Highly specific detection of Cryptosporidium spp. oocysts in human stool samples by undemanding and inexpensive phase contrast microscopy.

To compare phase contrast microscopy (PCM) of unstained slides for the detection of Cryptosporidium spp. oocysts with a commercially available enzyme immunoassay (EIA) for the detection of cryptosporidial antigen in human stool samples, we prospectively analysed by both methods 463 fresh human stool samples obtained from diarrhoeic patients between July and October 2014. Compared with the EIA, the sensitivity, specificity, positive and negative predictive value of PCM were 88.9 % (95 % confidence interval (CI), 66.0-98.1 %), 100 % (95 % CI, 99.0-100 %), 100 % (95 % CI, 77.3-100 %) and 99.6 % (95 % CI, 98.3-100 %), respectively. Additionally, we retrospectively examined with PCM 65 fixed stool samples that had been collected in 2010 from mostly asymptomatic Rwandan children <5 years of age; 14 of these samples had previously yielded positive results with a highly sensitive real-time (RT)-PCR. PCM detected cryptosporidia in 5/14 RT-PCR-positive samples, and notably, also in one of 51 RT-PCR-negative samples, which was subsequently confirmed by acid-fast staining. Positive and negative percent agreement of PCM with RT-PCR were 35.7 % (95 % CI, 16.2-61.4 %) and 98.0 % (95 % CI, 88.7-100 %), respectively. Positive PCM results were associated with higher RT-PCR cycle threshold values (p = 0.044). In conclusion, PCM offers a highly specific, undemanding and inexpensive method for the laboratory diagnosis of acute human cryptosporidiosis independent of the causative Cryptosporidium species.

Occurrence of different Borrelia burgdorferi sensu lato genospecies including B. afzelii, B. bavariensis, and B. spielmanii in hedgehogs (Erinaceus spp.) in Europe.

In order to determine whether European hedgehogs (Erinaceus europaeus and E. roumanicus) play a role in the epidemiological cycle of Borrelia burgdorferi sensu lato in Central Europe and Great Britain, tissue samples of hedgehogs from Germany (n=211), Austria (n=4), the Czech Republic (n=22), and the U.K. (n=32) were tested for the presence of these tick-borne pathogens. PCR for amplification of the B. burgdorferi s.l.-specific 5S-23S intergenic spacer region as well as the outer surface protein A (ospA) gene were used. B. burgdorferi s.l. DNA was detected in 35 of the 259 E. europaeus and in 2 of 10 E. roumanicus. B. burgdorferi prevalences in E. europaeus ranged from 0% (U.K.) to 37.5% (Czech Republic), for E. roumanicus from 0% (Czech Republic) to 50.0% (Austria). Sequencing revealed the occurrence of 3 different B. burgdorferi genospecies in E. europaeus: B. afzelii was the dominant genospecies, followed by B. bavariensis (previously B. garinii OspA serotype 4) and B. spielmanii, the latter was detected for the first time in Hamburg (Germany). B. afzelii and B. bavariensis were also found in E. roumanicus. Our results suggest that hedgehogs modulate the epidemiology of certain species of the B. burgdorferi s.l. complex, potentially affecting the distribution and abundance of individual B. burgdorferi s.l. genospecies in various habitats. We hypothesise that juvenile or individuals with low immune competence in particular, have a high reservoir potential for the 3 genospecies identified here.

Diagnosis of acute Q fever with emphasis on enzyme-linked immunosorbent assay and nested polymerase chain reaction regarding the time of serum collection.

A commercially available enzyme-linked immunosorbent assay (ELISA) (Virion/Serion [Wuerzburg, Germany]), an indirect fluorescent antibody test (IFAT) (BIOS/Focus [Cypress, CA]), and a nested polymerase chain reaction (PCR) were explored for diagnosis of acute Q fever in reference to time of serum collection. Serum samples of 22 patients with acute Q fever collected around the fifth day of illness were included. A sensitivity of 30% by ELISA and 80% by IFAT (P = 0.1) was found for the first 5 days of illness and 92% by ELISA and 83% by IFAT during the sixth and eleventh day. PCR revealed a positive result in 8 cases (36%) with 6 cases deriving from the first 5 days of illness. We conclude that ELISA aids especially in the diagnosis of Q fever after 5 days of illness. The benefit of PCR as an additional tool to ELISA was especially evident in the early days of serum sampling.

Prevalence of Coxiella burnetii and Rickettsia spp. in ticks and rodents in southern Germany.

