Case report: tick-borne encephalitis in two Dutch travellers returning from Austria, Netherlands, July and August 2011.

Tick-borne encephalitis (TBE) is not endemic in the Netherlands and diagnostics are seldom requested. Here, we report about the rare event of TBE in two Dutch travellers returning from Austria in July and August 2011. This report serves to create awareness among physicians to consider travel-related TBE in their differential diagnosis of patients with neurological disease returning from TBE virus endemic regions and to promote awareness among professionals advising travellers.

Prevalence, characterisation and clinical profiles of Shiga toxin-producing Escherichia coli in The Netherlands.

Detection of Shiga toxin-producing Escherichia coli (STEC) in The Netherlands is traditionally limited to serogroup O157. To assess the relative importance of STEC, including non-O157 serogroups, stool samples submitted nationwide for investigation of enteric pathogens or diarrhoea were screened with real-time PCR for the presence of the Shiga toxin genes. Patients were selected if their stool contained blood upon macroscopic examination, if they had a history of bloody diarrhoea, were diagnosed with haemolytic uraemic syndrome, or were aged <6 years (irrespective of the bloody aspect of the stool). PCR-positive stools were forwarded to a central laboratory for STEC isolation and typing. In total, 4069 stools were examined, with 68 (1.7%) positive PCR results. The highest prevalence was for stools containing macroscopic blood (3.5%), followed by stools from patients with a history of bloody diarrhoea (2.4%). Among young children, the prevalence (1.0%) was not significantly higher than among random, non-bloody, stool samples from diarrhoeal patients (1.4%). STEC strains were isolated from 25 (38%) PCR-positive stools. Eleven O-serogroups were detected, including five STEC O157 strains. As serogroup O157 represented only 20% of the STEC isolates, laboratories should be encouraged to use techniques enabling them to detect non-O157 serogroups, in parallel with culture, for isolation and subsequent characterisation of STEC strains for public health surveillance and detection of outbreaks.

The 13carbon urea breath test for the diagnosis of Helicobacter pylori infection in subjects with atrophic gastritis: evaluation in a primary care setting.

BACKGROUND: (13)Carbon urea breath testing is reliable to detect current infection with Helicobacter pylori but has been reported to be of limited value in selected patients with atrophic body gastritis or acid-lowering medication. AIM: To evaluate the accuracy of (13)carbon urea breath testing for H. pylori detection in 20 asymptomatic patients with histologically confirmed atrophic body gastritis in a primary care setting. METHODS: (13)Carbon urea breath testing and serology were compared with H. pylori culture of a corpus biopsy as reference test. RESULTS: All tests were in agreement in 12 patients, being all positive in six and all negative in six. One patient was positive for serology and culture but negative for (13)carbon urea breath testing, five patients had only positive serology and two patients had only positive (13)carbon urea breath testing. (13)Carbon urea breath testing showed an accuracy with culture of 85% and anti-H. pylori serology with culture of 75%. (13)Carbon urea breath testing carried out in patients with positive serology showed an accuracy of 92%. Receiver operating characteristic curve analysis of (13)carbon urea breath testing shows optimal discrimination at the prescribed cut-off value. CONCLUSIONS: (13)Carbon urea breath testing can be used as diagnostic H. pylori test in asymptomatic patients with atrophic body gastritis, preferably in addition to serology, to select subjects for anti-H. pylori therapy.

Analysis of variability in lymphocyte transformation tests.

To estimate the importance of a number of variables influencing the results of the lymphocyte transformation test (LTT), we analysed retrospectively the results of 250 LTTs on healthy control lymphocytes tested during the last 5 years as part of the routine LTT program. Prospectively, frozen cells from 3 donors were tested together on 5 different occasions. Differences in day-to-day test conditions had only minor influence. The influence of biologic variables was also negligible. The 2 most important factors were intrinsic cell differences, most probably due to damage during freezing, and genetic influences. To demonstrate minor differences between donors or between bleeding dates, repeated testing is necessary to eliminate these intrinsic variables and to allow statistical analysis.

LDH isoenzyme distribution in human T gamma lymphocytes.

