Evidence for broad cross-reactivity of the SARS-CoV-2 NSP12-directed CD4+ T-cell response with pre-primed responses directed against common cold coronaviruses.

Introduction: The nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC). Methods: Here, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at single-peptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs. Results: Surprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4+ T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0-25) and seronegative controls including pre-pandemic samples (mean: 12.71 responses; range: 0-21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4+ T-cell peptide specificities in COVID-19 patients were aa236-250 (37%) and aa246-260 (44%), whereas the peptide specificities aa686-700 (50%) and aa741-755 (36%), were the most frequently detected in seronegative controls. In CCC-specific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro. However, the NSP12 peptide-specific CD4+ T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection. Discussion: The results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs.

High prevalence of periodontal disease in patients with NASH- possible association of poor dental health with NASH severity.

INTRODUCTION AND OBJECTIVES: Recent translational research indicated a bidirectional relationship between NASH (non-alcoholic steatohepatitis) and periodontitis; however, few clinical cohorts have studied this in detail. Thus we investigated this assumed association in a well-defined cohort. MATERIALS AND METHODS: Data were generated prospectively for 132 patients (32 patients with NASH and 100 unselected, consecutively collected, anonymized controls from a local dental practice): detailed periodontal parameters, i.e., pocket-probing-depths (PPD), bleeding-on-probing (BOP), plaque-index, and utilization of dental care were assessed and correlated with relevant hepatic parameters (liver stiffness via fibroscan, AST, ALT, bilirubin, and MELD-score). Gingiva samples were tested for Porphyromonas gingvalis (P.g.) and Actinobacillus actinomyctemcomitans (A.a.) by PCR. RESULTS: 87.5% of NASH patients and 47% of controls suffered from moderate to severe periodontitis (p=0.01). Liver stiffness was significantly correlated with elevated PPD (p=0.02) and BOP (p=0.03). 34 % of the NASH patients did not make use of regular dental health care. In these patients, AST (p=0.04), MELD score (p<0.01), and liver stiffness (p=0.01) were significantly elevated compared to those who see a dentist regularly. The severity of NASH was not associated with the intraoral detection of P.g. and A.a. CONCLUSIONS: The present study suggests that NASH might be associated with periodontitis, irrespective of the intraoral presence of P.g. and A.a. Moreover, regular dental care utilization might mitigate the course of NASH, and patients should be reminded by their hepatologists of the importance of regular dental visits. Future studies should investigate the role of regular dental care and additional anti-inflammatory treatments of the oral cavity.

High-resolution analysis of individual spike peptide-specific CD4+ T-cell responses in vaccine recipients and COVID-19 patients.

Objectives: Potential differences in the breadth, distribution and magnitude of CD4+ T-cell responses directed against the SARS-CoV-2 spike glycoprotein between vaccinees, COVID-19 patients and subjects who experienced both ways of immunisation have not been comprehensively compared on a peptide level. Methods: Following virus-specific in vitro cultivation, we determined the T-cell responses directed against 253 individual overlapping 15-mer peptides covering the entire SARS-CoV-2 spike glycoprotein using IFN-γ ELISpot and intracellular cytokine staining. In vitro HLA binding was determined for selected peptides. Results: We mapped 955 single peptide-specific CD4+ T-cell responses in a cohort of COVID-19 patients (n = 8), uninfected vaccinees (n = 16) and individuals who experienced both infection and vaccination (n = 11). Patients and vaccinees (two-time and three-time vaccinees alike) had a comparable number of CD4+ T-cell responses (median 26 vs. 29, P = 0.7289). Most of these specificities were conserved in B.1.1.529 and the BA.4 and BA.5 sublineages. The highest magnitude of these in vitro IFN-γ CD4+ T-cell responses was observed in COVID-19 patients (median 0.35%), and three-time vaccinees showed a higher magnitude than two-time vaccinees (median 0.091% vs. 0.175%, P < 0.0001). Twelve peptide specificities were each detected in at least 40% of subjects. In vitro HLA binding showed promiscuous presentation by DRB1 molecules for several peptides. Conclusion: Both SARS-CoV-2 infection and vaccination prime broadly directed T-cell responses directed against the SARS-CoV-2 spike glycoprotein. This comprehensive high-resolution analysis of spike peptide specificities will be a useful resource for further investigation of spike-specific T-cell responses.

SARS-CoV-2 screening strategies for returning international travellers: Evaluation of a rapid antigen test approach.

BACKGROUND: International travel poses the risk of importing SARS-CoV-2 infections and introducing new viral variants into the country of destination. Established measures include mandatory quarantine with the opportunity to abbreviate it with a negative rapid antigen test (RAT). METHODS: A total of 1,488 returnees were tested for SARS-CoV-2 with both PCR and RAT no earlier than 5 days after arrival. We assessed the sensitivity and specificity of the RAT. Positive samples were evaluated for infectivity in vitro in a cell culture outgrowth assay. We tracked if participants who tested negative were reported positive within 2 weeks of the initial test. RESULTS: Potential infectiousness was determined based on symptom onset analysis, resulting in a sensitivity of the antigen test of 89% in terms of infectivity. The specificity was 100%. All positive outgrowth assays were preceded by a positive RAT, indicating that all participants with proven in vitro infectivity were correctly identified. None of the negative participants tested positive during the follow-up. CONCLUSIONS: RAT no earlier than the 5th day after arrival was a reliable method for detecting infectious travellers and can be recommended as an appropriate method for managing SARS-CoV-2 travel restrictions. Compliance to the regulations and a high standard of test quality must be ensured.

