Seroprevalence of SARS-CoV-2 in German secondary schools from October 2020 to July 2021: a longitudinal study.

PURPOSE: To quantify the number of SARS-CoV-2 infections in students and teachers in 14 Secondary schools in eastern Saxony, Germany. Seroprevalence of SARS-CoV-2 antibodies in study population. Number of undetected cases. METHODS: Serial seroprevalence study. RESULTS: The role of educational settings in the SARS-CoV-2 Pandemic is still controversial. Seroprevalence increases from 0.8 to 5.9% from October to December when schools remained open and to 12.2% in March/April during a strict lockdown with closed schools. The ratio of undetected to detected cases decreased from 0.76 to 0.44 during the study period. CONCLUSION: During the second and third wave of the pandemic in Germany, students and teachers are not overrepresented in SARS-CoV-2 infections. The percentage of undetected cases is moderate and decreases over time. The risk of contracting SARS-CoV-2 within the household is higher than contracting it in educational settings making school closures rather ineffective in terms of pandemic control measures or individual risk reduction in children and adolescents. TRIAL REGISTRATION: DRKS00022455 (July 23rd, 2020).

Legionella antibiotic susceptibility testing: is it time for international standardization and evidence-based guidance?

Legionella pneumophila, a Gram-negative bacillus, is the causative agent of Legionnaire's disease, a form of severe community-acquired pneumonia. Infection can have high morbidity, with a high proportion of patients requiring ICU admission, and up to 10% mortality, which is exacerbated by the lack of efficacy of typical empirical antibiotic therapy against Legionella spp. The fastidious nature of the entire Legionellaceae family historically required inclusion of activated charcoal in the solid medium to remove growth inhibitors, which inherently interferes with accurate antimicrobial susceptibility determination, an acknowledged methodological shortfall, now rectified by a new solid medium that gives results comparable to those of microbroth dilution. Here, as an international Legionella community (with authors representing various international reference laboratories, countries and clinical stakeholders for diagnosis and treatment of legionellosis), we set out recommendations for the standardization of antimicrobial susceptibility testing methods, guidelines and reference strains to facilitate an improved era of antibiotic resistance determination.

Prevalence and Transmission of Severe Acute Respiratory Syndrome Coronavirus Type 2 in Childcare Facilities: A Longitudinal Study.

OBJECTIVE: To evaluate the role of childcare facilities in the transmission of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) in a longitudinal study to gain further knowledge of SARS-CoV-2 prevalence, transmission, and spread among preschool children, their parents, and their caregivers. STUDY DESIGN: Children aged 1-6 years, their parents, and their caregivers in 14 childcare facilities in Dresden, Saxony/Germany were invited to participate in the KiTaCoviDD19-study between July 2020 and January 2021. Seroprevalence of SARS-CoV-2 antibodies was assessed up to 4 times during the study period in all participating adults, and demographic characteristics, as well as epidemiologic information on personal SARS-CoV-2 history were obtained. Samples for stool virus shedding of SARS-CoV-2 were analyzed by polymerase chain reaction every 2-4 weeks in all participating children. RESULTS: In total, 318 children, 299 parents and 233 childcare workers were enrolled. By January 2021, 11% of the participating adults were found to be seropositive, whereas the percentage of children shedding SARS-CoV-2 was 6.8%. Overall, we detected 17 children with SARS-CoV-2 virus shedding in 8 different childcare facilities. In 4 facilities, there were a maximum of 3 connected cases in children. Approximately 50% of SARS-CoV-2 infections in the children could not be connected to a secondary case in our study population. CONCLUSIONS: This study does not provide evidence of relevant asymptomatic ("silent") spread of SARS-CoV-2 in childcare facilities in both low- and high-prevalence settings. Our findings add to the evidence that childcare and educational settings do not have a crucial role in driving the SARS-CoV-2 pandemic.

Persistent presence of outer membrane epitopes during short- and long-term starvation of five Legionella pneumophila strains.

