Serological and molecular evidence for spotted fever group Rickettsia and Borrelia burgdorferi sensu lato co-infections in The Netherlands.

Only a few reported cases indicate that Rickettsia helvetica and Rickettsia monacensis can cause disease in humans. Exposure to these two spotted fever group (SFG) rickettsiae occurs through bites of Ixodes ricinus, also the primary vector of Lyme borreliosis in Europe. To date, it is unclear how often exposure to these two microorganisms results in infection or disease. We show that of all the Borrelia burgdorferi s.l.-positive ticks, 25% were co-infected with rickettsiae. Predominantly R. helvetica was detected while R. monacensis was only found in approximately 2% of the ticks. In addition, exposure to tick-borne pathogens was compared by serology in healthy blood donors, erythema migrans (EM)-patients, and patients suspected of Lyme neuroborreliosis (LNB). As could be expected, seroreactivity against B. burgdorferi sensu lato was lower in blood donors (6%) compared to EM patients (34%) and suspected LNB cases (64%). Interestingly, seroreactivity against SFG Rickettsia antigens was not detected in serum samples from blood donors (0%), but 6% of the EM patients and 21% of the LNB suspects showed anti-rickettsial antibodies. Finally, the presence of B. burgdorferi s.l. and Rickettsia spp. in cerebrospinal fluid samples of a large cohort of patients suspected of LNB (n=208) was investigated by PCR. DNA of B. burgdorferi s.l., R. helvetica and R. monacensis was detected in seventeen, four and one patient, respectively. In conclusion, our data show that B. burgdorferi s.l. and SFG rickettsiae co-infection occurs in Dutch I. ricinus and that Lyme borreliosis patients, or patients suspected of Lyme borreliosis, are indeed exposed to both tick-borne pathogens. Whether SFG rickettsiae actually cause disease, and whether co-infections alter the clinical course of Lyme borreliosis, is not clear from our data, and warrants further investigation.

Immunogenicity of the Q fever skin test.

OBJECTIVES: The Q fever skin test is used to measure cell-mediated immunity to Coxiella burnetii in pre-vaccination screening to exclude individuals with pre-existing immunity. We investigated whether this in-vivo test influences subsequent measurements of immune response. METHODS: We assessed the humoral and cellular immune responses before, and 6 and 12 months after skin testing in 63 individuals who were not vaccinated because of either a positive skin test or positive serology in screening. IgG anti-C. burnetii antibodies were measured using immune-fluorescence assay (IFA). The cellular immune response was assessed by measuring in-vitro C. burnetii-specific interferon (IFN)-γ production in blood. RESULTS: Of the 35 subjects with a positive skin test and negative serology, 15/35 (43%) showed seroconversion at 6 months, and 7/32 (22%) seropositivity at 12 months. The mean ± SE specific IFN-γ production in this group increased from 185 ± 88 pg/mL (at baseline) to 422 ± 141 pg/mL at 6 months (P = 0.009) and 223 ± 91 pg/mL at 12 months (P = 0.17). Of the 28 subjects with positive serology (and unknown skin test results), 21/28 (75%) showed an increase in IgG anti-phase I titres at 6 months, and 11/25 (44%) at 12 months. The mean ± SE specific IFN-γ production was significantly increased at 6 months, but not at 12 months. CONCLUSIONS: Q fever skin testing causes higher antibody titres and higher in-vitro IFN-γ to C. burnetii, and therefore affects subsequent Q fever diagnostics.

Limited humoral and cellular responses to Q fever vaccination in older adults with risk factors for chronic Q fever.

