Abstract: Francisella tularensis is the causative agent of tularemia, a severe zoonotic infectious disease. The natural reservoir of the bacteria is not yet known precisely, but it may persist for over a year in water or mud. It is naturally maintained and spread by various terrestrial and aquatic mammals, and outbreaks in humans are often paralleled by enzootics or epizootics. The reasons for this seem to be multifactorial, though not well understood. Therefore, for epidemiological and outbreak investigations, a rapid hand-held assay would be helpful. Here we described a column-based immunofiltration assay called ABICAP, which has several characteristics that permit its application under field conditions. The assay can be performed within 25 min, with a detection limit of 103 bacteria. Further validation of the assay included the analytical range, precision, detection limit and performance for different human, animal and environmental sample matrices, accuracy, recovery, reproducibility and stability. PRACTICAL APPLICATIONS The assay is an interesting tool applicable for the rapid detection of F. tularensis in various specimens under laboratory and field conditions. It can be used for the identification of reservoirs of F. tularensis in epidemiological studies, for the detection of sources of infection in outbreak situations of tularemia and in laboratory investigation of animal and human specimens. The methodological platform can be adapted to the detection of other microbiological agents and antigens.

Abstract:

Abstract:

Abstract:

Abstract: Das Kuhpockenvirus und seine Symptomatik sind vielen �rzten und Tierärzten unbekannt, denn die gängige Meinung ist: �Die Pocken sind ja ausgerottet�. Dies stimmt zwar für den Erreger der echten Menschenpocken, das Variola-Virus, aber keinesfalls für seine nahen Verwandten. Durch die Einstellung der Pockenschutzimpfung in den 1970er Jahren sind Personen bis zu einem Alter von ca. 35 Jahren auch nicht mehr gegen Infektionen mit Kuhpockenviren geschützt. Dies erklärt, warum vermehrt über die meist lokal begrenzten Infektionen berichtet wird. Bei Immunsupprimierten wurden auch schwere, generalisierte Verläufe bekannt. Mittlerweile wei� man, dass Nagetiere und nicht etwa Katzen oder gar Kühe das eigentliche Reservoir der Kuhpockenviren sind.

Abstract: Point of care testing (POCT) systems for rapid clinical-microbiological diagnostics of infectious diseases that are dangerous to public safety could assist the local responsible executives in their ethically delicate task to triage affected people after a major event causing damage, e.g., after a bioterrorist assault. Due to its potentially major influence on public health, POCT is of great importance and further development is of great concern. Because of the possible consequences for affected people arising from the test results, the strictest standards have to be set for POCT in this field. It is essential that the quality of POCT is associated with the quality of medical care. As a matter of principle, POCT has to meet the same conditions of quality as laboratory tests. Further development of rapid methods used for the detection of agents which could possibly be used in a bioterrorist assault is very complex, cost intensive and potentially dangerous. For the most part, the pre-analytic phase is demanding and, in this field, POCT is therefore not to be performed by inexperienced people. The power of the tests is often deficient. This generates a high immanent rate of false negative results at low levels of prevalence and may lead to a false estimation of the situation. Therefore, it has to be discussed whether further development in this field makes sense at all. Moreover, the medical professionals in Germany have very limited knowledge about B-agents and the identification of diseases caused by them. Consequently, it is doubtful whether specific POCT for the detection of B-agents would be used meaningfully. Another area for the use of POCT is the diagnostic investigation of exceptional epidemic events. There is ample experience with rapid diagnostics of influenza in pandemic situations. At low levels of prevalence at the beginning of an outbreak one can find extremely high rates of false negative results which gives a false sense of security and may lead to a false estimation of the situation, too. There is a lack of adequately sensitive and reliable commercialized rapid test systems for the detection of B-agents and diseases which are non-endemic in Germany. At present, hand-held test kits (lab-on-a-chip) are under way that may possibly be used as POCT in the future.

