDRplus, both of which were performed on sputa, were used to resolve discrepancies. The sensitivity of FluoroType for the detection of M. tuberculosis complex was 98% (95% confidence interval [CI], 95 to 99%) and 92% (95% CI, 84 to 96%) for smear-positive and smear-negative specimens, respectively (232/237 versus 90/98 specimens; P < 0.009). The sensitivity and specificity for smear-negative specimens were 100% and 97%, respectively, for rifampin resistance; 100% and 98%, respectively, for isoniazid resistance; and 100% and 100%, respectively, for MDR-TB. FluoroType identified 98%, 97%, and 97% of the rpoB, katG, and inhA promoter mutations, respectively. FluoroType has excellent sensitivity with sputa equivalent to that of MTBDRplus with the isolates and can provide rapid drug susceptibility testing for rifampin and isoniazid. In addition, the capacity of FluoroType to simultaneously identify virtually all mutations in the rpoB, katG, and inhA promoter may be useful for individualized treatment regimens.

Optimal processing of tongue swab samples for Mycobacterium tuberculosis detection by the Xpert MTB/RIF Ultra assay.

Tongue swabs represent a potential alternative to sputum as a sample type for detecting pulmonary tuberculosis (TB) using molecular diagnostic tests. The methods used to process tongue swabs for testing in the WHO-recommended Xpert MTB/RIF Ultra (Xpert Ultra) assay vary greatly. We aimed to identify the optimal method for processing diagnostic tongue swabs for subsequent testing by Xpert Ultra. We compared four methods for treating dry tongue swabs with Xpert Ultra sample reagent (SR) mixed with various concentrations of Tris-EDTA-Tween (TET), to treatment with SR alone or to a commonly used SR-free heat-inactivation protocol. In each condition, swabs obtained from volunteers without TB were placed into test buffer spiked with known amounts of Mycobacterium tuberculosis ( Mtb ) strain H37Rv-mc 2 6230. Swabs processed with 1:1 diluted SR buffer had the lowest Mtb limit of detection (LOD) at 22.7 CFU/700 µl (95% CI 14.2-31.2), followed by 2:1 diluted SR buffer at 30.3 CFU/700 µl (95% CI 19.9-40.7), neat SR at 30.9 CFU/700 µl (95% CI 21.5-40.3) and SR prefilled in the Xpert Ultra at 57.1 CFU/700 µl (95% CI 42.4-71.7). Swabs processed using the heat-based protocol had the highest LOD (77.6 CFU/700 µl; 95% CI 51.2-104.0). Similar findings were observed for LOD of RIF-susceptibility. Assay sensitivity using the 2:1 diluted SR buffer did not vary considerably in the presence of sputum matrix or phosphate buffer saline. Further studies are needed to assess the performance of this processing protocol in a clinical setting. Importance: Xpert MTB/RIF Ultra (Xpert Ultra) is approved by the World Health Organization for the diagnosis of tuberculosis (TB). This test is typically performed using sputum specimens obtained from people with presumptive TB. In order to inactivate Mtb and aid liquefaction, sputum must be mixed with Xpert SR prior to transfer into the Xpert Ultra. However, some people under evaluation for TB are unable to produce sputum. Alternative sample types for TB diagnosis would therefore be of value. Oral-swabs, including tongue-swabs have shown promise, but there are technical challenges associated with sample processing. In this study, several new tongue swab processing conditions were evaluated, utilizing SR, either neat or diluted in buffer. The ability of Xpert Ultra to detect TB was improved under these conditions compared with the previously published heat-processing method (1-3), processing steps were simplified, and technical challenges were overcome.

Accuracy of cobas MTB and MTB-RIF/INH for Detection of Mycobacterium tuberculosis and Drug Resistance.

