Evaluation of the FluoroType Mycobacteria assay for identification of mycobacteria.

Accurate species identification is a prerequisite for successful management of tuberculosis and non-tuberculous mycobacterial (NTM) diseases. The novel FluoroType Mycobacteria assay combines three established GenoType DNA strip assays (CM, AS, and NTM-DR), allowing detection of Mycobacterium tuberculosis and 32 NTM species/subspecies in a single assay with automatic detection and result analysis. We evaluated the clinical performance of the FluoroType assay and its feasibility in replacing the GenoType Mycobacterium CM assay as the initial method for mycobacterial identification. A total of 191 clinical mycobacterial cultures were analyzed in this study: 180 identified for one mycobacterial species, 6 for multiple, and 5 for no mycobacterial species. Positive percent agreement (PPA) for the FluoroType assay was 87.8% (n = 158), with full agreement for 23/29 species. Weakest PPA was observed for Mycobacterium gordonae (50%, n = 9/18), Mycobacterium interjectum (40%, n = 2/5), and Mycobacterium intracellulare (42%, n = 5/12). Clinical and mixed cultures containing multiple mycobacterial species gave equally single species and genus level identifications (n = 30). No cross-reactivity with non-mycobacterial species was observed (n = 22). In a separate in silico analysis of 2016-2022 HUS area (Finland) register data (n = 2,573), the FluoroType assay was estimated to produce 18.8% (n = 471) inadequate identifications (genus/false species) if used as the primary identification method compared to 14.2% (n = 366) with the GenoType CM assay. The FluoroType assay was significantly more convenient in terms of assay workflow and result interpretation compared to the entirely manual and subjective GenoType CM assay. However, the feasibility of the assay should be critically assessed with respect to the local NTM species distribution. IMPORTANCE: This study is the first clinical evaluation report of the novel FluoroType Mycobacteria assay. The assay has the potential to replace the established GenoType NTM product family in identification of culture-enriched mycobacteria. However, our research results suggest that the assay performs suboptimally and may not be feasible for use in all clinical settings.

Evaluation of the Roche-SD Biosensor rapid antigen test: Antigen is not reliable in detecting SARS-CoV-2 at the early stage of infection with respiratory symptoms.

We evaluated a rapid antigen test against SARS-CoV-2 virus (Roche-SD Biosensor; RSDB-RAT) in children and adults with respiratory symptoms compared to those with nonrespiratory symptoms or asymptomatic. Also the performance of RSDB-RAT with respect to the duration of respiratory symptoms was assessed. A viral cross-reactivity panel was included. RSDB-RAT was reliable in detecting SARS-CoV-2 in children and adults if the respiratory symptoms had endured 1 to 7 days. If the respiratory symptoms had lasted less than 1 day, the sensitivity was significantly lower. No cross-reactivity with other respiratory viruses was observed.

Incidence Trends for SARS-CoV-2 Alpha and Beta Variants, Finland, Spring 2021.

Severe acute respiratory syndrome coronavirus 2 Alpha and Beta variants became dominant in Finland in spring 2021 but had diminished by summer. We used phylogenetic clustering to identify sources of spreading. We found that outbreaks were mostly seeded by a few introductions, highlighting the importance of surveillance and prevention policies.

Comparison of 2 fully automated tests detecting antibodies against nucleocapsid N and spike S1/S2 proteins in COVID-19.

Automated assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in coronavirus disease 2019 (COVID-19) diagnostics have recently come available. We compared the performance of the Elecsys® Anti-SARS-CoV-2 and LIAISON® SARS-CoV-2 S1/S2 IgG tests. The seroconversion panel comprised of 120 samples from 13 hospitalized COVID-19 patients. For the sensitivity and specificity testing, samples from COVID-19 outpatients >15 days after positive nucleic acid amplification test (NAAT) result (n = 35) and serum control samples collected before the COVID-19 era (n = 161) were included in the material. Samples for the detection of possible cross-reactions were also tested. Based on our results, the SARS-CoV-2 antibodies can be quite reliably detected 2 weeks after NAAT positivity and 3 weeks after the symptom onset with both tests. However, since some COVID-19 patients were positive only with Elecsys®, the antibodies should be screened against N-antigen (Elecsys®) and reactive samples confirmed with S antigen (LIAISON®), but both results should be reported. In some COVID-19 patients, the serology can remain negative.

Evaluation of a semi-automated Seegene PCR workflow with MTB, MDR, and NTM detection for rapid screening of tuberculosis in a low-prevalence setting.

In areas of low tuberculosis (TB) prevalence, laboratory diagnosis of TB may essentially cover non-tuberculous mycobacteria (NTM) in addition to Mycobacterium tuberculosis (MTB). In this study, a semi-automated PCR workflow distinguishing MTB and NTM (Anyplex™ MTB/NTMe, Seegene) and subsequently detecting MTB isoniazid/rifampicin resistance (Allplex™ MTB/MDRe, Seegene) was evaluated for replacing smear microscopy of acid-fast bacilli as the rapid screening method for TB. With 279 clinical samples, 47 cultures positive for MTB and 76 for NTM, the Anyplex™ MTB/NTMe assay and smear microscopy showed equal sensitivities (49.6% vs 50.8%, respectively) but Anyplex™ MTB/NTMe was more sensitive for MTB (63.8% vs 25.6%) than for NTM (40.8% vs 64.5%). Allplex™ MTB/MDRe showed a slightly higher sensitivity of 68.1% for MTB (32/47 positive, n = 222). Antibiotic resistance profiles were correctly identified for all MTB isolates (one MDR isolate). Specificity was 100% for both assays. Anyplex™ MTB/NTMe detected all the 18 NTM species present in the study. The analytical performance of the evaluated high-throughput workflow was relatively weak compared to culture but potentially adequate as a rapid screening method analogous to smear microscopy with additional differentiation between TB, MDR-TB, and NTM.

