Molecular detection of cosaviruses in a patient with acute flaccid paralysis and in sewage samples in Germany.

Cosaviruses (CoSV) were first identified in stool samples collected from non-polio acute flaccid paralysis (AFP) cases and their healthy contacts in Pakistan in 2003. The clinical importance of CoSV remains unclear as data on epidemiology are scarce and no routine diagnostic testing is done. In this study, we characterized human CoSV (HCoSV) in a child with non-polio AFP and in sewage samples collected in Berlin, Germany. Using unbiased high-throughput sequencing and specific PCR, we characterized a HCoSV-D in stool samples of a three-year-old child hospitalized in Germany with non-polio AFP and travel history to Pakistan. The shedding pattern and absence of other relevant pathogens suggests that HCoSV-D may have been involved in the genesis of AFP. The HCoSV-RNA concentration was high, with 2.57 × 106 copies per mL fecal/suspension, decreasing in follow-up samples. To investigate the possibility of local circulation of HCoSV, we screened Berlin sewage samples collected between 2013 and 2018. Molecular testing of sewage samples has shown the presence of CoSV in several parts of the world, but until now not in Germany. Of our sewage samples, 54.3 % were positive for CoSV, with up to three viral species identified in samples. Phylogenetically, the German sequences clustered intermixed with sequences obtained globally. Together, these findings emphasize the need for further clinical, epidemiological, environmental, pathogenicity and phylogenetic studies of HCoSV.

A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Preoperative platelet transfusions to reverse antiplatelet therapy for urgent non-cardiac surgery: an observational cohort study.

Essentials An increasing number of patients requiring surgery receive antiplatelet therapy (APT). We analyzed 181 patients receiving presurgery platelet transfusions to reverse APT. No coronary thrombosis occurred after platelet transfusion. This justifies a prospective trial to test preoperative platelet transfusions to reverse APT. SUMMARY: Background Patients receiving antiplatelet therapy (APT) have an increased risk of perioperative bleeding and cardiac adverse events (CAE). Preoperative platelet transfusions may reduce the bleeding risk but may also increase the risk of CAE, particularly coronary thrombosis in patients after recent stent implantation. Objectives To analyze the incidence of perioperative CAE and bleeding in patients undergoing non-cardiac surgery using a standardized management of transfusing two platelet concentrates preoperatively and restart of APT within 24-72 h after surgery. Methods A cohort of consecutive patients on APT treated with two platelet concentrates before non-cardiac surgery between January 2012 and December 2014 was retrospectively identified. Patients were stratified by the risk of major adverse cardiac and cerebrovascular events (MACCE). The primary objective was the incidence of CAE (myocardial infarction, acute heart failure and cardiac troponine T increase). Secondary objectives were incidences of other thromboembolic events, bleedings, transfusions and mortality. Results Among 181 patients, 88 received aspirin, 21 clopidogrel and 72 dual APT. MACCE risk was high in 63, moderate in 103 and low in 15 patients; 67 had cardiac stents. Ten patients (5.5%; 95% CI, 3.0-9.9%) developed a CAE (three myocardial infarctions, four cardiac failures and three troponin T increases). None was caused by coronary thrombosis. Surgery-related bleeding occurred in 22 patients (12.2%; 95% CI, 8.2-17.7%), making 12 re-interventions necessary (6.6%; 95% CI, 3.8-11.2%). Conclusion Preoperative platelet transfusions and early restart of APT allowed urgent surgery and did not cause coronary thromboses, but non-thrombotic CAEs and re-bleeding occurred. Randomized trials are warranted to test platelet transfusion against other management strategies.

Comparison of clinical and laboratory characteristics during two major paediatric meningitis outbreaks of echovirus 30 and other non-polio enteroviruses in Germany in 2008 and 2013.