BACKGROUND: Legionella pneumophila, the causative agent of Legionnaire's disease, may enter a viable but non-culturable (VBNC) state triggered by environmental stress conditions. Specific outer-membrane epitopes of L. pneumophila are used in many diagnostic applications and some of them are linked to important virulence-related factors or endotoxins. However, it is not clear how the presence and status of these epitopes are influenced by environmental stress conditions. In this study, changes of outer membrane epitopes for monoclonal antibodies (mAb) from the Dresden panel and the major outer membrane protein MOMP were analysed for five L. pneumophila strains during short- and long-term starvation in ultrapure water. RESULTS: With ELISA and single cell immuno-fluorescence analysis, we could show that for most of the investigated mAb-strain combinations the total number of mAb-stained Legionella cells stayed constant for up to 400 days. Especially the epitopes of mAb 3/1, 8/5, 26/1 and 20/1, which are specific for L. pneumophila serogroup 1 subtypes, and the mAb 9/1, specific for serogroup 6, showed long-term persistence. For most mAb- stained cells, a high percentage of viable cells was observed at least until 118 days of starvation. At the same time, we observed a reduction of the fluorescence intensity of the stained cells during starvation indicating a loss of epitopes from the cell surface. However, most of the epitopes, including the virulence-associated mAb 3/1 epitope were still present with high fluorescence intensity after 400 days of starvation in up to 50% of the starved L. pneumophila population. CONCLUSIONS: The results demonstrate the continuous presence of outer membrane epitopes of L. pneumophila during short-term and long-term starvation. Thus, culture-independent mAb-based diagnostic and detection tools, such as immuno-magnetic separation and microarray techniques are applicable for both L. pneumophila in the culturable and the VBNC state even after long-term starvation but nevertheless require careful testing before application. However, the mere presence of those epitopes is not necessarily an indication of viability or infectivity.

Epidemiological information is key when interpreting whole genome sequence data - lessons learned from a large Legionella pneumophila outbreak in Warstein, Germany, 2013.

IntroductionWhole genome sequencing (WGS) is increasingly used in Legionnaires' disease (LD) outbreak investigations, owing to its higher resolution than sequence-based typing, the gold standard typing method for Legionella pneumophila, in the analysis of endemic strains. Recently, a gene-by-gene typing approach based on 1,521 core genes called core genome multilocus sequence typing (cgMLST) was described that enables a robust and standardised typing of L. pneumophila. Methods: We applied this cgMLST scheme to isolates obtained during the largest outbreak of LD reported so far in Germany. In this outbreak, the epidemic clone ST345 had been isolated from patients and four different environmental sources. In total 42 clinical and environmental isolates were retrospectively typed. Results: Epidemiologically unrelated ST345 isolates were clearly distinguishable from the epidemic clone. Remarkably, epidemic isolates split up into two distinct clusters, ST345-A and ST345-B, each respectively containing a mix of clinical and epidemiologically-related environmental samples. Discussion/conclusion: The outbreak was therefore likely caused by both variants of the single sequence type, which pre-existed in the environmental reservoirs. The two clusters differed by 40 alleles located in two neighbouring genomic regions of ca 42 and 26 kb. Additional analysis supported horizontal gene transfer of the two regions as responsible for the difference between the variants. Both regions comprise virulence genes and have previously been reported to be involved in recombination events. This corroborates the notion that genomic outbreak investigations should always take epidemiological information into consideration when making inferences. Overall, cgMLST proved helpful in disentangling the complex genomic epidemiology of the outbreak.

Microbiological diagnosis and molecular typing of Legionella strains during an outbreak of legionellosis in Southern Germany.

