Diagnostic behaviour of general practitioners when suspecting Lyme disease: a database study from 2010-2015.

BACKGROUND: Due to the raised public awareness of Lyme Borreliosis (LB), its increased incidence and the increased availability of serological tests, the demand for diagnostic testing on LB has increased. This may affect the diagnostic behaviour of general practitioners (GPs). Aim of our study was to describe GPs' diagnostic behaviour when suspecting LB. METHODS: In this descriptive study from January 2010 to June 2015, we used the anonymized electronic medical records of 56,996 patients registered in 12 general practices in Amsterdam, The Netherlands. The target population was identified by means of an extensive search strategy, based on International Classification of Primary Care (ICPC-1) codes, free text and diagnostic test codes. All contacts related to LB were included in the analysis. RESULTS: 2311 patients were included, accounting for 3861 LB contacts and 2619 LB episodes. The distribution of LB contacts showed annual peaks during spring and summer. Serological testing was performed in 36.4% of LB episodes and was mostly requested in patients presenting with general symptoms (71.4%). Unnecessary testing often occurred and only 5.9% of the tests turned out to be positive by immunoblot. From January 2010 to June 2015, no significant differences were found in the number of requested serological tests. The level of serological testing during LB episodes differed significantly between the general practices (19.2% to 75.8%). CONCLUSIONS: Contrary to clinical guidelines, GPs regularly requested serology even when there was a low suspicion of LB. The development of an easy-to-use diagnostic algorithm may decrease overuse of diagnostic tests and thereby reduce overtreatment of LB.

Diarrhea, Urosepsis and Hemolytic Uremic Syndrome Caused by the Same Heteropathogenic Escherichia coli Strain.

We describe an 8-month-old girl with diarrhea, urosepsis and hemolytic uremic syndrome caused by Escherichia coli. Typing of cultured E. coli strains from urine and blood revealed the presence of virulence factors from multiple pathotypes of E. coli. This case exemplifies the genome plasticity of E. coli and the resulting heteropathogenic strains.

Epidemiology of Guillain-Barré Syndrome in Aruba.

The epidemiology of Guillain-Barré syndrome (GBS) in tropical areas is different compared with developed countries. We investigated the epidemiology of GBS on the Caribbean island of Aruba. Data were collected retrospectively from all 36 patients hospitalized with GBS between 2003 and 2011 in Aruba. We observed a seasonal distribution of GBS cases with a peak in February. The incidence rate (IR) fluctuated heavily between individual years. The overall IR was 3.93/100,000, which is higher than that observed in developed countries. Serological studies indicated a possible relation of GBS cases with dengue virus infections. We also observed a relation between the annual number of dengue cases in Aruba and the number of GBS cases in the same year. We conclude that the epidemiology of GBS in tropical areas can be different from temperate climate regions and that dengue may be a trigger for developing GBS.

Hepatitis E virus as a Cause of Acute Hepatitis in The Netherlands.

BACKGROUND: Recent studies indicate that 27% of Dutch blood donors have evidence of past infection with HEV. However, the low number of diagnosed HEV infections indicates either an asymptomatic course or under diagnosis. OBJECTIVES: We investigated whether HEV is a cause of acute hepatitis in Dutch patients and which diagnostic modality (serology or PCR) should be used for optimal detection. STUDY DESIGN: Serum samples were retrospectively selected from non-severely immuno-compromised patients from a university hospital population, suspected of having an infectious hepatitis. Criteria were: elevated alanine aminotransferase (ALT> 34 U/l) and request for antibody testing for CMV, EBV or Hepatitis A (HAV). RESULTS: All samples were tested for HEV using ELISA and PCR. Ninety patients/sera were tested, of which 22% were HEV IgG positive. Only one serum was IgM positive. HEV PCR was positive in two patients: one patient was both HEV IgM and IgG positive, the other patient was only IgG positive. Both HEV RNA positive samples belonged to genotype 3. Evidence of recent infection with CMV, EBV and HAV was found in 13%, 10% and 3% respectively. CONCLUSIONS: Although our study is limited by small numbers, we conclude that HEV is a cause of acute hepatitis in hospital associated patients in The Netherlands. Moreover, in our study population the prevalence of acute HAV (3%) was almost similar to acute HEV (2%). We propose to incorporate HEV testing in panels for acute infectious hepatitis. Negative results obtained for HEV IgM in a HEV PCR positive patient, indicates that antibody testing alone may not be sufficient and argues for PCR as a primary diagnostic tool in hospital associated patients. The high percentage of HEV IgG seropositivity confirms earlier epidemiological studies.