Coxiella burnetii, the causative agent of Q fever, and Rickettsia spp. are bacterial pathogens that can be transmitted by ticks of the genus Dermacentor (i.e., Dermacentor marginatus and D. reticulatus). In Germany, the occurrence of these ticks is currently limited to few areas. However, due to increasing temperatures, these vectors will likely extend their distribution in the future, and C. burnetii and Rickettsia spp. might spread with them. To assess the prospective risk of human infections by these agents, it is important to know their current distribution. We collected 666 adult Dermacentor spp. and 119 rodents, mainly Microtus arvalis, in 3 Q fever endemic areas in southern Germany. Ticks and rodent organ pools were screened by PCR for C. burnetii and Rickettsia spp. No evidence of C. burnetii infections could be found in ticks or rodents, suggesting that these animals do not play an essential role in the epidemiology of Q fever in Germany. Rickettsia raoultii and R. slovaca could be detected in 30.3% and 0.75% of all examined ticks, respectively. In contrast, no rickettsia infections could be found in any rodent samples. Both rickettsia species can cause tick-borne lymphadenopathy (TIBOLA), a usually mild human disease. Because of the possible transmission of these rickettsiae to humans, TIBOLA should be considered in the differential diagnosis of tick-borne diseases. Our data show that a spread of these rickettsiae is possible in Germany and that more studies on the distribution of these agents are necessary.

Spread of ticks and tick-borne diseases in Germany due to global warming.

Tick-transmitted diseases like tick-borne encephalitis and Lyme Borreliosis have been well known in Germany for decades. Global climate changes may influence the emergence and reemergence of diseases. Ongoing research now gives an additional focus on other tick-borne pathogens such as Coxiella burnetii, Rickettsia conorii, Anaplasma phagocytophilum and Babesia spp., the causative agents of Q-fever, Mediterranean spotted fever, Anaplasmosis and Babesiosis, respectively. The epidemiology of these pathogens was investigated on ticks as well as on rodents, the main hosts. Therefore adults of Dermacentor spp. (n = 862) and rodents (n = 119) were collected and examined for the existence of C. burnetii and Rickettsia spp. by polymerase chain reaction (PCR). In none of the ticks and rodents C. burnetii could be detected, in contrast to Rickettsia spp. where the infection rate in ticks was about 20%. Over and above that, nymphs and adults of Ixodes ricinus were also collected and investigated by PCR for A. phagocytophilum (n = 5,424), Rickettsia helvetica (n = 1,187) and Babesia spp. (n = 3,113). Thereby infection rates of 1%, 8.9% and 1%, respectively, could be determined. The prevalence in rodents was 5.3% for A. phagocytophilum and 0.8% for Babesia microti. None of the rodents was R. helvetica positive.

A super-spreading ewe infects hundreds with Q fever at a farmers' market in Germany.

BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identify notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study (cases were Q fever patients, controls were randomly selected Soest citizens) and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii (C. burnetii). RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16-24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3rd trimester and to test animals in petting zoos regularly for C. burnetii.

Serological diagnosis and follow-up of asymptomatic and acute Q fever infections.

During an outbreak of Q fever at a farmer's market in Soest (North Rhine-Westphalia, Germany) in 2003, we examined 263 serum samples of presumably infected persons for Q fever antibodies. One hundred and seventy-one of these patients were tested positive for acute Q fever infection. Furthermore, 29 persons of certain risk groups like pregnant women (n=11) or patients with valvular heart disease (n=18) were examined. Among these, in four pregnant women and two heart patients an acute but asymptomatic infection could be diagnosed. With 30 patients we performed a serological follow-up for 8-60 weeks. In our study, phase 2 (PH2)-IgM antibodies as a marker for acute infection were present in all 30 patients 3-4 weeks after onset of clinical signs and disappeared 3-4 months later. Six weeks to three months after clinical manifestation, all patients developed PH1-IgG antibodies in low levels with no clinical signs of chronic Q fever. Three patients, including one pregnant woman showed high-level titres and were treated for chronic Q fever. Eleven patients with low PH1-IgG antibodies and all three patients with high titres developed IgA antibodies from 10 weeks after clinical manifestation; therefore PH1-IgA cannot be used as the only serological marker for chronic Q fever. Chronic infections were indicated only by a continuous increase of PH1 antibodies and a high persistence of PH2-IgG. We therefore conclude that the exclusion of chronic Q fever infection by a single serological examination cannot be done. At least three consecutive tests should be performed, that is 3, 6, and 9 months after initial Q fever infection.

The Golden Agers and Tick-borne encephalitis. Conference report and position paper of the International Scientific Working Group on Tick-borne encephalitis.

The 7th meeting of the ISW TBE had the main topic "Tick-borne encephalitis in the Golden Agers". Data from 14 European countries were presented about incidence and clinical course of Tick borne encephalitis (TBE) in general and especially in the population over 50 years of age. With age immunity is impaired quantitatively and qualitatively, the reactions to vaccinations are generally slower, antibody titres reach lower values and decrease earlier. The incidence of the disease is increasing with age, also the clinical course is more severe, they suffer significantly more sequelae, need a longer rehabilitation and have a higher case fatality. Vaccination as the only efficient protection is needed in endemic areas, considering that mobility has increased very much. For the age group over 50 years regular booster vaccinations according to the recommended vaccination intervals or even shorter intervals are most important.

Tick-borne encephalitis in childhood--consensus 2004.

Tick-borne encephalitis (TBE) is a communicable disease caused by a flavi-virus, ticks being the main vectors. The nervous system is affected, four clinical features of different severity are observed: meningitis, meningoencephalitis, meningoencephalomyelitis, meningoradiculoneuritis. TBE is a preventable disease, which is rapidly becoming a growing public health problem in Europe. So far no causal treatment is possible but an efficient, safe vaccination is available. During the 6th meeting of the International Scientific Working Group on TBE with the main conference issue "Tick-borne encephalitis in childhood" an international consensus was achieved. In countries where TBE is endemic--and not prevented by immunization--both children and adults are affected. The disease in children is generally milder, although severe illness may occur and even lead to permanent impairment of the quality of life due to neuropsychological sequelae. Therefore immunization should be offered to all children living in or traveling to endemic areas.

Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation.

BACKGROUND: Noroviruses (NoV) have become one of the most commonly reported causative agents of large outbreaks of non-bacterial acute gastroenteritis worldwide as well as sporadic gastroenteritis in the community. Currently, reverse transcriptase polymerase chain reaction (RT-PCR) assays have been implemented in NoV diagnosis, but improvements that simplify and standardize sample preparation, amplification, and detection will be further needed. The combination of automated sample preparation and real-time PCR offers such refinements. METHODS: We have designed a new real-time RT-PCR assay on the LightCycler (LC) with SYBR Green detection and melting curve analysis (Tm) to detect NoV RNA in patient stool samples. The performance of the real-time PCR assay was compared with that obtained in parallel with a commercially available enzyme immunoassay (ELISA) for antigen detection by testing a panel of 52 stool samples. Additionally, in a collaborative study with the Baden-Wuerttemberg State Health office, Stuttgart (Germany) the real-time PCR results were blindly assessed using a previously well-established nested PCR (nPCR) as the reference method, since PCR-based techniques are now considered as the "gold standard" for NoV detection in stool specimens. RESULTS: Analysis of 52 clinical stool samples by real-time PCR yielded results that were consistent with reference nPCR results, while marked differences between the two PCR-based methods and antigen ELISA were observed. Our results indicate that PCR-based procedures are more sensitive and specific than antigen ELISA for detecting NoV in stool specimens. CONCLUSIONS: The combination of automated sample preparation and real-time PCR provided reliable diagnostic results in less time than conventional RT-PCR assays. These benefits make it a valuable tool for routine laboratory practice especially in terms of rapid and appropriate outbreak-control measures in health-care facilities and other settings.

Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in Southern Germany.

Tick-transmitted diseases like tick-borne encephalitis and Lyme borreliosis have been well known in Germany for decades. Ongoing research now gives an additional focus to a broad range of other bacteria and parasites in ticks like Anaplasma phagocytophilum, former Ehrlichia sp., Rickettsia sp. and Babesia sp. Knowledge about the prevalence of these infectious agents in ticks is an important prerequisite for risk assessment of human diseases. Therefore nymphs and adult Ixodes ricinus ticks were collected and examined for Anaplasma phagocytophilum (n = 5424 ticks), Rickettsia sp. (n = 1187), and Babesia sp. (n = 3113). For the detection of Anaplasma phagocytophilum, DNA from the 16S rDNA gene was amplified by nested PCR and hybridized with a DIG-labeled oligonucleotide probe. The examination of Rickettsia sp. was performed by single PCR. A partial sequence of the citrate synthase gene was amplified. As a target for the detection of Babesia sp., DNA from the 18S rDNA gene was amplified, also by single PCR. All positive PCR products were sequenced to control specificity. Anaplasma phagocytophilum was detected by PCR in n = 103 (1.9%) out of 5,424 examined ticks from 11 investigation areas. However, not all positive PCR products hybridized using DIG-labeled oligonucleotide probe. Thus, the result of sequencing indicated that only 1.0% (n = 54) belonged to Anaplasma phagocytophilum and nearly half of these PCR products (0.9%) were identified as Wolbachia sp. Rickettsia sp. in Ixodes ricinus ticks from 3 areas were found in n = 105 (8.9%) out of 1,187 ticks examined (range from 13.3% to 5.6%). Sequencing showed Rickettsia helvetica exclusively. In about 2.6% of Rickettsia-positive ticks, double infection with Anaplasma phagocytophilum was found. Babesia sp. was detected in n= 31 (1.0%) out of 3,113 ticks examined, which originated from 4 different areas. By sequencing, n = 28 (90.0%) were identified as Babesia divergens. Three of all Babesia-positive ticks were identified as harboring Babesia microti. The detection of Anaplasma phagocytophilum, Rickettsia sp. and Babesia sp. demonstrates their possible role as a source of human infection in Germany.

Salmonella in sesame seed products.

In the context of an international outbreak of multiresistant Salmonella Typhimurium DT 104 that was correlated to the consumption of halvah ("helva," an Asian candy made from sesame seed), we examined several sesame seed products for the occurrence of Salmonella. Of 117 ready-to-eat food items containing sesame, we isolated salmonellae from 11 (9.4%) samples. In addition to finding Salmonella Typhimurium DT 104 in the halvah involved in the outbreak, we also isolated different Salmonella Typhimurium strains out of halvah from other manufacturers and countries of origin, as well as Salmonella Offa, Salmonella Tennessee, and Salmonella Poona from sesame paste (tahini) and sesame seed, which is sold for raw consumption in cereals.