An explosive outbreak of Legionnaires' disease with 64 reported cases occurred in Ulm/Neu-Ulm in the South of Germany in December 2009/January 2010 caused by Legionella (L.) pneumophila serogroup 1, monoclonal (mAb) subtype Knoxville, sequence type (ST) 62. Here we present the clinical microbiological results from 51 patients who were diagnosed at the University hospital of Ulm, the results of the environmental investigations and of molecular typing of patients and environmental strains. All 50 patients from whom urine specimens were available were positive for L. pneumophila antigen when an enzyme-linked immunosorbent assay (EIA) was used following concentration of those urine samples that tested initially negative. The sensitivity of the BinaxNow rapid immunographic assay (ICA), after 15 min reading and after 60 min reading were 70% and 84%, respectively. Direct typing confirmed the monoclonal subtype Knoxville in 5 out of 8 concentrated urine samples. Real time PCR testing of respiratory tract specimens for L. pneumophila was positive in 15 out of 25 (60%) patients. Direct nested sequence based typing (nSBT) in some of these samples allowed partial confirmation of ST62. L. pneumophila serogroup 1, monoclonal subtype Knoxville ST62, defined as the epidemic strain was isolated from 8 out of 31 outbreak patients (26%) and from one cooling tower confirming it as the most likely source of the outbreak. While rapid detection of Legionella antigenuria was crucial for the recognition and management of the outbreak, culture and molecular typing of the strains from patients and environmental specimens was the clue for the rapid identification of the source of infection.

Rapid quantification method for Legionella pneumophila in surface water.

World-wide legionellosis outbreaks caused by evaporative cooling systems have shown that there is a need for rapid screening methods for Legionella pneumophila in water. Antibody-based methods for the quantification of L. pneumophila are rapid, non-laborious, and relatively cheap but not sensitive enough for establishment as a screening method for surface and drinking water. Therefore, preconcentration methods have to be applied in advance to reach the needed sensitivity. In a basic test, monolithic adsorption filtration (MAF) was used as primary preconcentration method that adsorbs L. pneumophila with high efficiency. Ten-liter water samples were concentrated in 10 min and further reduced to 1 mL by centrifugal ultrafiltration (CeUF). The quantification of L. pneumophila strains belonging to the monoclonal subtype Bellingham was performed via flow-based chemiluminescence sandwich microarray immunoassays (CL-SMIA) in 36 min. The whole analysis process takes 90 min. A polyclonal antibody (pAb) against L. pneumophila serogroup 1-12 and a monoclonal antibody (mAb) against L. pneumophila SG 1 strain Bellingham were immobilized on a microarray chip. Without preconcentration, the detection limit was 4.0 × 10(3) and 2.8 × 10(3) CFU/mL determined by pAb and mAb 10/6, respectively. For samples processed by MAF-CeUF prior to SMIA detection, the limit of detection (LOD) could be decreased to 8.7 CFU/mL and 0.39 CFU/mL, respectively. A recovery of 99.8 ± 15.9% was achieved for concentrations between 1-1000 CFU/mL. The established combined analytical method is sensitive for rapid screening of surface and drinking water to allow fast hygiene control of L. pneumophila.

Subtyping of the Legionella pneumophila "Ulm" outbreak strain using the CRISPR-Cas system.

In 2009/2010 an outbreak of Legionnaires' disease with 64 cases including four fatalities took place in the city of Ulm/Neu-Ulm in Germany. L. pneumophila serogroup 1, mAb type Knoxville, sequence type (ST) 62 was identified as the epidemic strain. This strain was isolated from eight patients and from a cooling tower in the city of Ulm. Based on whole genome sequencing data from one patient strain, we identified an Lvh type IV secretion system containing a CRISPR-Cas system. The CRISPR sequence contains 38 spacer DNA sequences. We used these variable DNA spacers to further subtype the outbreak strain as well as six epidemiologically unrelated strains of CRISPR-Cas positive ST62 strains isolated at various regions in Germany. The first 12 spacer DNAs of eight patient isolates and three environmental isolates from the suspected source of infection were analyzed and found to be identical. Spacer DNAs were identified in further six epidemiologically unrelated patient isolates of L. pneumophila of ST62 in addition to the 12 "core" spacers. The presence of new spacer DNAs at the 5' site downstream of the first repeat indicates that these CRISPR-Cas systems seem to be functional. PCR analysis revealed that not all L. pneumophila sg1 ST62 strains investigated exhibited a CRISPR-Cas system. In addition, we could demonstrate that the CRISPR-Cas system is localized on a genomic island (LpuGI-Lvh) which can be excised from the chromosome and therefore may be transferable horizontally to other L. pneumophila strains.

DNA-Microarray-based Genotyping of Clostridium difficile.

