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Background: To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology. Results: Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced. Conclusions: These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.

Background: Although infectious diseases are less common in high-income countries compared to low-income countries, they should still be seriously considered as a relevant public health issue. Some professions, such as healthcare workers, laboratory workers, and care providers may be at a particularly high risk of acquiring infections. In Germany, work-related infectious diseases are after skin diseases, the most common cause of occupational diseases reported to the Institution for Statutory Social Accident Insurance and Prevention in the Health Care and Welfare Services (BGW). An occupational disease, as defined by the WHO, is “any disease contracted primarily as a result of an exposure to risk factors arising from work activity”, although this definition varies between countries. In order for infections to be recognized as an occupational disease, either the identification of an index case is needed or it must be shown that the likelihood in which a particular case of illness was attributable to the occupation: the probability of causation must be greater than 50% (the “more-likely-than-not” rule). A general “rule-of-thumb” is to equate the probability of causation of 50% with a relative risk of disease equal to two (the “doubling of the risk”). This principle is used by many countries for the recognition of an occupational disease. Few studies have concentrated on the risk of infectious disease in daycare workers, who may be at higher risk than the general population due to their frequent and close contact to young children. Research questions: The primary aim of this review was to summarize the evidence on the relationship between being a daycare worker working with children and the possible increased risk for infections not preventable by vaccines. Furthermore, research gaps were to be identified. Finally, the implications for practice and health policy based on the evidence were to be described. Methods: For the systematic reviews with meta-analysis, the Medline and Embase databases were searched using search strings defined according to the Population, Exposure, Comparison, and Outcomes (PECO) applicable to the research questions in order to find studies on vaccine non-preventable infections in daycare workers published since 2000. The search hits were evaluated using predefined inclusion and exclusion criteria by two independent reviewers. A separate manual search was performed by reviewing the reference lists of key articles and systematic reviews. The “citation tracking factor” by Google scholar was used to find additional relevant studies. The resulting studies were extracted and were assessed in eight risk of bias domains for the judgement of study quality. With a meta-analysis, the pooled risk of infections for daycare workers compared to the general or a reference population was calculated. The quality of evidence was assessed by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE). Results: After evaluating the 6879 records, ten methodologically adequate studies were identified regarding parvovirus B19 infection (four studies) and cytomegalovirus (CMV) infection (six studies). No adequate studies on other infections were found. For parvovirus B19 infection, three cross-sectional studies and one retrospective cohort study were identified. The pooled parvovirus B19 seroprevalence in daycare workers was 70.3% (95% CI 59.5-80.4). Of three studies investigating the relative risk (RR) of parvovirus B19 infection on daycare workers, only one study evaluated seroconversion rates. There was an indication for an increased risk of parvovirus B19 infection for daycare workers compared to the unexposed population (RR = 1.12, 95% CI 0.98–1.27) using prevalence estimators. Furthermore, daycare workers had a higher parvovirus B19 seroconversion rate compared to the unexposed population (RR = 2.63, 95% CI 1.27–5.45) in the low risk of bias study. For CMV infection, five cross-sectional studies and one cohort study were included. The pooled CMV seroprevalence of daycare workers was 59.3% (95% CI 47.6-70.9). The four studies investigating risk of infection indicated an increased seroprevalence for daycare workers compared to a reference population (prevalence ratio, RR=1.54, 95% CI 1.33-1.77). No study evaluated CMV seroconversions for daycare workers. Conclusions: The findings suggest higher parvovirus B19 and cytomegalovirus seroprevalence for daycare workers compared to the general population. There is a need for longitudinal and higher-quality studies regarding infections not preventable by vaccines in daycare workers, as well as a need to study other infections for which daycare workers may be at higher risk. Nonetheless, when the actual occupational seroconversion risk is considered by taking into account the pre-occupational seroprevalences, the pooled relative risks for both parvovirus B19 and CMV infection are compatible with a doubled seroconversion risk corresponding to a probability of causation due to the occupation of at least 50%. Preventative efforts in the workplace are needed based on the legally required risk assessment at the workplace. Moreover, it is important to raise awareness of the potential risk of infection in women trying to conceive or during pregnancy. Recommendations to prevent infections in the day care center include using gloves and frequent handwashing after exposure to young children’s bodily fluids, cleaning surfaces, and avoiding intimate contact with young children if pregnant, although these measures alone may not completely protect the daycare worker from infection. Currently, in Germany, an employment ban for pregnant daycare workers depends on the federal state. To avoid occupational risks for pregnant daycare workers, scientific-based guidelines should be developed and applied consistently.

