Christina Schrader

PCR inhibitors – occurrence, properties and removal

The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of sample types. However, the method is prone to inhibiting substances, which may be present in the analysed sample and which may affect the sensitivity of the assay or even lead to false‐negative results. The PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action. Some of them are predominantly found in specific types of samples thus necessitating matrix‐specific protocols for preparation of nucleic acids before PCR. A variety of protocols have been developed to remove the PCR inhibitors. This review focuses on the general properties of PCR inhibitors and their occurrence in specific matrices. Strategies for their removal from the sample and for quality control by assessing their influence on the individual PCR test are presented and discussed.

Tick-borne encephalitis (TBE) in Germany - epidemiological data, development of risk areas and virus prevalence in field-collected ticks and in ticks removed from humans.

In Germany, 100-300 autochthonous clinical TBE cases have been recorded annually. There are high-risk areas in Bavaria and Baden-Wuerttemberg and ongoing low-risk areas in Hesse, Thuringia, and the Rhineland-Palatinate and single cases in Saxony. In order to be able to evaluate the epidemiological changes described here, it must be mentioned that a new definition of TBE risk areas was introduced on the district level in 1998 in Germany and in 2001 with the new Infection Protection Act (Infektionsschutzgesetz) which states that TBE is a notifiable disease. This led to the replacement of earlier surveillance systems and to many changes to data collection. In 1998 63 country and town districts were TBE risk areas, in 2001 79 and in 2002 86. There were new risk districts within Bavaria and Baden-Wuerttemberg and outside these regions in Thuringia, Hesse and the Rhineland-Palatinate. An interesting trend was observed in TBE epidemiology. The TBE incidence in Bavaria and Baden-Wuerttemberg has been stable on a high level for years; outside these areas it has steadily been climbing (Odenwald, Thuringia). On the basis of epidemiological data on TBE from the eastern part of Germany since 1960, it is obvious that major changes in virus activity in TBE risk areas also occurred in the past, the explanation of which has remained a matter for speculation. The epidemiological situation in the different risk areas for TBE in Germany was found to vary considerably, if one considers the surveillance data of the last 40 years. 1. Establishment of completely new low-risk areas. 2. Reactivation of formerly active areas with endemic latency. 3. High-risk areas with stable viral activity over long periods. 4. High-risk areas which have expanded and merged with low-risk areas. 5. High-risk areas which have developed into endemic areas or become inactive. High-risk TBE areas from 1960-1975 (i.e. Mecklenburg-Western Pomerania) have since completely disappeared. There were, at the same time, high-risk areas in Thuringia which had only become latent and have now obviously become active again. The Odenwald demonstrated growing virus activity in the 1990s. These changes in TBE activity in German risk areas over more than the last 40 years are presented schematically. This ongoing number of risk areas is certainly linked to the notification obligation and greater public awareness. Nevertheless, any effects of ecological and climatic changes on the natural foci cannot be ruled out nor can changes in human leisure behaviour. Local weather conditions also have a major effect on the TBE incidence. Warm and dry summers may cause low tick activities, rainy summers may lead to low exposure rates of human beings. Even changes in forms of agricultural production prompted by different political structures probably have an impact as do economic constraints which may lead to lower vaccination and higher exposure rates. Regular, systematic virus prevalence measurements from 1997 to 2002 in field-collected ticks in German high-risk areas do not indicate any risk increase nor do they suggest a downward trend. Studies on virus prevalence in questing versus partially engorged ticks indicate that we neither exactly know nor understand the real quantitative relations between the virus and the host. In a first study, virus prevalence in Ixodes ricinus removed from humans was examined. Humans which were exposed in some districts near Passau in Bavaria. In the autumn of 2001, virus prevalence of unengorged free-living nymphs (n = 820) in this area was 0.38 (0.08-1.1)% and of adults (n = 90) 1.17 (0.03-6.38)%. Surprisingly, virus prevalence in partially engorged ticks from the same area collected during the same period was significantly higher (nymphs, n = 86, 6.9% and adults, n = 129, 9.3%). Virus-positive partially engorged ticks were only found in districts known as risk areas. Nucleotide and deduced amino acid sequence data of the PCR products have confirmed the presence of virus prototype Neudoerfl only.