BACKGROUND: Clostridium difficile can cause antibiotic-associated diarrhea and a possibility of outbreaks in hospital settings warrants molecular typing. A microarray was designed that included toxin genes (tcdA/B, cdtA/B), genes related to antimicrobial resistance, the slpA gene and additional variable genes. RESULTS: DNA of six reference strains and 234 clinical isolates from South-Western and Eastern Germany was subjected to linear amplification and labeling with dUTP-linked biotin. Amplicons were hybridized to microarrays providing information on the presence of target genes and on their alleles. Tested isolates were assigned to 37 distinct profiles that clustered mainly according to MLST-defined clades. Three additional profiles were predicted from published genome sequences, although they were not found experimentally. CONCLUSIONS: The microarray based assay allows rapid and high-throughput genotyping of clinical C. difficile isolates including toxin gene detection and strain assignment. Overall hybridization profiles correlated with MLST-derived clades.

Epidemiological investigation and case-control study: a Legionnaires' disease outbreak associated with cooling towers in Warstein, Germany, August-September 2013.

Between 1 August and 6 September 2013, an outbreak of Legionnaires' disease (LD) with 159 suspected cases occurred in Warstein, North Rhine-Westphalia, Germany. The outbreak consisted of 78 laboratory-confirmed cases of LD, including one fatality, with a case fatality rate of 1%. Legionella pneumophila, serogroup 1, subtype Knoxville, sequence type 345, was identified as the epidemic strain. A case-control study was conducted to identify possible sources of infection. In univariable analysis, cases were almost five times more likely to smoke than controls (odds ratio (OR): 4.81; 95% confidence interval (CI): 2.33-9.93; p < 0.0001). Furthermore, cases were twice as likely to live within a 3 km distance from one identified infection source as controls (OR: 2.14; 95% CI: 1.09-4.20; p < 0.027). This is the largest outbreak of LD in Germany to date. Due to a series of uncommon events, this outbreak was most likely caused by multiple sources involving industrial cooling towers. Quick epidemiological assessment, source tracing and shutting down of potential sources as well as rapid laboratory testing and early treatment are necessary to reduce morbidity and mortality. Maintenance of cooling towers must be carried out according to specification to prevent similar LD outbreaks in the future.

Genome sequence and phenotypic analysis of a first German Francisella sp. isolate (W12-1067) not belonging to the species Francisella tularensis.

BACKGROUND: Francisella isolates from patients suffering from tularemia in Germany are generally strains of the species F. tularensis subsp. holarctica. To our knowledge, no other Francisella species are known for Germany. Recently, a new Francisella species could be isolated from a water reservoir of a cooling tower in Germany. RESULTS: We identified a Francisella sp. (isolate W12-1067) whose 16S rDNA is 99% identical to the respective nucleotide sequence of the recently published strain F. guangzhouensis. The overall sequence identity of the fopA, gyrA, rpoA, groEL, sdhA and dnaK genes is only 89%, indicating that strain W12-1067 is not identical to F. guangzhouensis. W12-1067 was isolated from a water reservoir of a cooling tower of a hospital in Germany. The growth optimum of the isolate is approximately 30°C, it can grow in the presence of 4-5% NaCl (halotolerant) and is able to grow without additional cysteine within the medium. The strain was able to replicate within a mouse-derived macrophage-like cell line. The whole genome of the strain was sequenced (~1.7 mbp, 32.2% G + C content) and the draft genome was annotated. Various virulence genes common to the genus Francisella are present, but the Francisella pathogenicity island (FPI) is missing. However, another putative type-VI secretion system is present within the genome of strain W12-1067. CONCLUSIONS: Isolate W12-1067 is closely related to the recently described F. guangzhouensis species and it replicates within eukaryotic host cells. Since W12-1067 exhibits a putative new type-VI secretion system and F. tularensis subsp. holarctica was found not to be the sole species in Germany, the new isolate is an interesting species to be analyzed in more detail. Further research is needed to investigate the epidemiology, ecology and pathogenicity of Francisella species present in Germany.