OBJECTIVES: In the Netherlands, people at risk for chronic Q fever were vaccinated against Coxiella burnetii with the inactivated whole cell vaccine Q-vax®. We aimed to measure the immune responses to C. burnetii six and twelve months after vaccination in this relevant population. METHODS: In 260 vaccinees, antibody responses were assessed by immunofluorescence assay (IFA), complement fixation test and ELISA. The cellular immune responses were assessed by measuring C. burnetii-specific interferon (IFN)-γ production in blood. Serological results of 200 individuals with past Q fever were used for comparison. RESULTS: At six months, 46% of vaccinees showed low IFA antibody titres and 67% had a positive IFN-γ assay; At twelve months, both were 60%. In contrast, individuals with a past Q fever were seropositive in 99.5% at six and twelve months, with relatively higher IFA titres. Interestingly, vaccinees with positive IFN-γ assay pre-vaccination, showed a higher seroconversion rate than IFN-γ negative vaccinees: 74% vs. 41% (p < 0.001). CONCLUSIONS: The immune response after Q-vax® vaccination is lower and restricted to a smaller proportion than found after past Q fever and than previously described after vaccination, suggesting decreased vaccine immunogenicity in this high-risk population. A positive IFN-γ assay before vaccination in seronegative vaccinees likely points to pre-existing immunity resulting in boosting by vaccination.

A prospective study among patients presenting at the general practitioner with a tick bite or erythema migrans in The Netherlands.

BACKGROUND: We performed a nationwide prospective study on the transmission risk for Borrelia to humans, investigating symptoms and serology at enrolment and three months after tick bites, and after standard treatment for erythema migrans (EM). Aiming to quantify the infection risk at point of care by physicians, we explored risk factors such as tick testing for Borrelia and assessment of the duration of the tick's blood meal. METHODS AND FINDINGS: Questionnaires, blood samples and ticks from patients who consulted one of 307 general practitioners for tick bites (n = 327) or EM (n = 283) in 2007 and 2008, were collected at enrolment and three months later at follow-up. Borrelia burgdorferi sensu lato DNA was detected in 29.3% of 314 ticks, using PCR/reverse line blot and real-time PCR on the OspA gene. Seroconversion in C6 ELISA, IgM or IgG immunoblots for Borrelia-specific antibodies was observed in 3.2% of tick bite cases. Fourteen tick bite cases had evidence of early Borrelia infection, of which EM developed among seven cases. The risk of developing EM after tick bites was 2.6% (95%CI: 1.1%-5.0%), and the risk of either EM or seroconversion was 5.1% (95%CI: 2.9%-8.2%). Participants with Borrelia-positive ticks had a significantly higher risk of either EM or seroconversion (odds ratio 4.8, 95%CI: 1.1-20.4), and of seroconversion alone (odds ratio 11.1, 95%CI: 1.1-108.9). A third (34%) of the cases enrolled with EM did not recall preceding tick bites. Three EM cases (1%) reported persisting symptoms, three months after standard antibiotic treatment for EM. CONCLUSIONS: One out of forty participants developed EM within three months after tick bites. The infection risk can be assessed by tick testing for Borrelia at point of care by physicians. However, further refining is needed considering sensitivity and specificity of tick tests, accuracy of tick attachment time and engorgement.

Short communication: prevalence of antibodies against Coxiella burnetii (Q fever) in children in The Gambia, West Africa.

OBJECTIVE: To estimate the prevalence of antibodies against Coxiella burnetii (Q fever) among children in eight villages in The Gambia, West Africa. METHODS: Sera of 796 children aged 1-15 years were tested for presence of antibodies against phase II of C. burnetii by ELISA. RESULTS: IgG and/or IgM phase II antibodies against C. burnetii were detectable in 8.3% (66/796) of all serum samples analysed with significant differences in seroprevalence between villages. Highest prevalence was found in the age group 1-4 years. CONCLUSIONS: Exposure to C. burnetii is considerable in the early years of life in The Gambia, and further studies are warranted to estimate the role of Q fever in acute febrile illness in The Gambia and elsewhere in Africa.

Specific interferon γ detection for the diagnosis of previous Q fever.