Les adénovirus (AdV) sont des virus à ADN se répliquant dans le noyau de la cellule hôte. Pour pouvoir se répliquer, ils détournent la machinerie cellulaire à leur profit. Au cours de l’entrée dans la cellule, les particules virales utilisent la machinerie de transport des microtubules pour rejoindre le noyau. Les AdV interagissent avec la dynéine, moteur moléculaire associé aux microtubules, pour être transportés vers le compartiment nucléaire. Ils se lient alors aux pores nucléaires, structures ancrées dans l’enveloppe nucléaire (EN). Une fois aux pores nucléaires, les capsides virales se désassemblent pour libérer et importer leur génome. Les mécanismes de détachement des microtubules, de translocation nucléaire et d’import du génome des AdV impliquent des facteurs de la machinerie de transport nucléocytoplasmique. Cependant, le mécanisme exact utilisé par les virus pour atteindre les pores nucléaires n’est pas clairement défini. Le transport nucléocytoplasmique est composé de différents facteurs et est hautement régulé dans les cellules. Le transport actif de cargos est dû à des facteurs d’import et d’export interagissant avec RanGTP. Le principal facteur d’export est CRM1 et il est connu pour être essentiel dans la translocation des AdV vers l’EN. L’inhibition de CRM1 par la Leptomycine B conduit à l’accumulation des AdV au centrosome, le principal Centre Organisateur des Microtubules (COMT) des cellules de mammifères. Nous avons donc étudié le rôle de CRM1 dans la libération du génome adénoviral. Nous avons analysé l’interaction des AdVs avec le COMT et nous avons observé que l’absence de facteurs cytoplasmiques ainsi que la perte d’intégrité des microtubules n’affectaient pas leur accumulation au COMT. En revanche, nous avons identifié et caractérisé un mutant de CRM1, qui reste fonctionnel pour l’export physiologique de cargo mais qui induit un retard important dans la translocation des AdV vers l’EN. Nous avons utilisé l’imagerie sur cellules vivantes pour analyser l’infection de l’AdV dans des cellules mitotiques et ceci a permis de révéler le rôle de CRM1 dans la libération du génome de ce virus. Nous avons également identifié un partenaire viral potentiel pour CRM1 parmi les protéines associées au génome viral, la Terminal Protein (TP). Cette protéine possède un signal d’export nucléaire et est un substrat de CRM1. Nos données soulignent le rôle de CRM1 comme un médiateur essentiel au désassemblage total de la capside adénovirale, qui favorise la libération du génome et son import.

Toxoplasma gondii ist weltweit einer der häufigsten zoonotischen Parasiten. Der obligat in-trazelluläre Gewebeparasit hat ein breites Wirtsspektrum als Zwischenwirte. Der Mensch infiziert sich häufig durch orale Aufnahme von Gewebezysten aus rohem oder unzureichend erhitztem Fleisch. Schweine und Hühner als fleischliefernde Tiere stellen eine wichtige Infek-tionsquelle für den Menschen dar. Ziel der Arbeit war die Verteilung und Parasitenbelastung von T. gondii in verschiedenen Geweben von infizierten Schweinen und Hühnern mit Hilfe quantitativer real-time PCR auf Basis des 529-bp-Fragmentes zu bestimmen. Experimentell wurden 10 Schweine und 12 Hühner mit unterschiedlicher Infektionsdosen von Toxoplasma-Oozysten infiziert. Anhand der 529-bp-PCR waren 90% der untersuchten Schweine und >90% der untersuchten Hühner Toxoplasma-DNA-positiv. In Schweinen gelang der Nach-weis von Toxoplasma-DNA in der Oberschenkelmuskulatur mit 70% und Bauchmuskulatur mit 60% am häufigsten. Gehirn und Vorderbeinmuskulatur waren mit jeweils 40%, Herz mit 30% und Zunge mit 10% Toxoplasma-DNA-positiv. In experimentell infizierten Hühnern wur-de T. gondii-DNA am häufigsten in Oberschenkelmuskulatur, Brustmuskulatur und Gehirn mit jeweils 50% und im Herz mit ungefähr 20% nachgewiesen. Die Quantifizierung des Erre-gers in den T. gondii-positiven Gewebeproben ergab eine Parasitenanzahl von 0,1 bis 4,1 in 25 mg Schweinegewebe und von 0,1 bis 4,9 in 25 mg Hühnergewebe, die unabhängig von der Gewebeart war. Es wurde in dieser Arbeit auch eine Reverse Transkriptase real-time PCR zur Bestimmung der Viabilität von Parasiten in den Gewebe infizierter Schweine und Hühner durch Nachweis von T. gondii-mRNA etabliert werden. Die Sensitivität dieser Metho-de war geringer als die der real-time PCR für T. gondii-DNA und konnte in experimentell infi-zierten Schweinen und Hühnern keine lebenden Parasiten detektieren. Die Ergebnisse die-ser Arbeit zeigen, dass Muskulatur von Schweinen und Hühnern bevorzugte Orte für die Persistenz von T. gondii in diesen fleischliefernden Tieren darstellen. Das ist ein wichtiger Hinweis, dass bei Verzehr von rohem oder nicht ausreichend erhitztem Fleisch oder Fleisch-produkten dieser Tiere ein potenzielles Infektionsrisiko für den Menschen besteht.

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