Legionella oakridgensis ATCC 33761 genome sequence and phenotypic characterization reveals its replication capacity in amoebae.

Legionella oakridgensis is able to cause Legionnaires' disease, but is less virulent compared to L. pneumophila strains and very rarely associated with human disease. L. oakridgensis is the only species of the family legionellae which is able to grow on media without additional cysteine. In contrast to earlier publications, we found that L. oakridgensis is able to multiply in amoebae. We sequenced the genome of L. oakridgensis type strain OR-10 (ATCC 33761). The genome is smaller than the other yet sequenced Legionella genomes and has a higher G+C-content of 40.9%. L. oakridgensis lacks a flagellum and it also lacks all genes of the flagellar regulon except of the alternative sigma-28 factor FliA and the anti-sigma-28 factor FlgM. Genes encoding structural components of type I, type II, type IV Lvh and type IV Dot/Icm, Sec- and Tat-secretion systems could be identified. Only a limited set of Dot/Icm effector proteins have been recognized within the genome sequence of L. oakridgensis. Like in L. pneumophila strains, various proteins with eukaryotic motifs and eukaryote-like proteins were detected. We could demonstrate that the Dot/Icm system is essential for intracellular replication of L. oakridgensis. Furthermore, we identified new putative virulence factors of Legionella.

A structural comparison of lipopolysaccharide biosynthesis loci of Legionella pneumophila serogroup 1 strains.

BACKGROUND: The lipopolysaccharide (LPS) is the major immuno-dominant antigen of all Legionella species including L. pneumophila. Its diversity is the basis for the classification of L. pneumophila into serogroups and monoclonal subgroups and is thought to be involved in strain specific virulence. The understanding of the genetic basis of the LPS-antigen is incomplete. Thus, we analyzed the genetic locus involved in LPS-biosynthesis of L. pneumophila serogroup 1 (Sg1) strains with the focus on strain specific gene composition. RESULTS: The LPS-biosynthesis loci of 14 L. pneumophila Sg1 strains comprise two distinct regions: A 15 kb region containing LPS-biosynthesis genes that can be found in all L. pneumophila strains and a Sg1-specific 18 kb region. The 15 kb region is highly conserved among Sg1 strains as reflected by high homologies of single ORFs and by a consistent ORF arrangement. In contrast, the Sg1 specific 18 kb region is variable and partially disrupted by phage related genes. We propose that the region spanning from ORF 6 to ORF 11 of the Sg1-specific region is likely involved in late LPS-modification. Due to the high variability of this small region and various combinations of single ORFs within this region a strain specific LPS-structure could be synthesized including modifications of legionaminic acid derivates. CONCLUSIONS: Our data clearly demonstrate that the gene structure of the LPS-biosynthesis locus of L. pneumophila Sg1 strains show significant interstrain variability. These data can be used for further functional analysis of the LPS synthesis to understand pathogenesis and reactivity with monoclonal antibodies. Moreover, variable but strain specific regions can serve as basis for the development of novel genotyping assays.

Could oral hygiene prevent cases of at-home-acquired Legionnaires' disease? - Results of a comprehensive case-control study on infection sources, risk, and protective behaviors.