BACKGROUND: Current practice for diagnosis of Q fever, caused by the intracellular pathogen Coxiella burnetii, relies mainly on serology and, in prevaccination assessment, on skin tests (STs), which both have drawbacks. In this study, C. burnetii-specific interferon γ (IFN-γ) production was used as a new diagnostic tool for previous Q fever, circumventing most of these drawbacks. Our aim was to compare this test to serology and ST. METHODS: One thousand five hundred twenty-five individuals from an endemic area with a risk for chronic Q fever were enrolled. IFN-γ production was measured after in vitro stimulation of whole blood with C. burnetii antigens. Various formats using different C. burnetii antigens were tested. Serology and ST were performed in all individuals. RESULTS: In all assay formats, C. burnetii-specific IFN-γ production was higher (P < .0001) in seropositive or ST-positive subjects than in seronegative and ST-negative subjects. Whole blood incubated for 24 hours with C. burnetii Nine Mile showed optimal performance. After excluding subjects with equivocal serology and/or borderline ST results, IFN-γ production was 449 ± 82 pg/mL in the positive individuals (n = 219) but only 21 ± 3 pg/mL in negative subjects (n = 908). Using Bayesian analysis, sensitivity and specificity (87.0% and 90.2%, respectively) were similar to the combination of serology and ST (83.0% and 95.6%, respectively). Agreement with the combination of serology and ST was moderate (84% concordance; κ = 0.542). CONCLUSIONS: Specific IFN-γ detection is a novel diagnostic assay for previous C. burnetii infection and shows similar performance and practical advantages over serology and ST. Future studies to investigate the clinical value in practice are warranted.

Comparison of the performance of IFA, CFA, and ELISA assays for the serodiagnosis of acute Q fever by quality assessment.

The indirect immunofluorescence assay (IFA) is considered the reference method for diagnosing Q fever, but serology is also performed by complement fixation assay (CFA) or enzyme-linked immunosorbent assay (ELISA). However, comparability between these assays is not clear, and therefore a quality assessment was performed. A total of 25 serum samples from negative controls, Q fever patients, and a serial diluted high-positive sample were analyzed in 10 Dutch laboratories. Six laboratories performed CFA, 5 performed IFA, and 5 performed ELISAs. Three international reference laboratories from Australia, France, and the USA also participated in this study. Qualitative values between laboratories using the same methods were within close range, and all 3 methods correctly identified acute Q fever patients. The IFA, ELISA, and CFA are all suitable serodiagnostic assays to diagnose acute Q fever, but the IFA remains an important tool in the follow-up of patients and in identifying patients at risk for developing chronic Q fever.

Evaluation and improvement of two PCR targets in molecular typing of clinical samples of Leishmania patients.

Leishmaniasis is a disease caused by the unicellular Leishmania parasite. World wide millions of people are affected by this vector born disease. The disease presents itself in different clinical manifestations which are caused by specific Leishmania species. The therapeutic strategy depends on the Leishmania species involved. It is important to detect Leishmania and subsequently type the infecting species in a sensitive way using PCR. Various targets have been proposed but two seem to be best suited, the ITS1 region and the mini-exon. There is, however, no consensus as to which of these two is best. The aim of this study was to compare both targets with our current method, a PCR on the 18S ribosomal RNA gene. The ITS1 PCR proved to be slightly more sensitive and more practical than the mini-exon. Nevertheless, the mini-exon is more polymorphic and is needed in subtyping Leishmania species belonging to the L. Viannia subgenus. The ITS1 method was adapted to use as a real-time PCR for diagnostic purposes. In addition, designing and testing a new primer set improved sensitivity of the PCR on the mini-exon.

Increase of imported Leishmaniasis in the Netherlands: a twelve year overview (1996-2007).

Surveillance data indicates that the number of cutaneous (CL), mucocutaneous (ML) and visceral leishmaniasis (VL) cases has increased globally and in Europe during the past decades. Leishmaniasis is only seen as an imported disease in the Netherlands. We investigated occurrence in the Netherlands through an analysis of Leishmania patients sent to our laboratory and to the nationwide network of the pathology departments between 1996 and 2007. The majority of patients suffered from CL, and an outbreak among military personnel stationed in endemic regions in 2005 was noted. ML was rarely found. However, the occurrence of VL has clearly increased. Physicians in non-endemic regions should be aware that leishmaniasis can be contracted as close as Southern Europe and that it is not limited to tropical and subtropical regions only.

[Decline of echinococcosis in the Netherlands; 1997-2008]. / Afname van echinokokkose in Nederland; 1997-2008.