Has CXCL13 an added value in diagnosis of neurosyphilis?

In patients with syphilis, central nervous system (CNS) involvement is often difficult to determine. In patients who also are infected with human immunodeficiency virus (HIV), this is even more challenging, as cerebrospinal fluid (CSF) pleocytosis can be attributed to HIV, syphilis, or both. Hence, this study investigated (i) CSF chemokine (C-X-C motif) ligand 13 (CXCL13) as a potential marker to diagnose neurosyphilis in HIV-infected individuals and (ii) the added value of CSF CXCL13 to conventional CSF biomarkers, such as the rapid plasma reagin test (RPR), in diagnosing neurosyphilis. We included 103 syphilis patients from two centers in The Netherlands: 47 non-HIV-infected patients and 56 HIV-infected patients. A positive CSF-RPR was regarded as the gold standard for neurosyphilis. CSF CXCL13 levels were significantly higher in neurosyphilis patients when neurosyphilis was diagnosed by CSF-RPR (P = 0.0002) than in the syphilis control group. The sensitivity and specificity of CSF CXCL13 (cutoff of 76.3 pg/ml) to diagnose neurosyphilis by using positive CSF-RPR as the gold standard were 50% and 90%, respectively. CSF CXCL13 had an added value to CSF-RPR positivity in 70% of HIV-positive patients and in 33% of HIV-negative patients. Our data show that CSF CXCL13 might be a potential additional marker in neurosyphilis when other markers are not conclusive. The added value of CSF CXCL13 measurement to the current neurosyphilis gold standard appears to benefit HIV-positive patients more than HIV-negative patients.

[Torticollis and seizures due to neuroborreliosis in a child]. / Een kind met torticollis en insulten bij neuroborreliose.

BACKGROUND: In children, neuroborreliosis often manifests itself as cranial neuritis (particularly facial palsy) or aseptic meningitis. Presentation with torticollis and simple partial seizures resulting from diffuse leptomeningeal inflammation is rare. CASE DESCRIPTION: A seven-year-old boy who had developed torticollis and partial seizures, lost weight and was complaining of tiredness was seen by a paediatric neurologist. A brain and spinal cord MRI showed diffuse leptomeningeal enhancement, in combination with a hyperintense cervical cord lesion. Laboratory testing of serum and cerebrospinal fluid confirmed the diagnosis of neuroborreliosis. The boy was treated with intravenous ceftriaxone for 30 days and made a full recovery. CONCLUSION: As illustrated by this case neuroborreliosis can manifest itself atypically with torticollis, seizures and diffuse leptomeningeal enhancement due to inflammation. If there is leptomeningeal enhancement on MRI then neuroborreliosis should be included in the differential diagnosis. In childhood neuroborreliosis can be successfully treated and the prognosis is good.

Host factors determine anti-GM1 response following oral challenge of chickens with Guillain-Barré syndrome derived Campylobacter jejuni strain GB11.

BACKGROUND: Anti-ganglioside antibodies with a pathogenic potential are present in C. jejuni-associated Guillain-Barré syndrome (GBS) patients and are probably induced by molecular mimicry. Immunization studies in rabbits and mice have demonstrated that these anti-ganglioside antibodies can be induced using purified lipo-oligosaccharides (LOS) from C. jejuni in a strong adjuvant. METHODOLOGY/PRINCIPAL FINDINGS: To investigate whether natural colonization of chickens with a ganglioside-mimicking C. jejuni strain induces an anti-ganglioside response, and to investigate the diversity in anti-ganglioside response between and within genetically different chicken lines, we orally challenged chickens with different C. jejuni strains. Oral challenge of chickens with a C. jejuni strain from a GBS patient, containing a LOS that mimics ganglioside GM1, induced specific IgM and IgG anti-LOS and anti-GM1 antibodies. Inoculation of chickens with the Penner HS:3 serostrain, without a GM1-like structure, induced anti-LOS but no anti-ganglioside antibodies. We observed different patterns of anti-LOS/ganglioside response between and within the five strains of chickens. CONCLUSIONS: Natural infection of chickens with C. jejuni induces anti-ganglioside antibodies. The production of antibodies is governed by both microbial and host factors.