Introduction: The "LeTriWa study" on community-acquired cases of Legionnaires' disease (LD) found that most cases likely acquired their infection at home (AHALD). However, which sources confer the infection is largely unknown. We therefore analyzed the data set from the LeTriWa study to find out if individual sources were associated with AHALD and if specific behavioral habits may increase or lower the risk for AHALD. Methods: During the study we had used two comparison groups: (i) controls matched for age group and hospital ("controls"), (ii) household members of cases with AHALD ("AHALD-HHM"). We inquired about exposure to water sources, such as showering or wearing dentures, as well as behavioral factors and habits related to oral hygiene. We took standardized household bathroom water and biofilm samples of both cases with AHALD and controls, and in addition from households of cases with AHALD only samples from suspect residential (non-)drinking water sources. We first conducted bivariate analyses for infection sources and behaviors, followed by multivariable analyses. Results: There were 124 cases with AHALD, 217 controls and 59 AHALD-HHM. In bivariate analyses using controls for comparison, wearing dentures was the only variable significantly positively associated (odds ratio (OR) = 1.7, 95% confidence interval (CI) = 1.1-2.7, p-value = 0.02). Behavioral factors such as showering, letting water run before use and not being alcohol abstinent were significantly negatively associated, smoking was significantly positively associated. In a multivariable analysis, we identified good oral hygiene as a preventive factor for both denture wearers (OR = 0.33, 95% CI = 0.13-0.83, p-value = 0.02) and non-denture wearers (OR = 0.32, 95% CI = 0.10-1.04, p-value = 0.06). Analyses of comparisons with AHALD-HHM showed similar effects but lacked statistical power. We identified Legionella in 16 residential (non-)drinking water sources, one of which was a PCR-positive scratch sample of dentures. Discussion: Wearing (inadequately cleaned) dentures or poor oral hygiene might confer an increased risk for AHALD, and oral hygiene may prevent AHALD. The hypothesis that Legionella in oral biofilm or dental plaque may be the cause of cases with AHALD should be examined further. If confirmed this may open new and simple avenues for the prevention of LD.

Comparative Genomics of Legionella pneumophila Isolates from the West Bank and Germany Support Molecular Epidemiology of Legionnaires' Disease.

Legionella pneumophila is an environmental bacterium and clinical pathogen that causes many life-threating outbreaks of an atypical pneumonia called Legionnaires' disease (LD). Studies of this pathogen have focused mainly on Europe and the United States. A shortage in L. pneumophila data is clearly observed for developing countries. To reduce this knowledge gap, L. pneumophila isolates were studied in two widely different geographical areas, i.e., the West Bank and Germany. For this study, we sequenced and compared the whole genome of 38 clinical and environmental isolates of L. pneumophila covering different MLVA-8(12) genotypes in the two areas. Sequencing was conducted using the Illumina HiSeq 2500 platform. In addition, two isolates (A194 and H3) were sequenced using a Pacific Biosciences (PacBio) RSII platform to generate complete reference genomes from each of the geographical areas. Genome sequences from 55 L. pneumophila strains, including 17 reference strains, were aligned with the genome sequence of the closest strain (L. pneumophila strain Alcoy). A whole genome phylogeny based on single nucleotide polymorphisms (SNPs) was created using the ParSNP software v 1.0. The reference genomes obtained for isolates A194 and H3 consisted of circular chromosomes of 3,467,904 bp and 3,691,263 bp, respectively. An average of 36,418 SNPs (min. 8569, max. 70,708 SNPs) against our reference strain L. pneumophila str. Alcoy, and 2367 core-genes were identified among the fifty-five strains. An analysis of the genomic population structure by SNP comparison divided the fifty-five L. pneumophila strains into six branches. Individual isolates in sub-lineages in these branches differed by less than 120 SNPs if they had the same MLVA genotype and were isolated from the same location. A bioinformatics analysis identified the genomic islands (GIs) for horizontal gene transfer and mobile genetic elements, demonstrating that L. pneumophila showed high genome plasticity. Four L. pneumophila isolates (H3, A29, A129 and L10-091) contained well-defined plasmids. On average, only about half of the plasmid genes could be matched to proteins in databases. In silico phage findings suggested that 43 strains contained at least one phage. However, none of them were found to be complete. BLASTp analysis of proteins from the type IV secretion Dot/Icm system showed those proteins highly conserved, with less than 25% structural differences in the new L. pneumophila isolates. Overall, we demonstrated that whole genome sequencing provides a molecular surveillance tool for L. pneumophila at the highest conceivable discriminatory level, i.e., two to eight SNPs were observed for isolates from the same location but several years apart.

FamilyCoviDD19: results of a cross-sectional study-long-term outcomes of infected and uninfected household members.