OBJECTIVE: To establish the prevalence of human echinococcosis in the Netherlands by using data from laboratories carrying out diagnostic procedures and data from pathology registries from 1997-2008. DESIGN: Descriptive. METHOD: Data on serological diagnostic tests for Echinococcus granulosus carried out from 1997 to 2008 were gathered from the National Institute for Public Health and the Environment (RIVM) in Bilthoven and Leiden University Medical Center (LUMC). Additionally, all echinococcosis patients registered on the pathology database of the Dutch pathological anatomy national automated archive (PALGA) were analysed. RESULTS: A total of 7314 serum samples from 5125 patients were examined for antibodies. Cyst material from 39 patients was examined using molecular methods. The number of serum samples sent in annually was stable at 550 to 600. Over the period investigated, 1997-2008, on serological investigation a total of 485 patients were found to have a positive result on IgG-ELISA. Of these, the diagnosis of echinococcosis was confirmed in 445 patients by further serological investigation (on average 37 new patients each year (range: 19-59)) and/or a positive PCR result. Over the duration of the study period the number of new patients decreased from over 40 to fewer than 30 patients per year. Going by the family name, 95.5% of the 445 patients were probably imported cases of disease. CONCLUSION: In the Netherlands, echinococcosis is primarily seen as an imported disease with the majority of patients originating from areas around the Mediterranean Sea where it is endemic. Each year there are nearly 30 confirmed cases.

Non-travel related Hepatitis E virus genotype 3 infections in the Netherlands; a case series 2004 - 2006.

BACKGROUND: Human hepatitis E virus (HEV) infections are considered an emerging disease in industrialized countries. In the Netherlands, Hepatitis E virus (HEV) infections have been associated with travel to high-endemic countries. Non-travel related HEV of genotype 3 has been diagnosed occasionally since 2000. A high homology of HEV from humans and pigs suggests zoonotic transmission but direct molecular and epidemiological links have yet to be established. We conducted a descriptive case series to generate hypotheses about possible risk factors for non-travel related HEV infections and to map the genetic diversity of HEV. METHODS: A case was defined as a person with HEV infection laboratory confirmed (positive HEV RT-PCR and/or HEV IgM) after 1 January 2004, without travel to a high-endemic country three months prior to onset of illness. For virus identification 148 bp of ORF2 was sequenced and compared with HEV from humans and pigs. We interviewed cases face to face using a structured questionnaire and collected information on clinical and medical history, food preferences, animal and water contact. RESULTS: We interviewed 19 cases; 17 were male, median age 50 years (25-84 y), 12 lived in the North-East of the Netherlands and 11 had preexisting disease. Most common symptoms were dark urine (n = 16) and icterus (n = 15). Sixteen ate pork >/= once/week and six owned dogs. Two cases had received blood transfusions in the incubation period. Seventeen cases were viremic (genotype 3 HEV), two had identical HEV sequences but no identified relation. For one case, HEV with identical sequence was identified from serum and surface water nearby his home. CONCLUSION: The results show that the modes of transmission of genotype-3 HEV infections in the Netherlands remains to be resolved and that host susceptibility may play an important role in development of disease.

Preexisting poliovirus-specific IgA in the circulation correlates with protection against virus excretion in the elderly.

BACKGROUND: Epidemiological studies have indicated that at least 10% of the Dutch elderly do not have poliovirus serotype-specific neutralizing antibody titers and might be at risk for poliovirus infection. Previously we established that memory immunity does not protect the elderly against poliovirus replication. In this study, we investigated whether preexisting immunoglobulin (Ig) A protects against poliovirus infection. METHODS: Elderly individuals (n = 383), divided into seronegative and seropositive groups, were challenged with monovalent oral poliovirus vaccine (mOPV), either serotype 1 or serotype 3. After challenge, poliovirus serotype-specific circulating and salivary IgA responses were measured by enzyme-linked immunosorbent assays, and poliovirus excretion in stool was measured. RESULTS: The majority of elderly persons without preexisting IgA excreted poliovirus in the stool. In contrast, most elderly persons seropositive for IgA did not excrete poliovirus. Significant inverse correlations were found between preexisting titers of poliovirus serotype-specific circulating IgA and virus excretion. Challenge with mOPV (re)induced IgA responses; low salivary IgA responses correlated with that in the circulation but not with virus excretion. CONCLUSIONS: These results indicate that preexisting IgA values in the circulation correlate with protection against poliovirus infection in the elderly. This further implies that persons without preexisting IgA might contribute to the circulation of poliovirus and therefore may threaten its eradication.