Amantadine resistance among porcine H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany between 1981 and 2001

This study was designed to gain insight into amantadine susceptibility of porcine influenza A viruses isolated in Germany between 1981 and 2001. The 12 studied H1N1, H1N2, and H3N2 porcine influenza virus strains were isolated in chicken eggs and passaged once in MDCK cells. Plaque reduction assays were applied to examine virus susceptibility to amantadine. Genotyping was used to confirm drug resistance. In the results of these antiviral studies, only 3 of the 12 isolates were shown to be amantadine-susceptible. All resistant strains contained the AA substitutions G16E, S31N, and R77Q in the membrane protein 2 (M2). Additionally, L27A was detected in two H1N1 strains. S31N and/or L27A are well-known amino acid substitutions in M2 that confer amantadine resistance. The role of the pig as an intermediate host of avian and human influenza A viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadine-resistant porcine influenza A viruses suggest a real risk of emergence of amantadine resistant human viruses. Therefore, drug susceptibility monitoring appears to be warranted for effective application of those drugs.

Characterization of tick-borne encephalitis (TBE) foci in Germany and Latvia (1997–2000)

Knowledge concerning the prevalence of the tick-borne encephalitis virus (TBEV) in wild living tick populations is very important for understanding the epidemiology of the disease and for immuno prophylactic strategy. In Germany high and low risk areas of TBE exist. In the years 1997-2000, 533 autochthonous clinical TBE cases were recorded, in the high-risk areas of Bavaria and Baden-Wuerttemberg 140 and 363, and in the low risk areas in Hesse (Odenwald) and Rhineland-Palatinate 22 and 8, respectively. Corresponding to these case reports we have measured the virus prevalence in free living ticks in these four risk areas and compared these findings with the situation in high-risk areas in Latvia. In the years 1997-2000, 2,797 clinical TBE cases were recorded in Latvia. For the studies in Germany, a total of 17,398 Ixodesricinus ticks (14,860 nymphs and 2,538 adults) were collected by flagging and examined for TBEV, in Latvia the corresponding numbers were 525 I. ricinus ticks (350 adults and 175 nymphs) and 281 I. persulcatus ticks (adults only). Information concerning annual and seasonal differences of the TBEV prevalence in natural TBE foci is not available in Germany. This paper is a continuation of the study (Süss et al., 1999), starting in 1997. We investigated every year, in May and September, the virus prevalence in ticks in high risk areas of Bavaria (8 foci) and Baden-Wuerttemberg (5 foci). A total of 15,400 ticks (13,100 nymphs and 2,300 adults) were examined for TBEV. The ticks were tested for the presence of TBEV-RNA using a sensitive, nested-RT-PCR. The virus prevalence in the Bavarian foci of the whole tick population ranged from 0.3 to 2.0% during these four years, in adults between 1.2 and 5.3% and in nymphs between 0.1 and 1.4%. In the high-risk areas of Baden-Wuerttemberg, in the Black Forest, the estimated virus prevalence rates of investigated ticks varied from 0.2 to 3.4%, in adults from 0 to 4.8%, and in nymphs from 0.2 to 3.4%. Using the same methods, we have also tested the low risk areas in the Odenwald (840 nymphs, 160 adults) and in Rhineland-Palatinate (920 nymphs, 78 adults). Ticks were collected in those areas where most TBE cases were registered. The virus prevalence in the Odenwald was 0% in adults and 0.5% in nymphs, whereas in ticks from Rhineland-Palatinate we have not found any positive PCR signal. Sequence data of the PCR products have shown that all strains in Germany were closely related to the central European virus prototype Neudoerfl. In I. ricinus ticks, collected in Riga county, the following virus prevalence rates were found: in females 2.4%, in males 3.7%, and in all adults 3.0%, in nymphs 2.4% and in the I. ricinus tick population examined 2.8%. The virus prevalence in I. persulcatus, collected in the eastern parts of Latvia was 6% in females, 4% in males and 5% in all adults. All the PCR products were sequenced and a phylogenetic tree was constructed. Studies in natural foci of TBE in Latvia have shown that I. ricinus carried the central European virus subtype (prototype Neudoerfl) whereas in I. persulcatus two strains have been found, the central European virus subtype (prototype Neudoerfl) and the Siberian virus subtype (prototype Vasilchenko). Sequences of the Far Eastern subtype have not been detected yet.