Evaluation of two tuberculosis PCR assays for routine use in a clinical setting of low population and low tuberculosis prevalence.

Today, there are numerous different molecular diagnostic assays for the detection of tuberculosis (TB), allowing the optimization of rapid detection of TB according to the clinical need. In this study, two high-throughput TB PCR assays with combined antimicrobial resistance detection, Anyplex™ II MTB/MDR (Seegene) and RealTime MTB + RealTime MTB RIF/INH Resistance (Abbott Molecular), were evaluated for routine use in a clinical setting of low population and low TB prevalence in Finland. The RealTime MTB assay was 100% concordant (22/22 positive, n = 169) with the reference methods (culture and Xpert MTB/RIF PCR assay, Cepheid). However, with a limitation of four separate PCR cycles per kit, the routine use in a low TB-prevalence setting would easily lead to wasting most of the RIF/INH Resistance reagents. The Anyplex™ II MTB/MDR assay usability was more adaptive to suit the clinical setting but the assay sensitivity was considerably lower (86%, 19/22 positive, n = 76) being closer to the sensitivity of smear microscopy. The findings of this study suggest that the evaluated high-throughput MTB/MDR assays are evidently suboptimal for routine use in a low population, low TB-prevalence setting. In addition, neither of the two assays covers non-tuberculous mycobacteria and could therefore not fully replace acid-fast staining as the initial screening method.

Priming of innate antimycobacterial immunity by heat-killed Listeria monocytogenes induces sterilizing response in the adult zebrafish tuberculosis model.

Mycobacterium tuberculosis remains one of the most problematic infectious agents, owing to its highly developed mechanisms to evade host immune responses combined with the increasing emergence of antibiotic resistance. Host-directed therapies aiming to optimize immune responses to improve bacterial eradication or to limit excessive inflammation are a new strategy for the treatment of tuberculosis. In this study, we have established a zebrafish-Mycobacterium marinum natural host-pathogen model system to study induced protective immune responses in mycobacterial infection. We show that priming adult zebrafish with heat-killed Listeria monocytogenes (HKLm) at 1 day prior to M. marinum infection leads to significantly decreased mycobacterial loads in the infected zebrafish. Using rag1-/- fish, we show that the protective immunity conferred by HKLm priming can be induced through innate immunity alone. At 24 h post-infection, HKLm priming leads to a significant increase in the expression levels of macrophage-expressed gene 1 (mpeg1), tumor necrosis factor α (tnfa) and nitric oxide synthase 2b (nos2b), whereas superoxide dismutase 2 (sod2) expression is downregulated, implying that HKLm priming increases the number of macrophages and boosts intracellular killing mechanisms. The protective effects of HKLm are abolished when the injected material is pretreated with nucleases or proteinase K. Importantly, HKLm priming significantly increases the frequency of clearance of M. marinum infection by evoking sterilizing immunity (25 vs 3.7%, P=0.0021). In this study, immune priming is successfully used to induce sterilizing immunity against mycobacterial infection. This model provides a promising new platform for elucidating the mechanisms underlying sterilizing immunity and to develop host-directed treatment or prevention strategies against tuberculosis.This article has an associated First Person interview with the first author of the paper.

ß-CA-specific inhibitor dithiocarbamate Fc14-584B: a novel antimycobacterial agent with potential to treat drug-resistant tuberculosis.

Inhibition of novel biological pathways in Mycobacterium tuberculosis (Mtb) creates the potential for alternative approaches for treating drug-resistant tuberculosis. In vitro studies have shown that dithiocarbamate-derived ß-carbonic anhydrase (ß-CA) inhibitors Fc14-594 A and Fc14-584B effectively inhibit the activity of Mtb ß-CA enzymes. We screened the dithiocarbamates for toxicity, and studied the in vivo inhibitory effect of the least toxic inhibitor on M. marinum in a zebrafish model. In our toxicity screening, Fc14-584B emerged as the least toxic and showed minimal toxicity in 5-day-old larvae at 300 µM concentration. In vitro inhibition of M. marinum showed that both compounds inhibited growth at a concentration of 75 µM. In vivo inhibition studies using 300 µM Fc14-584B showed significant (p > .05) impairment of bacterial growth in zebrafish larvae at 6 days post infection. Our studies highlight the therapeutic potential of Fc14-584B as a ß-CA inhibitor against Mtb, and that dithiocarbamate compounds may be developed into potent anti-tuberculosis drugs.

Adequate Th2-type response associates with restricted bacterial growth in latent mycobacterial infection of zebrafish.

Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2)-type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease.