Treatment and Outcome of Culture-Confirmed Mycobacterium marinum Disease.

Background: Mycobacterium marinum is a nontuberculous mycobacterium that causes skin and soft tissue infections. Treatment consists of multiple antibiotics, sometimes combined with surgical debridement. There is little evidence for the choice of antibiotics, the duration of treatment, and the role of susceptibility testing. Methods: We performed a retrospective cohort study of culture-confirmed M. marinum infections in the Netherlands in the 2011-2018 period. Clinical characteristics, in vitro susceptibility, extent of disease, treatment regimens, and outcomes were analyzed. Incidence was assessed from laboratory databases. Results: Forty cases of M. marinum infection could be studied. Antibiotic treatment cured 36/40 patients (90%) after a mean treatment duration of 25 weeks. Failure/relapse occurred in 3 patients, and 1 patient was lost to follow-up. Antibiotic treatment consisted of monotherapy in 35% and 2-drug therapy in 63%. Final treatment contained mostly ethambutol-macrolide combinations (35%). Eleven patients (28%) received additional surgery. We recorded high rates of in vitro resistance to tetracyclines (36% of isolates). Tetracycline resistance seemed correlated with poor response to tetracycline monotherapy. The annual incidence rate was 0.15/100 000/year during the study period. Conclusions: Prolonged and susceptibility-guided treatment results in a 90% cure rate in M. marinum disease. Two-drug regimens of ethambutol and a macrolide are effective for moderately severe infections. Tetracycline monotherapy in limited disease should be used vigilantly, preferably with proven in vitro susceptibility.

Increasing diagnostic possibilities using the geneLEAD VIII platform for detection of SARS-CoV-2.

At the time SARS-CoV-2 was identified as the cause of coronavirus disease 2019 (COVID-19) no in vitro diagnostic (IVD) tests were available since it was a new virus. Very shortly after the release of the genomic sequence of SARS-CoV-2, laboratory-developed tests (LDTs) were developed, made available and implemented in several laboratories in the Netherlands and globally. In this study, the performance of an E-gene Sarbeco specific real-time reverse-transcriptase PCR (RT-PCR) was verified on the open modus of the geneLEAD VIII sample-to-answer platform. The results obtained from 134 clinical samples, of which 63 had been tested positive, showed almost complete concordance compared to the same PCR on the routine diagnostic systems and that was validated according to the national reference standard. The only discordant sample tested positive using the routine diagnostic workflow with a cycle threshold (CT) value of 37.7, while the sample tested negative using the geneLEAD VIII workflow. In addition, good performance was achieved in analyzing a blinded SARS-CoV-2 external quality assurance (EQA) panel. Implementation of the geneLEAD VIII platform as routine diagnostic tool resulted in testing 871 clinical samples with 115 positive results. In conclusion, the geneLEAD VIII SARS-CoV-2 workflow presented in this study showed excellent diagnostic performance and with a rapid turnaround time of approximately two hours it proved a valuable option for STAT SARS-CoV-2 testing in the absence of (rapid, CE-IVD) point-of-care testing platforms.

Mycobacterium tuberculosis clinical isolates of the Beijing and East-African Indian lineage induce fundamentally different host responses in mice compared to H37Rv.

Substantial differences exist in virulence among Mycobacterium tuberculosis strains in preclinical TB models. In this study we show how virulence affects host responses in mice during the first four weeks of infection with a mycobacterial strain belonging to the Beijing, East-African-Indian or Euro-American lineage. BALB/c mice were infected with clinical isolates of the Beijing-1585 strain or the East-African Indian (EAI)-1627 strain and host responses were compared to mice infected with the non-clinical H37Rv strain of the Euro-American lineage. We found that H37Rv induced a 'classical' T-cell influx with high IFN-γ levels, while Beijing-1585 and EAI-1627 induced an influx of B-cells into the lungs together with elevated pulmonary IL-4 protein levels. Myeloid cells in the lungs appeared functionally impaired upon infection with Beijing-1585 and EAI-1627 with reduced iNOS and IL-12 expression levels compared to H37Rv infection. This impairment might be related to significantly reduced expression in the bone marrow of IFN-γ, TNF-α and IFN-ß in mice infected with Beijing-1585 and EAI-1627, which could be detected from the third day post infection onwards. Our findings suggest that increased virulence of two clinical isolates compared to H37Rv is associated with a fundamentally different systemic immune response, which already can be detected early during infection.

Improving treatment outcome assessment in a mouse tuberculosis model.