Viral meningitis is mainly caused by non-polio enteroviruses (NPEV). Large-scale data on the clinical characteristics between different outbreaks within the same region are lacking. This study aimed to analyse a possible influence of the circulating NPEV genotype on the disease outcome of affected children. A retrospective cohort study analysing two major outbreaks of NPEV meningitis in Germany in 2008 and 2013 was conducted in cooperation with the National Reference Centre for Poliomyelitis and Enteroviruses (NRC PE) and five German children's hospitals. A total of 196 patients with laboratory-confirmed NPEV meningitis were enrolled. In 2008, children with NPEV meningitis had significantly higher fever and showed more behavioural changes and less back pain. To better define typical findings in echovirus 30 (E-30) meningitis, patients were split into the following three groups: E-30 positive patients, patients with "Non E-30" infection and patients with "Untyped" NPEV infection. E-30 positive patients were significantly older and their disease course was more acute, with early admission to but also early discharge from hospital. E-30 positive patients showed a significantly higher rate of headache and meningism, and a lower rate of diarrhoea and clinically defined septicaemia when compared to the others. Regarding laboratory testing, E-30 positive patients presented with significantly elevated peripheral blood neutrophil counts when compared to patients with "Non E-30" or "Untyped" NPEV infection. In conclusion, E-30 meningitis in children shows a characteristic pattern of clinical features. To further characterise NPEV strains worldwide, continuous surveillance and typing of NPEV strains causing central nervous system disease is warranted.

[Modern treatment of an appendiceal mass]. / Moderne Therapie perityphlitischer Abszesse.

BACKGROUND: The recommendations for the treatment of an appendiceal mass are still controversial. The need for staged appendectomy is still under discussion. PATIENTS AND METHODS: In a retrospective study all patients with the diagnosis appendiceal mass (ICD 10 K35.32) treated in the surgical clinic of the University Clinic in Greifswald between June 1999 and June 2011 were analyzed. RESULTS: A total of 38 patients were included in the study whereby 17 patients (9 male and 8 female) were treated with drainage and antibiotics (group A) and 21 (14 male and 7 female) were treated by immediate surgery (group B). Nonsurgical therapy with drainage of the abscess was found to be efficient. Immediate surgical treatment was also effective and should be performed when signs of peritonitis exist. Interval appendectomy is not indicated.

Genetic analysis of poliovirus strains isolated from sewage in Poland.

The study describes genetic characterization of poliovirus (PV) strains isolated from sewage samples in Poland. The analyses were performed for the detection of any putative polio revertants and recombinants in three genomic regions by sequencing analysis. Thirty-six strains were analyzed. The analyzed strains were identified by neutralization assay as 7 strains of serotype P1, 10 strains of serotype P2, and 19 strains of serotype P3. Sewage isolates were sequenced in 5'UTR, VP1, and 3D genomic regions. All detected PVs were classified as vaccine strains on the basis of VP1 sequence. Mutational differences in the VP1 sequences of isolated viruses ranged from 0.0% to 0.4%, indicating a limited replication period. The genetic analysis of the 3D region showed that some strains have recombinant genomes. Nine strains were found as dipartite recombinants (seven strains--S3/S2, one strain--S2/S1, one strain--S3/S1), while one strain was found as tripartite recombinant (S3/S2/S1). No recombinants with non-PV enteroviruses were identified. None of wild-type PVs or vaccine-derived polioviruses (VDPVs) were detected. This study showed the absence of wild or VDPV circulation in the country and demonstrated the usefulness of environmental surveillance in addition to acute flaccid paralysis (AFP) surveillance in support of polio eradication initiatives.

Induction of M2-macrophages by tumour cells and tumour growth promotion by M2-macrophages: a quid pro quo in pancreatic cancer.

