Tularemia in Germany: the tip of the iceberg?

Tularemia is a rare, notifiable zoonosis in Germany. Since November 2004, several lines of evidence including outbreaks in humans or animals and confirmed infections in indigenous hare and rodent populations have indicated a re-emergence of tularemia in different German federal states. Unfortunately, reliable basic information on the seroprevalence in different geographical regions, permitting the identification of risk factors, does not exist. Combining a sensitive screening assay with a highly specific confirmative immunoblot test, we performed a serological investigation on 2416 sera from a population-based, cross-sectional health survey of the city population of Leutkirch, Baden-Wuerttemberg. A total of 56 sera gave positive results indicating a seroprevalence of 2.32%. Thus, the seroprevalence is tenfold higher than that previously reported in a nationwide study in 2004. Francisella tularensis can cause a wide variety of clinical syndromes including severe, sometimes fatal disease. Missing epidemiological data on its spatial and temporal distribution in an endemic country complicate an appropriate risk assessment necessary for public health authorities to be prepared for an adequate outbreak management. This is of special concern regarding the extraordinary potential of F. tularensis as an agent of bioterrorism. Our investigation performed in a presumed low-risk area demonstrated that tularemia might be seriously underestimated in Germany and probably in other central European countries as well.

Serological and epidemiological analysis of an outbreak of gastroenteritis among military recruits in Germany caused by Cryptosporidium parvum.

BACKGROUND: Cryptosporidium spp. cause enteritic disease worldwide. Besides those patients with an impaired immune system, the general population is also at risk. PATIENTS AND METHODS: Stool samples from participants of a military field exercise were tested for enteritic pathogens and sera were analyzed for Cryptosporidium-antibodies. All participants received a questionnaire for assessing possible risk factors. RESULTS: After a 5-day field training, 201 of a total of 450 soldiers (45%) developed acute gastroenteritis. Immediate microbiological analysis ruled out enteropathogenic bacteria and viruses as the cause of the disease. Only after hospitalization of one of the patients diagnostic procedures were expanded to the identification of parasites and Cryptosporidium parvum was identified. In addition, 14 fecal samples of 217 specimens were subsequently identified in a Cryptosporidium antigen ELISA. A serological analysis of 214 sera revealed 72% positive for specific IgG antibodies compared with 17% of a control group of soldiers who had not participated in the field training (relative risk 3.38; 95% CI 2.39-4.77; p < 0.001). Analysis of specific IgM levels was less conclusive. Epidemiological analysis of questionnaires correlated drinking of tap water, or consumption of various meals with gastroenteritis. However, the source of contamination could not be identified. CONCLUSION: Cryptosporidium spp. can cause acute enteritis even in healthy, young adults as demonstrated by this outbreak. Using serological methods, the extent of the outbreak could be estimated in a retrospective analysis.

Apparently non-specific results found using a norovirus antigen immunoassay for fecal specimens from neonates.

Norovirus is increasingly recognized as a frequent cause of non-bacterial gastroenteritis. Despite a 10-fold increase in the number of cases reported following the availability of enzyme immunoassays, there are no reports yet from preterm neonates. We report on a sudden clustering of antigen-positive enzyme immuno assays results in a level III neonatal intensive care unit, involving 22 of 43 infants screened. Although antigen-positive samples were significantly associated with bloody stools (P<0.001) and gastric residues (P<0.02), norovirus infection could not be confirmed by reverse-transcriptase polymerase chain reaction or electron microscopy. We question the validity of the so called norovirus-specific antigen assays and warn against overreacting to positive enzyme immunoassays results without reverse-transcriptase polymerase chain reaction confirmation especially in the neonatal setting.

[Re-evaluation of the risk areas for tick-borne encephalitis in Germany]. / "FSME-risikogebiete in Deutschland müssen neu definiert werden".

In the view of the authors, the present map showing the distribution of the high-risk areas for tick-borne encephalitis is no longer compatible with state of the art information. They therefore call for a re-evaluation of present definitions and a new risk map. Detection of the virus in ticks with the aid of PCR should be given the same weight as an individual clinical case. In future, a differentiation into low-risk, risk and high-risk areas should be avoided.

[The occurrence of Coxiella burnetii in sheep and ticks of the genus Dermacentor in Baden-Wuerttemberg]. / Untersuchungen zum Vorkommen von Coxiella burnetii bei Schafen und Zecken der Gattung Dermacentor in Baden-Württemberg.