Preclinical treatment outcome evaluation of tuberculosis (TB) occurs primarily in mice. Current designs compare relapse rates of different regimens at selected time points, but lack information about the correlation between treatment length and treatment outcome, which is required to efficiently estimate a regimens' treatment-shortening potential. Therefore we developed a new approach. BALB/c mice were infected with a Mycobacterium tuberculosis Beijing genotype strain and were treated with rifapentine-pyrazinamide-isoniazid-ethambutol (RpZHE), rifampicin-pyrazinamide-moxifloxacin-ethambutol (RZME) or rifampicin-pyrazinamide-moxifloxacin-isoniazid (RZMH). Treatment outcome was assessed in n = 3 mice after 9 different treatment lengths between 2-6 months. Next, we created a mathematical model that best fitted the observational data and used this for inter-regimen comparison. The observed data were best described by a sigmoidal Emax model in favor over linear or conventional Emax models. Estimating regimen-specific parameters showed significantly higher curative potentials for RZME and RpZHE compared to RZMH. In conclusion, we provide a new design for treatment outcome evaluation in a mouse TB model, which (i) provides accurate tools for assessment of the relationship between treatment length and predicted cure, (ii) allows for efficient comparison between regimens and (iii) adheres to the reduction and refinement principles of laboratory animal use.

Assessment of Bactericidal Drug Activity and Treatment Outcome in a Mouse Tuberculosis Model Using a Clinical Beijing Strain.

Mycobacterium tuberculosis Beijing strains are associated with lower treatment success rates in tuberculosis (TB) patients. In contrast, laboratory strains such as H37Rv are often used in preclinical tuberculosis models. Therefore, we explored the impact of using a clinical Beijing strain on treatment outcome in our mouse tuberculosis model. Additionally, the predictive value of bactericidal activity on treatment outcome was assessed. BALB/c mice were infected with a Beijing strain and treated with one of 10 different combinations of conventional anti-TB drugs. Bactericidal activity was assessed by determining reductions in mycobacterial load after 7, 14, and 28 days and after 2, 3, and 6 months of treatment. Treatment outcome was evaluated after a 6-month treatment course and was based on lung culture status 3 months posttreatment. None of the anti-TB drug regimens tested could achieve 100% treatment success. Treatment outcome depended critically on rifampin. Four non-rifampin-containing regimens showed 0% treatment success compared to success rates between 81 and 95% for six rifampin-containing regimens. Bactericidal activity was predictive only for treatment outcome after 3 months of treatment. Our data advocate the use of multiple mycobacterial strains, including a Beijing strain, to increase the translational value of mouse TB models evaluating treatment outcome. Additionally, our findings support the notion that bactericidal activity in the first 2 months of treatment, as measured in clinical phase IIa/b trials, has limited predictive value for tuberculosis treatment outcome, thus emphasizing the need for better parameters to guide future phase IIII trials.

Interactions between Type 1 Interferons and the Th17 Response in Tuberculosis: Lessons Learned from Autoimmune Diseases.

The classical paradigm of tuberculosis (TB) immunity, with a central protective role for Th1 responses and IFN-γ-stimulated cellular responses, has been challenged by unsatisfactory results of vaccine strategies aimed at enhancing Th1 immunity. Moreover, preclinical TB models have shown that increasing IFN-γ responses in the lungs is more damaging to the host than to the pathogen. Type 1 interferon signaling and altered Th17 responses have also been associated with active TB, but their functional roles in TB pathogenesis remain to be established. These two host responses have been studied in more detail in autoimmune diseases (AID) and show functional interactions that are of potential interest in TB immunity. In this review, we first identify the role of type 1 interferons and Th17 immunity in TB, followed by an overview of interactions between these responses observed in systemic AID. We discuss (i) the effects of GM-CSF-secreting Th17.1 cells and type 1 interferons on CCR2+ monocytes; (ii) convergence of IL-17 and type 1 interferon signaling on stimulating B-cell activating factor production and the central role of neutrophils in this process; and (iii) synergy between IL-17 and type 1 interferons in the generation and function of tertiary lymphoid structures and the associated follicular helper T-cell responses. Evaluation of these autoimmune-related pathways in TB pathogenesis provides a new perspective on recent developments in TB research.

Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis.

Immune-modulating drugs that target myeloid-derived suppressor cells or stimulate natural killer T cells have been shown to reduce mycobacterial loads in tuberculosis (TB). We aimed to determine if a combination of these drugs as adjunct immunotherapy to conventional antibiotic treatment could also increase therapeutic efficacy against TB. In our model of pulmonary TB in mice, we applied treatment with isoniazid, rifampicin, and pyrazinamide for 13 weeks alone or combined with immunotherapy consisting of all-trans retinoic acid, 1,25(OH)2-vitamin D3, and α-galactosylceramide. Outcome parameters were mycobacterial load during treatment (therapeutic activity) and 13 weeks after termination of treatment (therapeutic efficacy). Moreover, cellular changes were analyzed using flow cytometry and cytokine expression was assessed at the mRNA and protein levels. Addition of immunotherapy was associated with lower mycobacterial loads after 5 weeks of treatment and significantly reduced relapse of disease after a shortened 13-week treatment course compared with antibiotic treatment alone. This was accompanied by reduced accumulation of immature myeloid cells in the lungs at the end of treatment and increased TNF-α protein levels throughout the treatment period. We demonstrate, in a mouse model of pulmonary TB, that immunotherapy consisting of three clinically approved drugs can improve the therapeutic efficacy of standard antibiotic treatment.