Comparison of two extraction methods for viruses in food and application in a norovirus gastroenteritis outbreak

Noroviruses are important causes of gastroenteritis; however, due to a lack of sensitive detection methods, the distinct role of contaminated food in norovirus outbreaks remains unclear. Two published virus extraction procedures combined with real-time RT-PCR for the detection of norovirus from food inoculated experimentally were compared. The elution-precipitation method was most efficient in all food matrices tested showing detection limits of 20 RT-PCRU for lettuce and ham, and 200 RT-PCRU for raspberries. The average recovery rates were 23%, 7% and 24% for lettuce, raspberries and ham, respectively. The ultrafiltration method yielded detection limits of 200 RT-PCRU for lettuce and ham, and 2000 RT-PCRU for raspberries; recovery rates were 9%, 7%, 3%, respectively. Subsequently, food items implicated in a norovirus outbreak were examined by the elution-precipitation method. Virus recovery rates determined by using MS2 phage ranged from 1 to 20% depending on the food matrix. However, norovirus could not be detected in the food items examined. This negative result may be explained by a low virus titer and heterogeneous virus distribution, or by random selection of food samples that contained no norovirus. Both, proper sampling and virus extraction from foods may be improved further to identify vehicles of infection.

Genetic Characterization of a Porcine H1N2 Influenza Virus Strain Isolated in Germany

We examined selected influenza virus strains from various outbreaks of respiratory diseases among pigs in Germany. Besides H3N2 and H1N1 subtypes presently circulating in Europe, an H1N2 influenza virus was isolated in 2000. Sequence analysis of hemagglutinin, neuraminidase and matrix genes indicated that this virus is a reassortant of human H1N1 and human H3N2 strains with an internal gene, the M gene of avian origin. It is a result of a multiple reassortment event.

Molecular Epidemiology of Porcine H3N2 Influenza A Viruses Isolated in Germany between 1982 and 2001

We examined influenza virus strains of the subtype H3N2 from outbreaks of respiratory diseases in swine herds in Germany. Four different clusters can be distinguished when comparing parts of the HA1 gene from porcine H3N2 isolates analyzed between 1982 and 2001. Comparison between these clusters reveals a bp homology of less than 90%. In contrast, the homology within the clusters is between 93.7 and 100%. Each of these clusters was confined to a specific time period. For the NA gene an additional cluster is formed by the porcine H1N2 isolate. The findings that different subtypes and drift variants are circulating in the German pig population explain the emergence of new influenza virus variants and the need for continued surveillance of swine.

Durch Zecken übertragene humanpathogene und bisher als apathogen geltende Mikroorganismen in Europa. Teil I: Zecken und Viren

ZusammenfassungFrühsommer-Meningoenzephalitis (FSME) und Lyme-Borreliose als die wesentlichen Krankheiten, die in Europa durch Zecken übertragen werden, nehmen in ihrer Bedeutung ständig zu, was mit sehr vielen verschiedenen Faktoren, u. a. auch dem Klimawandel, im Zusammenhang steht. In Zecken können aber noch viele weitere humanpathogene Viren, Bakterien und Parasiten gefunden werden. In der vorliegenden Übersicht wird das Wissen über humanpathogene und auch bisher als apathogen geltende Viren in Europa sowie über die verschiedenen Spezies vektorkompetenter Zecken zusammengestellt. In diesen Zecken findet man Viren aus 6 Familien mit 8 Genera und 35 Spezies,wobei die Familien der Flavi-, Bunya- und Reoviridae mit den Genera Flavivirus, Nairovirus und Orbivirus die meisten Vertreter stellen. Aufgrund ihrer besonderen Bedeutung werden das Omsker Hämorrhagische Fieber, die Louping-ill-Krankheit, die Frühsommer-Meningoenzephalitis und das Krim-Kongo Hämorrhagische Fieber detailliert besprochen. Obwohl das West-Nil-Virus primär von verschiedenen Mückenarten auf den Menschen übertragen wird, erfolgt hier eine ausführliche Besprechung, weil West-Nil-Fieber ein anschauliches Beispiel für eine neu auftretende und sich rasch ausbreitende „Vector-borne disease“ in den USA bzw. eine wiederkehrende Krankheit in Europa ist. Zudem zeichnet sich für das Virus neben dem Mücken-Vogel-Kreislauf auch noch ein solcher zwischen Vogel und Zecke ab. Die vorliegende Übersicht macht aber auch in aller Klarheit deutlich, dass die durch Zecken übertragenen Viren in Europa, mit Ausnahme des FSME-Virus, ein leider auch besonders in Deutschland vernachlässigter Forschungsgegenstand sind.AbstractThe importance of tick-borne encephalitis (TBE) and Lyme borreliosis—the main European tick-borne diseases—is steadily growing. This fact is due to many different factors including climate changes. However, across Europe many other human pathogenic viruses, bacteria, and parasites are found in ticks. This article gives an overview of tick-borne human pathogenic viruses present in Europe as well as those considered nonpathogenic . It also deals with the various species of vector-competent ticks. Among these ticks, viruses from 6 families encompassing 8 genera and 35 species are found,whereby the families Flaviviridae, Bunyaviridae, and Reoviridae with the genera flavivirus, nairovirus, and orbivirus are the most prevalent. Because of their special importance Omsk hemorrhagic fever, louping-ill disease, tick-borne encephalitis, and Crimean-Congo hemorrhagic fever are discussed in detail. Although West Nile virus is mainly transmitted to man by mosquitoes, it is discussed in detail, because West Nile fever has become a good example for a new and rapidly spreading vector-borne disease in the USA and for a recurrent one in Europe. Secondly, besides the mosquito-bird cycle an interesting tickbird cycle is emerging. The overview clearly demonstrates that in Europe (particularly in Germany) tick-borne viruses, with the exception of TBE, constitute an extremly ignored research subject. This lack of attention is in contrast to their importance and results in a lack of necessary scientific data.