Seroepidemiology of hepatitis E virus in patients with non-A, non-B, non-C hepatitis in Hungary.

Many cases of acute hepatitis remain undiagnosed and the hepatitis E virus (HEV) is emerging in industrialized countries. The aim of this study was to assess the role HEV as causative agent in acute non-A, non-B, and non-C hepatitis patients in Hungary. 10.5% of the 264 acute non-A, non-B, and non-C hepatitis patients tested had anti-HEV IgG and 1.9% had anti-HEV IgM as tested by ELISA. After confirmation by Western blot 6.1% of the acute non-A, non-B, and non-C hepatitis patients had anti-HEV IgG antibodies only and 1.1% of the patients had both IgG and IgM. All 19 patients that were positive for anti-HEV IgG and/or IgM tested negative for HEV RNA by PCR. Only a small proportion of the acute hepatitis cases in the southwest of Hungary are assumed to be attributed to HEV infection, however, hepatitis E should be considered along with hepatitis A, B, and C in the diagnosis of acute hepatitis.

Probiotic bacteria stimulate virus-specific neutralizing antibodies following a booster polio vaccination.

BACKGROUND: Orally ingested probiotic bacteria may modulate the immune response and increase antibody titers against enteric infections by bacteria or viruses. Even though positive effects of probiotics on respiratory tract infections have been reported, overall only few studies have examined effects on virus infections concerning organs other than the gastrointestinal tract. AIM OF THE STUDY: It was the aim of the study to investigate whether and how probiotics affect the immune response to a standardized enterovirus challenge (polio) and infections not limited to the gastrointestinal tract in healthy adults. METHODS: In a randomized, controlled and double-blind study 64 volunteers consumed for 5 weeks chemically acidified clotted milk without bacteria or with 10(10)/serving (Lactobacillus rhamnosus ) GG or Lactobacillus acidophilus CRL431 added. In the second week subjects were vaccinated orally against polio 1, 2 and 3. Polio virus neutralizing serum activity, the primary parameter, was determined by the standard neutralization test (WHO) before and three times after vaccination. Polio-specific IgA, IgG and IgM were detected by ELISAs. RESULTS: Probiotics increased poliovirus neutralizing antibody titers (NT) and affected the formation of poliovirus-specific IgA and IgG in serum. The maximum increase after immunization was about 2, 2.2, or 4-fold higher, respectively, for NT, IgG or, IgA, in volunteers consuming probiotics instead of placebo. No consistent difference was noted between bacterial strains. CONCLUSIONS: Probiotics induce an immunologic response that may provide enhanced systemic protection of cells from virus infections by increasing production of virus neutralizing antibodies.

Immunoglobulin a as a serological marker for the (silent) circulation of poliovirus in an inactivated poliovirus-vaccinated population.

Poliovirus-specific immunoglobulin A (IgA) is detected after infection with wild-type virus or vaccination with live attenuated oral poliovirus (OPV) but not after vaccination with inactivated poliovirus (IPV). We examined whether the presence of IgA in serum can be used as a marker for poliovirus circulation in IPV-vaccinated populations in The Netherlands. In seronegative persons challenged with OPV, the sensitivity of this marker was 76%-86%. Results from a serosurvey showed a high seroprevalence (63%-73%) of IgA in the population born before vaccination was introduced in The Netherlands, which reflects natural exposure. The start of the vaccination program in 1957 corresponded to a reduction in the IgA seroprevalence in both vaccinated (2.1%-4.5%) and nonvaccinated groups (8.3%-11.7%). The presence of IgA-positive persons in the population could largely be explained by the occurrence of episodes of proven poliovirus circulation. We propose to use the detection of poliovirus-specific IgA as a tool to monitor virus circulation in IPV-vaccinated and nonvaccinated populations, to aid the poliovirus eradication process.