Abstract:

Abstract:

Abstract: PURPOSE: To investigate a possible relation between antiepileptic drugs (AEDs) and the fatty acid composition of membranes. METHODS: Fatty acid (FA) composition of erythrocytes was studied in children with epilepsy receiving AED monotherapy. Children taking valproate (VPA, n = 28), carbamazepine (CBZ, n = 17), or phenobarbitone (PB, n = 14) were compared with healthy controls (n = 25). FAs were measured by capillary-gas chromatography (GC-FID). RESULTS: Significant changes (p < 0.05) in the FA composition of membranes were found. In children treated with VPA, C13:0 was decreased (8.2 +/- 2% vs. 10.7 +/- 4% in controls) and C14:0 increased (1.4 +/- 0.5% vs.1 +/- 0.5% in controls). C17:0 again was reduced (9.9 +/- 4% vs. 13.2 +/- 6% in controls), whereas the long-chained acids were enhanced: C18:2n-6 (6 +/- 2.4% vs. 3.9 +/- 2.5% in controls), and C20:4n-6 (1.9 +/- 1.7% vs. 1.4 +/- 0.5% in controls). The nonidentified FAs also increased with VPA therapy: 2.5 +/- 0.8% versus 1.7 +/- 0.9% in controls. Children treated with CBZ showed only minimal changes of FA composition: C13:0 was decreased compared with controls (8 +/- 2% vs. 10.7 +/- 4%). No changes were seen in patients taking PB. The mean corpuscular volume (MCV) showed important differences between the study groups: MCV was 84.7 +/- 6.0 fl during VPA therapy (p < 0.001) and 85.7 +/- 4.1 fl with CBZ (p < 0.001). During PB, the MCV increased to 82.87 +/- 3.29 fl compared with controls (78.73 +/- 4.92 fl; p < 0.01). CONCLUSIONS: VPA therapy is associated with changes of the FA composition of membranes, which is not the case with PB therapy. The implications of this finding remain to be established.

Abstract:

Abstract: Introduction: European hantaviruses are rodent-borne zoonotic pathogens that can cause haemorrhagic fever with renal syndrome (HFRS) of different severity. In Germany the predominant hantavirus species is Puumala virus. However, Tulavirus (TULV) has been described as the possible cause of fever, renal syndrome, and pneumonia in a German patient (Klempa et al., 2003, Schultze et al. 2002). Despite this report, the role of TULV in human infections as well as the spatial and temporal occurrence of TULV in Germany are quite unclear . Methods: The objective of our study was to investigate the prevalence of TULV in natural reservoir hosts in several regions of Germany including also military training areas in South-West Germany. RT-PCRs targeting the S-and M-segments of TULV were used to screen the lungs of trapped Microtus species. Assays based on recombinant TULV nucleocapsid protein were used to investigate hantavirus-specific antibodies in the rodent sera. Results: In some of the areas in central and south-eastern Germany (e.g. Göttingen, Sigmaringen) a high TULV prevalence with up to 7% was observed in common voles. At two different trapping sites in the federal state Brandenburg and in Göttingen TULV was not only found in its designated host, the common vole, but also in field voles. RT-PCR amplification and subsequent phylogenetic analysis of S- and M-segment genomic sequences demonstrated significant differences between TULV originating from different regions in Germany. A nt-sequence divergence of about 20% was observed in S- and M-segments between the TULV strains. Conclusions: Our investigations demonstrated a broad geographical distribution of TULV in Germany. In addition, genetically different TULV were found to presently circulate in voles in several regions of Germany. Embedded in the German-wide network �Rodent-borne pathogens� future rodent trapping activities are dedicated to monitor the population density of rodents as well as the prevalence and molecular variation of TULV in both Microtus species of different populations.