[Tick-borne human pathogenic microorganisms found in Europe and those considered nonpathogenic. Part II: Bacteria, parasites and mixed infections].

ZusammenfassungDie Bedeutung von durch Zecken übertragenen Krankheiten hat in den letzten Jahren objektiv und auch subjektiv deutlich zugenommen. Diese Tatsache wurde bereits im Teil I dieser Veröffentlichung bei der Darstellung der durch Zecken in Europa übertragenen Viren, insbesondere hinsichtlich der Frühsommer-Meningoenzephalitis, deutlich. Im vorliegenden Teil II werden die in Europa durch Zecken übertragenen Bakterien und Parasiten, ihre Bedeutung und ihre Vektoren vorgestellt sowie die Klinik, Diagnostik, Prophylaxe und Therapie der daraus resultierenden Erkrankungen dargelegt. Dabei steht die Lyme-Borreliose als bedeutungsvollste durch Zecken übertragene bakterielle Krankheit des Menschen naturgemäß an erster Stelle. Es werden neben grundlegenden Informationen auch wichtige praxisrelevante Hinweise gegeben. Anschließend werden nach dem gleichen Schema die weiteren bisher bekannten bakteriellen Erkrankungen besprochen: Rückfallfieber, Tularämie, die Ehrlichiosen und die Rickettsiosen, einschließlich des Q-Fiebers. Die durch Zecken übertragenen zoonotischen Babesien sind Parasiten, deren hohe veterinärmedizinische Bedeutung bereits seit weit über 100 Jahren bekannt ist, deren humanmedizinische Relevanz in Europa aber erst seit 1957 zunehmend klarer wird. Dass Mehrfach- und Mischinfektionen durch Zeckenstiche induziert werden können,weiß man seit vielen Jahren. In Kenntnis der z. T. hohen Erregerprävalenzen in Zecken waren solche Mehrfachinfektionen auch zu erwarten. Erst in den letzten Jahren zeigt sich aber, dass Doppel- und Mehrfachinfektionen des Menschen nach Zeckenstich viel weiter verbreitet sind, als bisher angenommen wurde. Dies erfordert z. B. bei unklaren Anamnesen ein neues diagnostisches und therapeutisches Denken. Insgesamt wird aber auch erkennbar, dass für eine effektive Zurückdrängung der durch Zecken übertragenen Krankheiten noch erhebliche Forschungsarbeit zu leisten ist.AbstractThe importance of tick-borne diseases has significantly increased objectively and subjectively during the last few years. This fact was demonstrated by the description of tick-borne viruses, in particular with respect to tickborne encephalitis published in part I. Here in part II, tick-borne bacteria and parasites will be discussed as well the significance of these agents, their vectors, clinical course, diagnostics, prophylaxis, and therapy. Naturally, Lyme borreliosis, one of the most important tick-borne bacterial illnesses of humans, is the center of our interest. In addition to basic understanding, critical practice-relevant advice regarding all agents is presented. Similarly all tick-borne bacterial diseases such as relapsing fever, tularemia, ehrlichiosis, and rickettsiosis including Q fever will be discussed. Tick-borne zoonotic babesiae are parasites whose veterinary importance has been known for the last 100 years but whose relevance for human medicine only became evident in 1957. The fact that multiple and mixed infections caused by ticks are possible has been known for years. Taking into account such a high prevalence of the infectious agents in ticks, such multiple infections were to be expected. During the last few years it has become evident that double and multiple infections of humans caused by tick bites occur far more frequently than has been known so far. As a result, in cases of unclear anamnesis,new diagnostic and therapeutic approaches should be taken. In general one can say that considerable additional scientific research is necessary to effectively reduce the incidence of tick-borne diseases.