Sialylation of Campylobacter jejuni lipo-oligosaccharides is associated with severe gastro-enteritis and reactive arthritis.

We used various genotyping methods to identify bacterial genetic markers for development of arthritic symptoms following Campylobacter enteritis. We genotyped a collection of population derived Campylobacter strains, with detailed information on clinical characteristics, including arthritic symptoms. Besides using whole genome screening methods, we focused on the lipo-oligosaccharide (LOS) gene locus in which marker genes for developing post-Campylobacter neurological disease are present. Patients with arthritic symptoms were more frequently infected with Campylobacter jejuni strains with a class A LOS locus. We also found that patients who were infected with a C. jejuni strain containing sialic acid-positive LOS (class A, B or C) more frequently had bloody diarrhoea and a longer duration of symptoms. Furthermore, the IgM antibody response against Campylobacter was stronger in patients with a sialic acid containing LOS. Ganglioside auto-antibodies were observed in a small number of patients following infection with a class C strain. We conclude that sialylation of C. jejuni LOS is not only a risk factor for development of post-infectious symptoms, but is also associated with increased severity of enteric disease.

Immunity to Campylobacter: its role in risk assessment and epidemiology.

Acquired immunity is an important factor in the epidemiology of campylobacteriosis in the developing world, apparently limiting symptomatic infection to children of less than two years. However, also in developed countries the highest incidence is observed in children under five years and the majority of Campylobacter infections are asymptomatic, which may be related to the effects of immunity and/or the ingested doses. Not accounting for immunity in epidemiological studies may lead to biased results due to the misclassification of Campylobacter-exposed but apparently healthy persons as unexposed. In risk assessment studies, health risks may be overestimated when immunity is neglected.

Origin of ganglioside complex antibodies in Guillain-Barré syndrome.

The origin of antibodies to ganglioside complexes, as new immunotargets for Guillain-Barré syndrome (GBS), is unknown. This was investigated in 21 GBS patients from which Campylobacter jejuni was isolated. Two of these patients had serum IgG to the GM1/GD1a complex and two other patients had IgG to the GQ1b/GD1a complex. These pairs of patients were clinically distinct. These antibodies all cross-reacted to lipo-oligosaccharides (LOS) from the autologous C. jejuni strain. Previous mass spectrometry studies on these LOS showed the presence of oligosaccharides with a similar structure, further supporting the hypothesis that in these patients LOS induced the ganglioside complex antibodies.

PCR-restriction fragment length polymorphism analysis of Campylobacter jejuni genes involved in lipooligosaccharide biosynthesis identifies putative molecular markers for Guillain-Barré syndrome.

Molecular mimicry of Campylobacter jejuni lipooligosaccharides (LOS) by gangliosides in peripheral nerve tissue probably triggers the Guillain-Barré syndrome due to the induction of cross-reactive antibodies. PCR-restriction fragment length polymorphism analysis of C. jejuni genes involved in the biosynthesis of LOS demonstrated that specific genes were associated with the expression of ganglioside mimics and the development of neuropathy.

Structural characterization of Campylobacter jejuni lipooligosaccharide outer cores associated with Guillain-Barre and Miller Fisher syndromes.