Abstract: ntroduction: European hantaviruses are rodent-borne zoonotic pathogens that can cause haemorrhagic fever with renal syndrome (HFRS) of different severity. In Germany the majority of human cases is caused by the hantavirus species Puumala virus (PUUV). About 140-240 clinically apparent human hantavirus infections have annually been reported during 2001-2004. After a strongly increased total number of cases in 2005 (about 450), in 2006 only a total of about 70 cases were reported. In 2004/2005 a drastically increased number of cases were observed in some administrative districts of South-East Germany (2004) and North-West Germany (2005). In contrast, this year more than1400 clinically apparent human hantavirus infections have been reported until October, implying a 3 up to 10 fold increase of the number of infections in comparison to the previous years. Methods: The objective of our study was to identify and characterise hantavirus strains involved in this increased occurrence of Nephropathia epidemica (NE) in humans in Germany including also military training areas in South-West Germany. The serological investigations were based on a novel recombinant nuucleocapsid protein of a PUUV strain originating from Lower Bavaria. Results: In all investigated regions in Baden-Wuerttemberg, North Rhine Westphalia, Lower Saxony and Bavaria a high PUUV prevalence of up to 76% was observed in trapped bank voles. RT-PCR amplification and subsequent phylogenetic analysis of S- and M-genome sequences demonstrated significant differences between PUUV strainsoriginating from the different regions in Germany. A nucleotide-sequence divergence of about 16% was observed in S- and M-segments between the PUUV strains. Conclusions: Our investigations demonstrated that different PUUV strainsare presently circulating in bank voles in Germany. Embedded in the German-wide network �Rodent-borne pathogens� future rodent trapping activities are dedicated to monitor the population density of rodents as well as the prevalence and molecular variation of PUUV in these rodent populations. These studies will allow conclusions on the evolution of PUUV and changes in their distribution which might result in a risk assessment for human infections in the investigated regions.

Abstract: During the last few years, an area in Eastern Bavaria developed to the most active tick-borne encephalitis (TBE) focus in Bavaria with about 10% of all cases of that German State in the two districts of Amberg und Schwandorf. In 2005, in one little village in the district of Amberg 4 severe cases of TBE occurred. Further examination resulted in the detection of one fatal TBE case both in 2003 and 2004. Sampling of ticks was undertaken in autumn 2005. Ticks were tested for TBE virus. Single adult ticks and pools of 5 nymphs and larvae each were processed for virus isolation and detection of TBE virus by real-time RT-PCR. A total of 367 ticks was collected. One single tick was found positive in real-time RT-PCR, and virus was isolated from the PCR-positive tick in Vero cells. The complete nucleotide sequence of the isolate was determined. To exclude nucleotide changes through cell culture, the sequence of the E protein was confirmed by re-sequencing directly from the ground tick suspension. The isolate showed 5 amino acid changes in the E protein compared to strain Neudoerfl, 3 of which were unique for all known TBE strains published so far: D375E, I405T, I452T. All 3 changes were near or in the clusters B and C of the E protein according to the model of Heinz, Vienna, which are known to be of importance for virus pathogenicity. To the strains 267 and Hypr, 7 and 8 amino changes were shown in the E protein, respectively. Regarding the complete virus genome, 34 amino acid changes (strain Neudoerfl), 35 amino acid changes (strain 263), and 44 amino acid changes (strain Hypr) were detected. A new strain of TBE virus was isolated and sequenced in a Bavarian area where severe human cases and fatal cases of TBE occurred. By nucleotide and amino acid comparison it was shown to be a virus of the western subtype of the virus. Unique amino acid changes in the E protein of TBE virus were detected near or in clusters important for pathogenicity. Thus, this virus strain warrants further characterization of its biological properties including in vivo studies of its pathogenicity.