Durch Zecken ubertragene humanpathogene und bisher als apathogen geltende Mikroorganismen in Europa. Teil II: Bakterien, Parasiten und Mischinfektionen

ZusammenfassungDie Bedeutung von durch Zecken übertragenen Krankheiten hat in den letzten Jahren objektiv und auch subjektiv deutlich zugenommen. Diese Tatsache wurde bereits im Teil I dieser Veröffentlichung bei der Darstellung der durch Zecken in Europa übertragenen Viren, insbesondere hinsichtlich der Frühsommer-Meningoenzephalitis, deutlich. Im vorliegenden Teil II werden die in Europa durch Zecken übertragenen Bakterien und Parasiten, ihre Bedeutung und ihre Vektoren vorgestellt sowie die Klinik, Diagnostik, Prophylaxe und Therapie der daraus resultierenden Erkrankungen dargelegt. Dabei steht die Lyme-Borreliose als bedeutungsvollste durch Zecken übertragene bakterielle Krankheit des Menschen naturgemäß an erster Stelle. Es werden neben grundlegenden Informationen auch wichtige praxisrelevante Hinweise gegeben. Anschließend werden nach dem gleichen Schema die weiteren bisher bekannten bakteriellen Erkrankungen besprochen: Rückfallfieber, Tularämie, die Ehrlichiosen und die Rickettsiosen, einschließlich des Q-Fiebers. Die durch Zecken übertragenen zoonotischen Babesien sind Parasiten, deren hohe veterinärmedizinische Bedeutung bereits seit weit über 100 Jahren bekannt ist, deren humanmedizinische Relevanz in Europa aber erst seit 1957 zunehmend klarer wird. Dass Mehrfach- und Mischinfektionen durch Zeckenstiche induziert werden können,weiß man seit vielen Jahren. In Kenntnis der z. T. hohen Erregerprävalenzen in Zecken waren solche Mehrfachinfektionen auch zu erwarten. Erst in den letzten Jahren zeigt sich aber, dass Doppel- und Mehrfachinfektionen des Menschen nach Zeckenstich viel weiter verbreitet sind, als bisher angenommen wurde. Dies erfordert z. B. bei unklaren Anamnesen ein neues diagnostisches und therapeutisches Denken. Insgesamt wird aber auch erkennbar, dass für eine effektive Zurückdrängung der durch Zecken übertragenen Krankheiten noch erhebliche Forschungsarbeit zu leisten ist.AbstractThe importance of tick-borne diseases has significantly increased objectively and subjectively during the last few years. This fact was demonstrated by the description of tick-borne viruses, in particular with respect to tickborne encephalitis published in part I. Here in part II, tick-borne bacteria and parasites will be discussed as well the significance of these agents, their vectors, clinical course, diagnostics, prophylaxis, and therapy. Naturally, Lyme borreliosis, one of the most important tick-borne bacterial illnesses of humans, is the center of our interest. In addition to basic understanding, critical practice-relevant advice regarding all agents is presented. Similarly all tick-borne bacterial diseases such as relapsing fever, tularemia, ehrlichiosis, and rickettsiosis including Q fever will be discussed. Tick-borne zoonotic babesiae are parasites whose veterinary importance has been known for the last 100 years but whose relevance for human medicine only became evident in 1957. The fact that multiple and mixed infections caused by ticks are possible has been known for years. Taking into account such a high prevalence of the infectious agents in ticks, such multiple infections were to be expected. During the last few years it has become evident that double and multiple infections of humans caused by tick bites occur far more frequently than has been known so far. As a result, in cases of unclear anamnesis,new diagnostic and therapeutic approaches should be taken. In general one can say that considerable additional scientific research is necessary to effectively reduce the incidence of tick-borne diseases.