This study examined the occurrence of Coxiella burnetii (C. burnetii), the infectious agent of Q-fever, in sheep and sheep-ticks in Baden-Wuerttemberg, Germany, as a possible source of infection in Q-fever outbreaks. Using PCR, we examined a total of 1066 Dermacentor ticks from 23 herds and 49 samples of tick excrement from 18 herds for C. burnetii. We found the infectious agent in one non-engorged tick and in one sample of tick excrement from the same herd, in Efringen-Kirchen (district Loerrach). Sequencing the PCR-products confirmed the amplifications as specific for C. burnetii. Further serological tests of random samples of the four districts of Baden-Wuerttemberg showed a seroprevalence from 0 to 1.4% using complement fixation test (CFT), as well as a 0.9 to 10.2% seroprevalence, using ELISA test. Serum samples from a Q-fever-suspicious herd resulted, however, in 6% (CFT) and 53% (ELISA) positive reactions. A comparison between CFT and ELISA showed both a correlation of the two test methods that increased with higher CFT titration levels and positive reactions using ELISA for 9.4% of the serums that had tested negative using CFT. The results of the present study reveal that ticks and their excrements are important vectors of transmission of Q-fever in Baden-Wuerttemberg. Investigations on C. burnetii using PCR as well as serological surveys of sheep are important instruments for diagnosis and disease control of Q-fever.

Neotrombicula autumnalis (Acari, Trombiculidae) as a vector for Borrelia burgdorferi sensu lato?

Larvae of the trombiculid mite Neotrombicula autumnalis were collected at 18 sites in and around Bonn, Germany, to be screened for infection with Borrelia burgdorferi s.l. by means of PCR. Questing larvae numbering 1380 were derived from the vegetation and 634 feeding ones were removed from 100 trapped micromammals including voles, mice, shrews and hedgehogs. In a laboratory infection experiment, a further 305 host-seeking larvae from the field were transferred onto Borrelia-positive mice and gerbils, and examined for spirochete infection at various intervals after repletion. In three cases borrelial DNA could be amplified from the mites: (1) from a larva feeding on a wild-caught greater white-toothed shrew (Crocidura russula), (2) from a pool of four larvae feeding on a B. garinii-positive laboratory mouse, and (3) from a nymph that had fed on a B. afzelii-positive laboratory gerbil as a larva. In the first case, borrelial species determination by DNA hybridization of the PCR product was only possible with a B. burgdorferi complex-specific probe but not with a species-specific one. In the second case, probing showed the same borrelial genospecies (B. garinii) as the laboratory host had been infected with. In the latter case, however, DNA hybridization demonstrated B. valaisiana while the laboratory host had been infected with B. afzelii. Subsequent DNA sequencing confirmed much higher similarity of the PCR product to B. valaisiana than to B. afzelii indicating an infection of the mite prior to feeding on the laboratory host. The negligible percentage of positive mites found in this study suggests that either the uptake of borrelial cells by feeding trombiculids is an extremely rare event or that ingested spirochetes are rapidly digested. On the other hand, the results imply a possible transstadial and transovarial transmission of borreliae once they are established in their trombiculid host. However, unless the transmission of borreliae to a given host is demonstrated, a final statement on the vector competence of trombiculid mites is not possible.

[Epidemiology of hantaviruses in Baden-Wurttemberg]. / Epidemiologie von Hantaviren in Baden-Württemberg.

Hantavirus, originally named after the Hantaan River in Korea, is the aetiologic agent for the Hemorrhagic Fever with Renal Syndrome (HFRS) in the asian region, in the Americas for the Hantavirus Pulmonary Syndrome (HPS). In Middle Europe hantaviruses are responsible for the "Nephropathia Epidemica" (NE), a mild form of HFRS. Hantaviruses belong to the family of Bunyaviridae. Like other members of this family their genome consists of three segments of single stranded RNA (ss-RNA) leading to various subtypes, strongly associated with different rodent hosts. There are two major groups, the hantaan lineage harbored by murine rodents and the Puumala lineage harbored by arvicolidae ("old world") and sigmodontidae ("new world"). Infected rodents may develop chronic infections for months or even life-long and may shed infectious virus with urine and feces. The primary mode of infection of man occurs by inhaling contaminated aerosols or soil particles. The collection of epidemiologic data in the state of Baden-Württemberg was realized in three different steps: Collection and localisation of clinical cases (n = 62): A concentration of clinical cases in the middle of the state was found. The examination of the seroprevalence of exposed persons: By the examination of 4000 sera from forest workers, a seroprevalence with an average of 2.1% was found. In the districts of Reutlingen and Tübingen seroprevalences up to 9% were found. This leads to the assumption that there are endemic areas. Epidemiologic studies of reservoir hosts: Serologic surveys of rodents (n = 1150) in the described areas yielded to a seroprevalence up to 10-30%. Virus carriers were determined with RT-PCR and nested-PCR testing. The prevalence in the rodent population showed an average of 10%. The isolated subtypes were all identified as members of the Puumala-lineage. The origin of sporadic infections with Hantavirus of the Hantaan-lineage in Baden-Wuerttemberg is still unknown.