This study evaluated the performance of cobas MTB and cobas MTB-RIF/INH for the diagnosis of tuberculosis and detection of rifampicin (RIF) and isoniazid (INH) resistance. Adults presenting with pulmonary tuberculosis symptoms were recruited in South Africa, Moldova, and India. Performance of cobas MTB was assessed against culture, whereas cobas MTB-RIF/INH was assessed using phenotypic drug susceptibility testing and whole-genome sequencing as composite reference standards. Xpert MTB/RIF (Xpert) or Xpert MTB/RIF Ultra (Ultra) was used as a comparator. The overall sensitivity and specificity of cobas MTB were 95% (95% CI, 93%-96%) and 96% (95% CI, 95%-97%). Among smear-negatives, the sensitivity of cobas MTB was 75% (95% CI, 66%-83%). Among participants tested with both cobas MTB and Xpert, sensitivity was 96% (95% CI, 94%-97%) for cobas MTB and 95% (95% CI, 93%-97%) for Xpert. Among participants tested with both cobas MTB and Ultra, sensitivity was 88% (95% CI, 81%-92%) for cobas MTB and 89% (95% CI, 83%-93%) for Ultra. Sensitivity and specificity of cobas MTB-RIF/INH for RIF and INH detection were 90% (95% CI, 84%-94%) and 100% (95% CI, 99%-100%), and 89% (95% CI, 84%-93%) and 99.5% (95% CI, 98%-100%), respectively. The cobas MTB and cobas MTB-RIF/INH assays exhibited high performance in a diverse population and present a suitable option for molecular detection of tuberculosis and RIF and INH resistance.

Determining cost and placement decisions for moderate complexity NAATs for tuberculosis drug susceptibility testing.

BACKGROUND: Access to drug resistant testing for tuberculosis (TB) remains a challenge in high burden countries. Recently, the World Health Organization approved the use of several moderate complexity automated nucleic acid amplification tests (MC-NAAT) that have performance profiles suitable for placement in a range of TB laboratory tiers to improve drug susceptibility tests (DST) coverage. METHODS: We conducted cost analysis of two MC-NAATs with different testing throughput: Lower Throughput (LT, < 24 tests per run) and Higher Throughput (HT, upto 90+ tests per run) for placement in a hypothetical laboratory in a resource limited setting. We used per-test cost as the main indicator to assess 1) drivers of cost by resource types and 2) optimized levels of annual testing volumes for the respective MC-NAATs. RESULTS: The base-case per test cost of $18.52 (range: $13.79 - $40.70) for LT test and $15.37 (range: $9.61 - $37.40) for HT test. Per test cost estimates were most sensitive to the number of testing days per week, followed by equipment costs and TB-specific workloads. In general, HT NAATs were cheaper at all testing volume levels, but at lower testing volumes (less than 2,000 per year) LT tests can be cheaper if the durability of the testing system is markedly better and/or procured equipment costs are lower than that of HT NAAT. CONCLUSION: Assuming equivalent performance and infrastructural needs, placement strategies for MC-NAATs need to be prioritized by laboratory system's operational factors, testing demands, and costs.

Feasibility, Ease-of-Use, and Operational Characteristics of World Health Organization-Recommended Moderate-Complexity Automated Nucleic Acid Amplification Tests for the Detection of Tuberculosis and Resistance to Rifampicin and Isoniazid.

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.

Diagnostic performance of GENEDIA W and ActiveXpress+ COVID-19 antigens tests among symptomatic individuals in Peru and The United Kingdom.

OBJECTIVES: In order to generate independent performance data regarding accuracy of COVID-19 antigen-based rapid diagnostic tests (Ag-RDTs), prospective diagnostic evaluation studies across multiple sites are required to evaluate their performance in different clinical settings. This report describes the clinical evaluation the GENEDIA W COVID-19 Ag Device (Green Cross Medical Science Corp., Chungbuk, Korea) and the ActiveXpress+ COVID-19 Complete Testing Kit (Edinburgh Genetics Ltd, UK), in two testing sites Peru and the United Kingdom. METHODS: Nasopharyngeal swabs collected from 456 symptomatic patients at primary points of care in Lima, Peru and 610 symptomatic participants at a COVID-19 Drive-Through testing site in Liverpool, England were analyzed by Ag-RDT and compared to RT-PCR. Analytical evaluation of both Ag-RDTs was assessed using serial dilutions of direct culture supernatant of a clinical SARS-CoV-2 isolate from the B.1.1.7 lineage. RESULTS: For GENEDIA brand, the values of overall sensitivity and specificity were 60.4% [95% CI 52.4-67.9%], and 99.2% [95% CI 97.6-99.7%] respectively; and for Active Xpress+ the overall values of sensitivity and specificity were 66.2% [95% CI 54.0-76.5%], and 99.6% [95% CI 97.9-99.9%] respectively. The analytical limit of detection was determined at 5.0 x 102 pfu/ml what equals to approximately 1.0 x 104 gcn/ml for both Ag-RDTs. The UK cohort had lower median Ct values compared to that of Peru during both evaluations. When split by Ct, both Ag-RDTs had optimum sensitivities at Ct<20 (in Peru; 95% [95% CI 76.4-99.1%] and 100.0% [95% CI 74.1-100.0%] and in the UK; 59.2% [95% CI 44.2-73.0%] and 100.0% [95% CI 15.8-100.0%], for the GENDIA and the ActiveXpress+, respectively). CONCLUSIONS: Whilst the overall clinical sensitivity of the Genedia did not meet WHO minimum performance requirements for rapid immunoassays in either cohort, the ActiveXpress+ did so for the small UK cohort. This study illustrates comparative performance of Ag-RDTs across two global settings and considers the different approaches in evaluation methods.