INTRODUCTION: More effective therapies are required to improve survival of pancreatic cancer. Possible immunologic targets include tumour associated macrophages (TAMs), generally consisting of M1- and M2-macrophages. We have analysed the impact of TAMS on pancreatic cancer in a syngeneic orthotopic murine model. METHODS: 6606PDA murine pancreatic cancer cells were orthotopically injected into C57BL6 mice. Tumour growth was monitored using MRI. Macrophages were depleted by clodronate liposomes. Tumours including microvessel density were evaluated using immunohistochemistry, immunofluorescence and/or cytometric beads assays. Naïve macrophages were generated employing peritoneal macrophages. In vitro experiments included culturing of macrophages in tumour supernatants as well as tumour cells cultured in macrophage supernatants using arginase as well as Griess assays. RESULTS: Clodronate treatment depleted macrophages by 80% in livers (p = 0.0051) and by 60% in pancreatic tumours (p = 0.0169). MRI revealed tumour growth inhibition from 221.8 mm(3) to 92.3 mm(3) (p = 0.0216). Micro vessel densities were decreased by 44% (p = 0.0315). Yet, MCP-1-, IL-4- and IL-10-levels within pancreatic tumours were unchanged. 6606PDA culture supernatants led to a shift from naïve macrophages towards an M2-phenotype after a 36 h treatment (p < 0.0001), reducing M1-macrophages at the same time (p < 0.037). In vivo, M2-macrophages represented 85% of all TAMs (p < 0.0001). Finally, culture supernatants of M2-macrophages induced tumour growth in vitro by 63.2% (p = 0.0034). CONCLUSIONS: This quid pro quo of tumour cells and M2-macrophages could serve as a new target for future immunotherapies that interrupt tumour promoting activities of TAMs and change the iNOS-arginase balance towards their tumoricidal capacities.

[Disease-adapted closure of the appendicular stump in laparoscopic appendectomy]. / Stadiengerechte Versorgung der Appendixbasis im Rahmen der laparoskopischen Appendektomie.

The frequency of laparoscopic appendectomy has dramatically increased with it now being the standard procedure for acute appendicitis in many hospitals in Germany. Hence, these hospitals require the personnel, the technique as well as the expertise to guarantee a top standard and quality of appendectomies at all times. Only this will meet the requirements of the high case load and the socio-economical importance of appendectomies. The closure of the appendicular stump is a critical step of this procedure. The three most commonly used techniques consist of the endo-loop, the clip and the endo-stapler. The endo-stapler has the advantage of offering closure and transection of the appendix in one step; it can be employed in all cases of appendicular inflammation; and it allows a partial caecal resection. However, it is quite expensive. The clip and the endo-loop offer the same cost-effectiveness, yet the clip appears to be simpler in use compared to the loop. Also, sharing this feature with the stapler, it offers the possibility of closing and transecting the appendix before dissecting the mesoappendix. A disadvantage of the clip not being shared with the loop is the limitation of clips to appendicular diameters of only up to 16 mm. In summary, we propose a cost-effective disease-adapted closure of the appendicular stump employing the clip in standard appendectomies, with the endo-loop being a good alternative. If clip or loop cannot be applied the stapler is the technique of choice. In particular, it should be used if the base of the appendix is inflamed. Adopting this pathway helps to control the still somewhat higher costs of laparoscopic appendectomies compared to classical open appendectomies.

Surgical trauma and postoperative immune dysfunction.

BACKGROUND: In postoperative sepsis, mortality is increased due to the surgically induced immune dysfunction. Further causes of this traumatic effect on the immune system include burn injuries and polytrauma, as well as endogenous traumata like stroke. Several animal models have been defined to analyse the characteristics of trauma-induced immune suppression. This article will correlate our results from animal studies and clinical observations with the recent literature on postoperative immune suppression. METHODS: The previously described model of surgically induced immune dysfunction (SID) was performed in mice by laparotomy and manipulation of the small intestine in the antegrade direction. Blood samples were collected 6 and 72 h following SID to analyse the white blood cell count and corticosterone levels. To assess the postoperative immune status in humans, we analysed expression of HLA-DR on monocytes of 118 patients by flow cytometry prior to and 24, 48 and 72 h after surgery. RESULTS: The postoperative immune suppression in our SID model is characterised by lymphocytopenia and significantly increased corticosterone levels in mice dependent on the degree of surgical trauma. This is comparable to the postoperative situation in humans: major and especially long-lasting surgery results in a significantly reduced expression of HLA-DR on circulating monocytes. Previous studies describe a similar situation following burn injury and endogenous trauma, i.e. stroke. CONCLUSIONS: We suggest the completion of our previously published sepsis classification due to the immune status at the onset of sepsis: type A as the spontaneously acquired sepsis and type B as sepsis in trauma-induced pre-existing immune suppression.