Handling of laundry in nursing homes in Frankfurt am Main, Germany, 2016 - laundry and professional clothing as potential pathways of bacterial transfer.

Background: In accordance with the German Infection Protection Act, the treatment and handling of laundry was checked by the Public Health Department in 2016 in all Frankfurt nursing homes with special focus on the staff's clothing. Methods: On-site visits and surveys were conducted in all 44 nursing homes in Frankfurt/Main, Germany, and random microbiological examinations of 58 reprocessed and 58 already worn protective gowns were performed to determine the numbers of the colony forming units (cfu) and microbiological differentiation of the pathogen species. Results: 41 (93%) of the 44 homes tested had contracted a certified laundry service. 23 (52%) of the homes also ran a laundry of their own; in 21 of these, laundry was reprocessed and disinfected in an industrial washing machine. Regular technical or microbiological tests were carried out in 16 or 12 of the home-owned laundries, respectively. Only 31 homes (70%) provided uniforms for their employees. The staff's clothing was processed in 25 homes by the external laundry, in 9 homes by the internal laundry, and in 12 homes, the nursing staff had to do this privately at their own home. Used coats exhibited significantly higher contamination than freshly prepared ones (median: 80 vs. 2 cfu/25 cm2; P 95 percentile: 256 cfu vs. 81 cfu/25 cm2). Clothing prepared in private homes showed significantly higher contamination rates than those washed in the certified external laundry or in the nursing homes themselves (Median: 16 cfu/25 cm2 vs. 0.5-1 cfu/25 cm2). Conclusion: Considering various publications on pathogen transfers and outbreaks due to contaminated laundry in medical facilities, the treatment of laundry, in particular the uniforms, must be given more attention, also in nursing homes for the elderly. The private reprocessing of occupational clothing by the employees at home must be rejected on hygienic principles, and is furthermore prohibited by law in Germany.

Prevention and control of catheter-associated urinary tract infections - implementation of the recommendations of the Commission for Hospital Hygiene and Infection Prevention (KRINKO) in nursing homes for the elderly in Frankfurt am Main, Germany.

INTRODUCTION: Urinary tract infections range among the most frequent infections not only in hospital patients but also in residents of long-term care facilities for the elderly. Urinary catheters are the greatest risk factor for urinary tract infections. In the guidance paper on the "prevention of infections in nursing homes" (2005) as well as in the updated recommendations for the "prevention and control of catheter-associated urinary tract infections" (2015), the Commission for Hospital Hygiene and Infection Prevention (KRINKO) has recommended adequate preventive measures. In 2015, the implementation of these KRINKO recommendations was investigated. METHOD: All of Frankfurt's 40 nursing homes were evaluated using a checklist based on the KRINKO recommendations. The evaluation included assessing the availability of operating instructions, appropriate indications for the placement of catheters etc. Age, sex and duration of catheterization, as well as current and previous infections within the past 6 months were documented for every resident with a catheter. RESULTS: In 35 (87.5%) of the nursing homes, operating instructions for the handling of urinary tract catheters were available. The decision as to whether a catheter is indicated is made by physicians, while its placement is often delegated to the nursing service. Typically, silicon catheters are used. In three-quarters of the nursing homes, regular intervals of 4-6 weeks for changing catheters were reported. On the respective survey day, 7.3% of the residents were catheterized. On the survey day, 3.6% (4.2%) and in the previous 6 months a total of 28% (28.9%) of the residents had a urinary tract infection (prevalence of antibiotic therapy in parentheses). Ciprofloxacin was used most often followed by cefuroxime and cotrimoxazole. DISCUSSION: In the current evaluation, fewer nursing home residents were catheterized than in previous years and the rate of urinary tract infections was low. This indicates an increasingly cautious and apparently appropriate usage of urinary tract catheters. Also, the prevalence of antibiotic therapy was low for residents with urinary tract catheters (4.2%). However, broad spectrum antibiotics are still preferentially administered (particularly quinolones), which may favor the high rate of colonization with ESBL-producing bacteria and 3MRGN. Given this background, a coordinated approach including resistance-based antibiotic stewardship appears increasingly important in nursing homes and other health care facilities.

Nicotinic acid adenine dinucleotide phosphate (NAADP) degradation by alkaline phosphatase.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous second messenger providing a Ca(2+) trigger in a wide range of cell types. However, its metabolism is not well understood. Here, we demonstrate the presence of endogenous NAADP in HeLa cells. CD38, a promiscuous enzyme described to be involved in NAADP metabolism, was not detectable in HeLa cells. In cell-free extracts of HeLa cells, NAADP was degraded to nicotinic acid adenine dinucleotide (NAAD). The enzyme was enriched in membranes (10,000 × g pellet) and displayed characteristics typical of alkaline phosphatase (AP), e.g. pH optimum at 8-9 and sensitivity to the inhibitors L-homoarginine and L-leucine. Importantly, NAADP at physiological concentrations (50-100 nM) was degraded to NAAD. Expression of AP isoenzymes was analyzed in HeLa cells. Based on the results together with inhibitor studies, the placental AP isoform emerged as the best candidate for NAADP degradation in HeLa cells. In contrast to HeLa cells, Jurkat T cells or HEK293 cells did not express any AP isoenzymes and did not display any NAADP 2'-phosphatase activity. Finally, the placental AP isoform was expressed heterologously in HEK293 cells, resulting in reconstitution of NAADP 2'-phosphatase activity in cell-free extracts. On the basis of the results, we provide evidence for AP as the metabolizing enzyme of NAADP in cells that do not express CD38.