Molecular mimicry between lipooligosaccharides (LOS) of Campylobacter jejuni and gangliosides in peripheral nerves plays a crucial role in the pathogenesis of C. jejuni-related Guillain-Barré syndrome (GBS). We have analyzed the LOS outer core structures of 26 C. jejuni strains associated with GBS and its variant, Miller Fisher syndrome (MFS), by capillary electrophoresis coupled with electrospray ionization mass spectrometry. Sixteen out of 22 (73%) GBS-associated and all 4 (100%) MFS-associated strains expressed LOS with ganglioside mimics. GM1a was the most prevalent ganglioside mimic in GBS-associated strains (10/22, 45%), and in eight of these strains, GM1a was found in combination with GD1a mimics. All seven strains isolated from patients with ophthalmoplegia (GBS or MFS) expressed disialylated (GD3 or GD1c) mimics. Three out of 22 GBS-associated strains (14%) did not express sialylated ganglioside mimics because their LOS locus lacked the genes necessary for sialylation. Three other strains (14%) did not express ganglioside mimics because of frameshift mutations in either the cstII sialyltransferase gene or the cgtB galactosyltransferase gene. It is not possible to determine if these mutations were already present during C. jejuni infection. This is the first report in which mass spectrometry combined with DNA sequence data were used to infer the LOS outer core structures of a large number of neuropathy-associated C. jejuni strains. We conclude that molecular mimicry between gangliosides and C. jejuni LOS is the presumable pathogenic mechanism in most cases of C. jejuni-related GBS. However, our findings suggest that in some cases, other mechanisms may play a role. Further examination of the disease etiology in these patients is mandatory.

Co-infection with two different Campylobacter jejuni strains in a patient with the Guillain-Barré syndrome.

Campylobacter jejuni is the predominant cause of antecedent infection in Guillain-Barré syndrome (GBS) or Miller Fisher syndrome (MFS). C. jejuni probably triggers GBS or MFS through molecular mimicry between bacterial sialylated lipo-oligosaccharides (LOS) and gangliosides in peripheral nerve tissue. We investigated whether co-infections with multiple C. jejuni strains occur in GBS or MFS patients and we further characterized these strains. PFGE analysis of 83 C. jejuni isolates from single primary colonies from stool cultures of 13 patients with GBS or MFS revealed co-infection with two different strains in one patient (8%). We showed that only strain GB5.1 contained an LOS biosynthesis gene locus that is associated with neuropathy. The patient serum strongly reacted with the LOS of strain GB5.1 and not with the LOS of strain GB5.2. Mass spectrometry revealed that both strains expressed a non-sialylated outer core structure in their LOS. The patient serum contained anti-asialo-GM2 antibodies that cross-reacted with the LOS of strain GB5.1. This study demonstrates that co-infection with multiple C. jejuni strains occurs in GBS patients. Consequently, not all C. jejuni strains isolated from the faeces of a GBS patient are involved in the pathogenesis of GBS per se. Furthermore, this is the first report in which cross-reactivity of antibodies to asialo-GM2 and to the LOS of a C. jejuni strain from a GBS patient has been demonstrated. This finding suggests that molecular mimicry with non-sialylated structures may also be involved in the pathogenesis of GBS.

Diagnostic value of anti-GM1 ganglioside serology and validation of the INCAT-ELISA.

The Inflammatory Neuropathy and Treatment (INCAT) group developed a standardized ELISA method for the detection of serum anti-GM1 antibodies. The diagnostic value of anti-GM1 antibodies determined by this method has not yet been established in large groups of patients. We assessed the reproducibility, sources of variation, optimal cut-off values and evaluated the diagnostic relevance of the INCAT-ELISA in various groups of patients and controls (N=1232). The coefficient of variance was 11.2% for IgM and 3.8% for IgG. High IgG titers were only found in Guillain-Barré syndrome (GBS) and other inflammatory polyneuropathies. High IgM titers were associated with GBS and multifocal motor neuropathy. Low IgM titers had no additional diagnostic value. The INCAT-ELISA is a reliable test with additional diagnostic value in specific clinical situations.

The crucial role of Campylobacter jejuni genes in anti-ganglioside antibody induction in Guillain-Barre syndrome.

Molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissue is considered to induce cross-reactive antibodies that lead to Guillain-Barre syndrome (GBS), an acute polyneuropathy. To determine whether specific bacterial genes are crucial for the biosynthesis of ganglioside-like structures and the induction of anti-ganglioside antibodies, we characterized the C. jejuni LOS biosynthesis gene locus in GBS-associated and control strains. We demonstrated that specific types of the LOS biosynthesis gene locus are associated with GBS and with the expression of ganglioside-mimicking structures. Campylobacter knockout mutants of 2 potential GBS marker genes, both involved in LOS sialylation, expressed truncated LOS structures without sialic acid, showed reduced reactivity with GBS patient serum, and failed to induce an anti-ganglioside antibody response in mice. We demonstrate, for the first time, to our knowledge, that specific bacterial genes are crucial for the induction of anti-ganglioside antibodies.

The Guillain-Barré syndrome: a true case of molecular mimicry.

Molecular mimicry between microbial antigens and host tissue forms an attractive hypothetical mechanism for the triggering of autoimmune disease by preceding infections. Recent crucial reviews state that molecular mimicry, as the causative mechanism, remains unproven for any human autoimmune disease. However, the peripheral neuropathy Guillain-Barré syndrome (GBS) is largely overseen in this debate. Based on recent evidence, we argue that GBS should be considered as an excellent paradigm and an attractive model for elucidation of both host and microbial aspects of molecular mimicry.

Evidence for acquisition of the lipooligosaccharide biosynthesis locus in Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barré syndrome, by horizontal exchange.

Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barré syndrome, has been shown to be genetically closely related to the completely sequenced strain C. jejuni NCTC 11168 by various molecular typing and serotyping methods. However, we observed that the lipooligosaccharide (LOS) biosynthesis genes strongly diverged between GB11 and NCTC 11168. We sequenced the LOS biosynthesis locus of GB11 and found that it was nearly identical to the class A LOS locus from the C. jejuni HS:19 Penner serotype strain (ATCC 43446). Analysis of the DNA sequencing data showed that a horizontal exchange event involving at least 14.26 kb had occurred in the LOS biosynthesis locus of GB11 between galE (Cj1131c in NCTC 11168) and gmhA (Cj1149 in NCTC 11168). Mass spectrometry of the GB11 LOS showed that GB11 expressed an LOS outer core that mimicked the carbohydrate portion of the gangliosides GM1a and GD1a, similar to C. jejuni ATCC 43446. The serum from the GB11-infected patient was shown to react with the LOS from both GB11 and ATCC 43446 but not with that from NCTC 11168. These data indicate that the antiganglioside response in the GB11-infected patient was raised against the structures synthesized by the acquired class A LOS locus.

Risk factors associated with Campylobacter jejuni infections in Curaçao, Netherlands Antilles.

A steady increase in the incidence of Guillain-Barré syndrome (GBS) with a seasonal preponderance, almost exclusively related to Campylobacter jejuni, and a rise in the incidence of laboratory-confirmed Campylobacter enteritis have been reported from Curaçao, Netherlands Antilles. We therefore investigated possible risk factors associated with diarrhea due to epidemic C. jejuni. Typing by pulsed-field gel electrophoresis identified four epidemic clones which accounted for almost 60% of the infections. One hundred six cases were included in a case-control study. Infections with epidemic clones were more frequently observed in specific districts in Willemstad, the capital of Curaçao. One of these clones caused infections during the rainy season only and was associated with the presence of a deep well around the house. Two out of three GBS-related C. jejuni isolates belonged to an epidemic clone. The observations presented point toward water as a possible source of Campylobacter infections.

Expansion of human gammadelta T cells after in vitro stimulation with Campylobacter jejuni.

Campylobacter jejuni is currently the prime cause of food-borne bacterial gastro-enteritis. An important complication of C. jejuni enteritis is Guillain-Barré syndrome (GBS), an immune-mediated disorder of peripheral nerve tissue. Because little is known about T cell reactivity to C. jejuni, we have analyzed the in vitro immune response of normal individuals against five isolates of C. jejuni representing five different serotypes. We found a preferential expansion of peripheral blood gammadelta T cells after exposure to crude sonicates of all five C. jejuni serotypes. Expansion of gammadelta T cells was dependent on the presence of CD4+/alphabeta+ T cells in the cultures or addition of exogenous IL-2 or IL-15. C. jejuni stimulation was mediated via the TCR and appeared to be induced by a non-proteinaceous bacterial antigen, most likely of phosphoantigenic origin.