Postoperative immune suppression in visceral surgery: characterisation of an intestinal mouse model.

BACKGROUND: Postoperatively acquired immune dysfunction is associated with a higher mortality rate in case of septic complications. As details of this severe clinical problem are still unknown, animal models are essential to characterise the mechanisms involved. METHODS: Mice were laparotomised and the small intestine was pressed smoothly in antegrade direction. For extension of trauma, the intestine was manipulated three times consecutively. Following this, the ex vivo cytokine release of splenocytes was determined. The degree of surgical trauma was analysed by detection of HMGB1 and IL-6 in serum and by neutrophil staining in the muscularis mucosae. RESULTS: We adapted the previously described animal model of intestinal manipulation to provide a model of surgically induced immune dysfunction. Following intestinal manipulation, the mice showed elevated serum levels of HMGB1 and IL-6 and increased infiltration of granulocytes into the muscularis mucosae. Ex vivo cytokine release by splenocytes was suppressed in the postoperative period. The degree of suppression correlated with the extent of surgical trauma. CONCLUSIONS: In this study, we describe a surgically induced immune dysfunction animal model, in which a significant surgical trauma is followed by an immune dysfunction. This model may be ideal for the characterisation of the postoperative immune dysfunction syndrome.

[Definition and management of wound infections]. / Definition und Management der Wundinfektion.

Surgical site infections (SSI) in the postoperative period represent the sword of Damocles in surgery. In spite of the medical progress in recent years these infections cannot always be avoided and occur in 25% of all nosocomial infections in Germany. They also generate up to 50% of the required costs in this context. The consequences vary from extended duration of hospitalization to elevated mortality. The degree of contamination of surgical wounds is of great importance as well as the patient's immune status and comorbidities. Prevention of infected surgical wounds is essential and important measures should begin even prior to the surgical procedure. In addition, during and following the surgical procedure several standards have to be followed. Rapid confirmation of diagnosis and correct management of surgical site infections are essential for the course of the disease. This study provides information on development, prevention and therapy of surgically infected wounds.

Stimulation of TLR7 prior to polymicrobial sepsis improves the immune control of the inflammatory response in adult mice.

OBJECTIVE: The role of Toll-like receptor 7 (TLR7), so far regarded as a receptor for viral RNA, was evaluated in a murine sepsis model. MATERIAL: We used the colon ascendens stent peritonitis model (CASP) in female C57B/6 mice. R-848 (1.5 µg/g body weight) was injected intravenously prior to sepsis induction. METHODS: We determined levels of cytokines by CBA detection kit. Different cell populations were isolated from the spleen by magnetic cell separation and the expression of TLR7 was visualized by immunofluorescence staining. Bacterial load of organs was quantified by incubating suspensions on agar in colony forming units. RESULTS: R-848 application per se led to elevated cytokine levels in serum, spleen and peritoneal cavity. Expression of TLR7 on splenocytes was upregulated following CASP. Bacterial clearance in polymicrobial sepsis was significantly increased in spleen and peritoneum of mice pre-treated with the TLR7-agonist. Cytokine release was regulated in the peritoneum and spleen. Furthermore, apoptosis in thymus and spleen during polymicrobial sepsis was significantly decreased following TLR7 agonist application. CONCLUSIONS: TLR7 seems to be essential for pathogen defence not only in viral but also in bacterial infections. Pharmacological stimulation of this receptor prior to induction of sepsis improves the host's capacity to cope with pathogens.

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network.

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

Phylogenetic evidence for a recent spread of two populations of human enterovirus 71 in European countries.