Abstract: Background: Since the war in Afghanistan started data on prevalence or incidence of infectious diseases are rare. However the knowledge on the occurrence of infectious diseases is of importance as it is a prerequisite for the implementation of prophylactic measures to prevent infection. Methods: We tested the sera of 140 residents of the northern Afghan city Kunduz for the prevalence of antibodies against flaviviruses (Dengue-, West Nile-, tick-borne encephalitis-viruses), alphaviruses (Sindbis virus ), bunyaviruses (Sandfly Naples-, Sandfly Sicilian-, Crimean Congo hemorrhagic fever viruses), and hantaviruses (Puumala- , Seoul-, Hantaan viruses). For testing commercial ELISA assays, commercial and in-house indirect immunofluorescence and neutralization tests (differentiation between West Nile virus and Japanese encephalitis virus) were used. Results: 31/140 sera (22.1%) reacted positive against West Nile virus. By plaque reduction neutralization test specific antibodies against WNV in comparison to Japanese encephalitis virus could be confirmed. 5/140 sera (3.6 %) were found specifically positive for dengue virus. None of the sera showed specific reaction for tick-borne encephalitis. 3/140 sera (2.1%) reacted against Sindbis virus. 13/140 sera (9.2%) were positive for sandfly fever virus, subtype Naples, while all sera were negative for the Sicilian subtype of sandfly fever virus. 6/140 sera (4.2%) exhibited reactivity against Crimean Congo hemorrhagic fever virus. 8/140 sera (5.7%) showed reactivity against hantaviruses. 3/140 sera (2.1%) of them showed higher titers in immunofluorescence for Puumala virus, while one serum each (0.7%) exhibited higher titers for Seoul virus and Hantaan virus. Two sera were indeterminate for the 3 hantaviruses tested. Conclusions: To the best of our knowledge, these are the first data available on the prevalence of antibodies against alpha-, flavi-, bunya-, and hantaviruses during the last decades. The exceptionally high prevalence rates for West Nile virus and Sandfly Naples virus imply intense virus circulation in the region tested. Except for tick borne encpehalitis virus and Japanes Encephalitis virus, antibodies against all other viruses tested were found in prevalence rates of 0.7% up to 22.1%. These results warrant further studies including isolation of arthropod-borne as well as rodent-borne viruses and the characterization of their pathogenic potential and medical importance.

Abstract: Objectives: West Nil virus (WNV) is an emerging pathogen causing increasing numbers of epidemics and human cases in Europe, Africa, Asia and America. While WNV was detected in France, Slovak and Czech Republic, there is no evidence for circulation of this flavivirus in Germany thus far. Seroepidemiological studies for a particular flavivirus infection are hampered because of cross-reacting antibodies against other autochthonous or travel-related and vaccination-induced flavivirus antibodies, e.g. tick borne encephalitis virus (TBEV) Dengue virus (DENV), Japanese encephalitis virus (JEV). Hence two commercially available ELISA assays for the specific detection of antibodies against WNV were compared using well defined sera from German soldiers. Methods: 715 sera from soldiers of different parts of Germany were tested for IgG antibodies (AB) against WNV using a commercial ELISA (Focus Diagnostics, USA). Reactive sera were retested in a second WNV AB ELISA, against DENV AB and TBEV AB (all Euroimmun, Germany). Positive sera were also tested for avidity (Euroimmun, Germany) to detect possible reduction of cross reactivity of AB. All tests were conducted according to the manufacturers� instructions. Results: 142/715 sera (19.9%) reacted positive in the WNV AB ELISA (Focus Diagnostics). Re-testing by WNV AB ELISA (Euroimmun) showed positive results in 73 of these 142 sera. However 123/142 sera reacted positive in TBEV AB ELISA and 59/142 sera showed positive results in DENV AB ELISA. Results of only 6/715 sera (0.8%) were consistent with past WNV or DENV infection. These results are consistent with data of earlier studies and it can be assumed that these 6 soldiers acquired a WNV or DENV during travelling in endemic areas. The use of WNV AB avidity test did not reduce cross-reactivity. Conclusions: Our data do not provide any evidence for autochthonous human WNV infection in Germany. However, huge differences in sensitivity and specificity of two commercial ELISA assays were found with the ELISA (Focus Diagnostics) showing the lowest specificity. The second ELISA (Euroimmun) showed higher specificity. The use of avidity testing had no effect on cross-reactivity at all. Only 6/715 sera showed evidence of past infection with WNV or DENV. In seroepidemiological studies or in serological diagnosis of WNV infections or vaccinations against other flaviviruses have to be excluded using tests with higher specificity, i.e. immunofluorescence and neutralization test.