Head-to-head comparison of nasal and nasopharyngeal sampling using SARS-CoV-2 rapid antigen testing in Lesotho.

OBJECTIVES: To assess the real-world diagnostic performance of nasal and nasopharyngeal swabs for SD Biosensor STANDARD Q COVID-19 Antigen Rapid Diagnostic Test (Ag-RDT). METHODS: Individuals ≥5 years with COVID-19 compatible symptoms or history of exposure to SARS-CoV-2 presenting at hospitals in Lesotho received two nasopharyngeal and one nasal swab. Ag-RDT from nasal and nasopharyngeal swabs were performed as point-of-care on site, the second nasopharyngeal swab used for polymerase chain reaction (PCR) as the reference standard. RESULTS: Out of 2198 participants enrolled, 2131 had a valid PCR result (61% female, median age 41 years, 8% children), 84.5% were symptomatic. Overall PCR positivity rate was 5.8%. The sensitivity for nasopharyngeal, nasal, and combined nasal and nasopharyngeal Ag-RDT result was 70.2% (95%CI: 61.3-78.0), 67.3% (57.3-76.3) and 74.4% (65.5-82.0), respectively. The respective specificity was 97.9% (97.1-98.4), 97.9% (97.2-98.5) and 97.5% (96.7-98.2). For both sampling modalities, sensitivity was higher in participants with symptom duration ≤ 3days versus ≤ 7days. Agreement between nasal and nasopharyngeal Ag-RDT was 99.4%. CONCLUSIONS: The STANDARD Q Ag-RDT showed high specificity. Sensitivity was, however, below the WHO recommended minimum requirement of ≥ 80%. The high agreement between nasal and nasopharyngeal sampling suggests that for Ag-RDT nasal sampling is a good alternative to nasopharyngeal sampling.

Designing molecular diagnostics for current tuberculosis drug regimens.

Diagnostic development must occur in parallel with drug development to ensure the longevity of new treatment compounds. Despite an increasing number of novel and repurposed anti-tuberculosis compounds and regimens, there remains a large number of drugs for which no rapid and accurate molecular diagnostic option exists. The lack of rapid drug susceptibility testing for linezolid, bedaquiline, clofazimine, the nitroimidazoles (i.e pretomanid and delamanid) and pyrazinamide at any level of the healthcare system compromises the effectiveness of current tuberculosis and drug-resistant tuberculosis treatment regimens. In the context of current WHO tuberculosis treatment guidelines as well as promising new regimens, we identify the key diagnostic gaps for initial and follow-on tests to diagnose emerging drug resistance and aid in regimen selection. Additionally, we comment on potential gene targets for inclusion in rapid molecular drug susceptibility assays and sequencing assays for novel and repurposed drug compounds currently prioritized in current regimens, and evaluate the feasibility of mutation detection given the design of existing technologies. Based on current knowledge, we also propose design priorities for next generation molecular assays to support triage of tuberculosis patients to appropriate and effective treatment regimens. We encourage assay developers to prioritize development of these key molecular assays and support the continued evolution, uptake, and utility of sequencing to build knowledge of tuberculosis resistance mechanisms and further inform rapid treatment decisions in order to curb resistance to critical drugs in current regimens and achieve End TB targets.Trial registration: ClinicalTrials.gov identifier: NCT05117788..

Diagnostic performance of four lateral flow immunoassays for COVID-19 antibodies in Peruvian population.

Serological 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. We performed a retrospective diagnostic evaluation study to independently determine the diagnostic accuracy of 4 different antibody-detection LFIA tests: Now Check (Bionote), CareStart (Access bio), Covid-19 BSS (Biosynex) and OnSite (CTK Biotech). 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. A 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. All 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.