Human enterovirus 71 (EV-71) is a cause of seasonal epidemics of hand, foot and mouth disease, and of less common but severe neurological manifestations. Uncertainty persists regarding the circulation of virus populations in several geographical areas and the timescale of their dissemination. We determined EV-71 sequences at loci 1D (VP1 capsid protein) and 3CD (non-structural proteins) in 86 strains recovered in Austria, France and Germany and performed an evolutionary genetic study of extant virus populations. Phylogenetic analyses positioned 78 of the 86 sequences within two clades among subgenogroups C1 and C2. A minor sequence cluster was assigned to subgenogroup C4. Analyses incorporating the available sequences estimated the substitution rate in genogroup C at 3.66 x 10(-3) and 4.46 x 10(-3) substitutions per site year(-1) for loci 1D and 3CD, respectively, assuming a relaxed molecular-clock model for sequence evolution. Most of the 'European' strains belonged to clades C1b and C2b, which originated in 1994 [95 % confidence interval (CI), 1992.7-1995.8] and 2002 (95 % CI, 2001.6-2003.8), respectively. Estimates of divergence times for locus 3CD were consistent with those measured for locus 1D. Intertwining between clades representing EV-71 subgenogroups and clades corresponding to other enterovirus types (notably early coxsackievirus A prototype strains) in the 3CD phylogeny is highly indicative of ancestral recombination events. Incongruent phylogenetic patterns estimated for loci 1D and 3CD show that a single tree cannot model the epidemic history of circulating EV-71 populations. The evolutionary timescale of genogroup C estimated for both loci was measured only in decades, indicating recent dissemination.

Evaluation of poliovirus antibody titers in orally vaccinated semi-captive chimpanzees in Uganda.

BACKGROUND: To understand immunological responses in chimpanzees vaccinated with live-attenuated vaccine (oral polio vaccine; OPV), serum neutralizing antibodies against poliovirus types 1, 2, and 3 were investigated over time. METHODS: The neutralizing antibody titers against poliovirus types 1, 2, and 3 were determined by microneutralization test using 100 ID(50) of poliovirus types 1, 2, and 3 (Sabin strains). RESULTS: Neutralizing antibodies against poliovirus types 1, 2, and 3 were detected in 85.7%, 71.4%, and 65% of the serum from 42 chimpanzees tested 9 years post-vaccination. The neutralizing antibody titers in chimpanzees were similar to the documented levels in human studies as an indicator of vaccine efficacy. CONCLUSIONS: This study reveals persistence of neutralizing antibodies in chimpanzees for at least 9 years after vaccination with OPV. This first study in chimpanzees provides useful information for the evaluation of the success of vaccination with OPV in other captive apes.

Rotavirus surveillance in europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data.

BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

Group A rotavirus genotypes in Germany during 2005/2006.

During the 2005/2006 winter season a total of 802 group A positive rotavirus specimens of patients from different regions throughout Germany were genotyped. Amplicons from a one-tube RT-PCR were typed by analysis of their (type-specific) size using type-specific primers, fluorescent consensus primers and a capillary sequencer for detection. While G1P[8] was predominant (45.8%), G9P[8] has emerged as the second most frequent genotype combination (37.7%). The distribution of genotypes was heterogeneous, regional frequencies regarding G1 and G9 were ranging from 15.0 to 89.3% and from 7.1 to 67.7%, respectively. Furthermore, a few human rotavirus G10P[6] and G10P[8] infections were observed.

[The German Health Interview and Examination Survey for Children and Adolescents (KiGGS): state of immunity against poliomyelitis in German children]. / Kinder- und Jugendgesundheitssurvey (KiGGS): Immunitätslage gegen Poliomyelitis.

One of the goals of the WHO is the worldwide eradication of poliomyelitis in the coming years. Europe was declared poliofree in 2002, but increasing migration may lead to a come-back of circulating polioviruses. A high level of population immunity protects against imported wild viruses from endemic areas. The first seroprevalence data since the switch from live to inactivated vaccine in Germany (OPV to IPV) are provided. A serum panel was tested (n=2,046) in order to study the serological status against poliomyelitis. The microneutralization test on RD cells was used. Overall, neutralizing antibodies against poliovirus types 1, 2 and 3 were detected in 97.4%, 97.6%, and 93.6% of samples, respectively. Of the test persons, 91.7% had antibodies against all three virus types. Only 26 children simultaneously lacked neutralizing antibodies for all three serotypes (1.3%). No significant correlation between gender, region (East/West)), migration status (with/without migration background) and antibody prevalence to polioviruses was found. The seroprevalence of antibodies against all three types of polioviruses indicates a very high level of population immunity in German children. It must be maintained through consequently performed vaccination programmes.