Abstract: Die vorliegende Studie untersuchte den Zusammenhang zwischen der antiepileptischen Monotherapie mit Valproinsäure, Carbamazepin und Phenobarbital und der Fettsäurezusammensetzung der Membran bei Kindern mit Epilepsie. Die Erythrocytenmembran diente als Modell fu�r neuronale Membranen. Zusätzlich wurde das mittlere korpuskuläre Volumen (MCV) der Erythrocyten als Globalparameter möglicher Membranveränderungen erfa�t. Der Hintergrund dieser Studie war es, Hinweise auf mögliche Wirkmechanismen dieser Antiepileptika zu gewinnen, die u�ber �nderungen der Fettsäuren in der Membran wirken könnten. Die Aufklärung solcher Wirkmechanismen könnte fu�r die Entwicklung neuer Antiepileptika von Interesse sein. Die insgesamt 84 Probanden wurden in drei Therapiegruppen nach der Medikation aufgeteilt. In der Valproinsäuregruppe wurden 29, in der Carbamazepingruppe 16 und in der Phenobarbitalgruppe 13 Probanden untersucht. Eine Kontrollgruppe mit 26 Probanden wurde ebenfalls erfa�t. Die Membranen der Erythrocyten wurden isoliert und gereinigt. Die Fettsäuren der Membran wurden mittels einer Ein-Schritt-Reaktion extrahiert und in die jeweiligen Fettsäuremethylester u�berfu�hrt. Der Anteil der einzelnen Fettsäuren wurde gaschromatographisch bestimmt und prozentual bezogen auf die Gesamt-Fettsäuren ausgewertet. Die Werte der Phenobarbital-Gruppe unterschieden sich nur wenig von den Werten der Kontrollgruppe. Die Patienten unter Valproinsäure und unter Carbamazepin zeigten dagegen deutliche Unterschiede im Vergleich zur Kontrollgruppe. Der Anteil an Linolsäure (C 18: 2n-6) war in der Valproinsäure-Gruppe mit durchschnittlich 5,97% höher als in der Kontrollgruppe mit 3,67% (p<0,01), ebenso der fu�r die Arachidonsäure (C 20: 4n-6) mit 6,81% im Vergleich zu 4,24% in der Kontrollgruppe (p<0,01), sowie fu�r die Myristinsäure (C 14:0) mit 1,45% im Vergleich zu 1,1% (p<0,05). Bei den Fettsäuren mit einer Kettenlänge von unter 13 C-Atomen war der Anteil in der Kontrollgruppe mit 25,76% im Vergleich zur Valproinsäure-Gruppe mit 18,74% signifikant erhöht (p<0,05). Damit fand sich unter der Valproat-Therapie eine Umverteilung von den kurzkettigen hin zu den langkettigen Fettsäuren. Auch in der Carbamazepin-Gruppe war der Anteil der Myristinsäure (C 14:0) mit 1,45% im Vergleich zu 1,1%, fu�r die Stearinsäure (C 18:0) mit 14,93% im Vergleich zu 11,69% und fu�r die Linolsäure (C 18: 2n-6) mit 5,36% im Vergleich zu 3,67% in der Kontrollgruppe erhöht. In der Phenobarbital-Gruppe traten lediglich minimale, nicht signifikante Unterschiede im Vergleich zur Kontrollgruppe bei der �lsäure (C 18: 1n-9) mit 7,42% im Vergleich zu 9,54% und bei der Eicosensäure (C 20: 1n-9) mit 0,6% im Vergleich zu 1,71% in der Kontrollgruppe auf. Auch bezu�glich des MCV verhielten sich die Valproat- und die Carbamazepingruppe gleichsinnig. In der Carbamazepin-Gruppe betrug das mittlere MCV 85,07 fl, in der Valproinsäure-Gruppe 84,73 fl (p<0,001 im Vergleich zur Kontrollgruppe). In der Phenobarbital-Gruppe lag das MCV dagegen lediglich bei 82,87 fl, in der Kontrollgruppe bei 78,73 fl. Diese Differenz blieb auch altersbereinigt bestehen. Zusammenfassend wurde gezeigt, da� die Fettsäurenzusammensetzung von Erythrocytenmembranen von der Valproinsäure und Carbamazepin im beschriebenen Sinne beeinflu�t und dieser Befund durch die entsprechende Abweichung des MCV untermauert wird.