Detection and molecular typing of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks and in different patient samples from southwest Germany.

The prevalence of different genospecies of Borrelia burgdorferi sensu lato in infected ticks could be a determinant for the risk of acquiring Lyme borreliosis (LB) and its clinical presentation. A total of 7373 ticks and 2761 samples from LB patients from the same area in southwest Germany were analyzed by PCR to assess the frequency of the occurrence of LB-associated genospecies. Fifteen percent of the tick samples and 19% of the human samples were found positive for the presence of B. burgdorferi sensu lato. Further identification of 1106 B. burgdorferi sensu lato positive tick samples by reverse line blotting and 125 positive patient samples by nested PCR using species-specific primers revealed the occurrence of B. afzelii, B. burgdorferi sensu stricto, B. garinii and B. valaisiana. Both single-species and mixed infections were noted and a similar distribution of the different genospecies was found in ticks compared with human samples. It was also the purpose of this study to obtain more information about a possible correlation between the distribution of Borrelia species and clinical syndromes of LB. Skin biopsies of 59 patients with acrodermatitis chronica atrophicans and cerebrospinal fluid samples from 78 patients with possible neuroborreliosis were analyzed. In conclusion, the distribution of the different genospecies in ticks is the decisive factor for the occurrence of the different Borrelia genospecies in samples from LB patients. Borrelia afzelii is the predominant genospecies in all kind of samples from the observed area and there seems to be no association of particular Borrelia genospecies with distinct clinical manifestations of LB.

Long-term seropositivity against Echinococcus multilocularis in an epidemiological follow-up study in southwestern Germany (Römerstein).

BACKGROUND: Out of 2,560 participants in an epidemiological survey of alveolar echinococcosis (AE) performed in 1996 in southwestern Germany, 47 participants had tested seropositive in one of two crude antigen screening ELISAs and were inconspicuous on hepatic ultrasound. PATIENTS AND METHODS: Out of these 47 seroreactors, 36 attended a follow-up examination 30 months after the primary examination, including ultrasound and serology with various Echinococcus multilocularis-specific antigens. RESULTS: No lesion suspicious for AE was detected in any participant. Serology showed only minor changes as compared to the earlier results. CONCLUSION: Persistent seropositivity without detectable hepatic lesions could be interpreted as an early sign of sonographically not yet detectable AE, immunity to E. multilocularis or unspecific serological reactivity. For seropositive and clinically inconspicuous inhabitants of areas endemic for AE follow-up examinations at intervals of 2-3 years seem to be adequate.

[Surveillance of group illnesses with Norwalk-like viruses (NLV) in Baden-Württemberg. Isolation and detection of Norwalk-like viral RNA from fecal samples with RT-PCR]. / Verfolgung von Gruppenerkrankungen mit Norwalk-like-Viren (NLV) in Baden-Württemberg. Isolierung und Detektion von Norwalk-like-Virus-RNA aus Stuhlproben mittels RT-PCR.

Between the turn of the years 98/99 and 99/2000, stool samples from various health departments in the south of Germany were sent to the LGA (Landesgesundheitsamt Baden-Württemberg). Different testings as bacteriological routines for enterobacteriaceae and virological antigen screenings of the stool samples for adeno-, astro- and rotaviruses lead to negative results. Additional RT-PCR tests for the presence of Norwalk-RNA showed nearly 40% of positive samples. From 14 group infections between december 98 and january 2000, 286 samples in total were sent to the LGA, 118 of them gave positive results for the norwalk agent. For the verification of the analysis, some random samples were sent to the Robert-Koch-Institut, div. of molecular virology at Berlin, where the results were confirmed.