OBJECTIVE: In this study, we aimed to compare long-term physical and mental health outcome between SARS-CoV-2 infected and uninfected household members to differentiate between infection-related and pandemic-related outcomes after about two and a half years of the pandemic. Furthermore, possible differences in the outcome of adults and children and young people (CYP) were of interest. DESIGN: In a cross-sectional study design, we compared the long-term physical and mental health outcome of between infected and uninfected as well as between adult and CYP (household members). SETTING: The FamilyCoviDD19 study-a serology study in households-was initially conducted to evaluate virus transmission in a close contact setting focusing on households with children and adolescents in Germany. At least 1 year after initial infection in the respective households, a follow-up took place in which the prevalence and type of possible long-term consequences were surveyed on the basis of self-reported information on physical and mental health. PARTICIPANT: In this study, a total of 533 household members of 146 families participated and responded to our survey, including 296 (55.5%) adults and 237 (44.5%) CYP. RESULT: The difference in frequency of reported symptoms between infected and uninfected individuals was very moderate, suggesting that the vast majority of reported symptoms were not attributable to a previous SARS-CoV-2 infection. However, regardless of age and infection status, this study showed overall high rates of self-reported symptoms with CYP having fewer long-term sequelae than adults one year after infection. Furthermore, over 50% of those reporting symptoms were not affected in their daily life, with CYPs reporting an even lower percentage compared with adults. CONCLUSION: CYP are at reduced risk not only to develop symptomatic infection or severe disease courses (previous analyses) but also to develop infection-associated long-term sequelae (this study). Independent of infection CYP reported high rates of neurocognitive, pain, somatic and mood symptoms, which makes the influence of the pandemic itself-including pandemic control measures-decisive.

The N-acylneuraminate cytidyltransferase gene, neuA, is heterogenous in Legionella pneumophila strains but can be used as a marker for epidemiological typing in the consensus sequence-based typing scheme.

Sequence-based typing (SBT) is the internationally recognized standard method for genotyping Legionella pneumophila. To date all strains of serogroup 1 (SG1) and some of SGs 2 to 14 yield a seven-allele profile and can be assigned a sequence type (ST). However, for some strains belonging to SGs 2 to 14, the targeted region of the neuA gene could not be amplified using the published standard primers. We determined the DNA sequence of a neuA gene homolog located in the lipopolysaccharide synthesis locus of strain Dallas-1E. By using newly designed degenerate consensus primers based on the neuA homolog in strains Dallas-1E, Philadelphia-1, Paris, Lens, and Corby, we were able to obtain DNA sequences for all 48 non-SG1 strains which were untypeable by the standard method. Our data show that the neuA gene is present in all L. pneumophila strains but differs significantly in some non-SG1 strains at both the DNA and amino acid levels. The new primers can be used to amplify and sequence the neuA gene in all strains and can substitute for the standard primers. This offers the possibility of assigning an ST to all strains of L. pneumophila.

SARS-CoV-2 seroprevalence in students and teachers: a longitudinal study from May to October 2020 in German secondary schools.

OBJECTIVE: To quantify the number of SARS-CoV-2 infections in secondary schools after their reopening in May 2020. DESIGN: Repeated SARS-CoV-2 seroprevalence study after the reopening of schools and 4 months later. SETTING: Secondary school in Dresden, Germany. PARTICIPANTS: 1538 students grades 8-12 and 507 teachers from 13 schools. INTERVENTIONS: Serial blood sampling and SARS-CoV-2 IgG antibody assessment. PRIMARY AND SECONDARY OUTCOME MEASURE: Seroprevalence of SARS-CoV-2 antibodies in study population. Number of undetected cases. RESULTS: 1538 students and 507 teachers were initially enrolled, and 1334 students and 445 teachers completed both study visits. The seroprevalence for SARS-CoV-2 antibodies was 0.6% in May/June and the same in September/October. Even in schools with reported COVID-19 cases before the lockdown of 13 March, no clusters could be identified. Of 12 persons with positive serology five had a known history of confirmed COVID-19; 23 out of 24 participants with a household history of COVID-91 were seronegative. CONCLUSIONS: Schools do not play a crucial role in driving the SARS-CoV-2 pandemic in a low-prevalence setting. Transmission in families occurs very infrequently, and the number of unreported cases is low in this age group. These observations do not support school closures as a strategy fighting the pandemic in a low-prevalence setting. TRIAL REGISTRATION NUMBER: DRKS00022455.