The lactate dehydrogenase (LDH) isoenzyme distribution was measured in T gamma+ lymphocytes from normal individuals. T gamma+ lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The B:A subunit ratio was clearly lower in the T gamma+ lymphocytes. Phenotyping of the T gamma+ lymphocytes showed a vast majority of OKT11+, OKM1+, OKT3- lymphocytes. In one experiment, T gamma+ lymphocytes were enriched by OKT3 depletion of T lymphocytes. The low B:A ratio was also found in these cells indicating that the LDH pattern is not the consequence of an in vitro activation by immune complexes. As the T gamma+ lymphocytes were considerably enriched in cells having the characteristics of NK cells according to their phenotyping, morphology and NK cell activity, we may assume that NK cells have a low B:A ratio.

Development of an antibody-capture IgM-enzyme-linked immunosorbent assay for diagnosis of acute Epstein-Barr virus infections.

An anti-EBV IgM-ELISA was developed using the antibody-capture principle, to be used for the diagnosis of acute infectious mononucleosis (IM). The test was based on anti-human IgM-coated microtiter plates; nuclei of EBV producer cells were used for antigen; conjugate was prepared by labeling sheep anti-EBV IgG with horseradish peroxidase. The specificity of the anti-EBV IgM-ELISA was studied with a panel of sera from acute infections with hepatitis A virus, rubella virus, Toxoplasma gondii and cytomegalovirus, and sera positive for rheumatoid factors, positive for antinuclear antibodies, as well as with sera from normal blood donors and pregnant women. Specificity in these panels was 98.4%. In a clinical study with 449 sera from patients with IM-like symptoms, 109 of 109 confirmed patients were detected by the anti-EBV IgM-ELISA. Specificity of the anti-EBV IgM-ELISA in this clinical study was 99.7%. The anti-EBV IgM-ELISA detected several acute EBV patients who had negative heterophile antibody titers.

[Intravenously-administered immunoglobulins as first-choice agent in juvenile dermatomyositis]. / Intraveneus toegediend immunoglobuline als middel van eerste keuze bij juveniele dermatomyositis.

Dermatomyositis is an acquired disease characterised by symmetric predominantly proximal muscle weakness of the arms and legs, and misery. It may be associated with myalgia and there is often a characteristic rash. The mainstay of therapy is corticosteroids. Recently efficacy of intravenous immunoglobulin (IVIg) in chronic refractory dermatomyositis was reported. Because corticosteroids can cause serious side effects, we treated a seven-year-old girl suffering from dermatomyositis with IVIg as initial therapy. After two courses of IVIg infusions at a dose of 0.4 g/kg/day for five consecutive days, the patient made a rapid and complete recovery. This case shows that IVIg may be effective as initial therapy in patients with dermatomyositis. Whether IVIg is really a better treatment than corticosteroids should be investigated in a randomised study.

Distribution of mononuclear cells during pregnancy.

Leucocyte and lymphocyte counts, the distribution of erythrocyte rosette-forming cells (E-RFC), erythrocyte-antibody-complement rosette-forming cells (EAC-RFC), cells bearing surface membrane immunoglobulin (SMIg), latex-ingesting cells and the PHA stimulation index of lymphocytes were evaluated in both pregnant and non-pregnant women. In the gravid female leucocytosis due to neutrophilia is seen, and the PHA-induced transformation of maternal lymphocytes in the absence of autologous plasma remains unaltered. There was a rise in the percentage of EAC-RFC and cells bearing SMIg during a pregnancy, but after monocyte adsorption on plastic dishes of the mononuclear cells, obtained after Ficoll-Hypapue centrifugation, this increase is abolished. There is a significant increase of the number of latex-ingesting cells during pregnancy. These findings indicate a significant increase of the monocytes during pregnancy.

Enzyme analysis of autologous rosette-forming cells.

Autologous rosette-forming cells (ARFC) have been considered to be post-thymic precursor cells. Since thymocytes and peripheral T lymphocytes differ considerably in lactate dehydrogenase isoenzyme pattern and in activity of the enzymes of the purine metabolism, we investigated the enzyme profile in the ARFC. The L-lactate: NAD+ oxi-reductase analysis showed an isoenzyme pattern that closely resembled the pattern found in peripheral T lymphocytes and was totally different from the thymocytes. The levels of adenosine deaminase and purine nucleoside phosphorylase were identical to those found in the peripheral T lymphocytes and different from thymocytes. In our hands, the ARFC-enriched suspension contained predominantly OKT4+ and T mu+ lymphocytes. We propose that ARFC are a heterogeneous population encompassing all known subsets and cannot be considered a separate homogeneous entity.

Ability of enzyme-linked immunosorbent assays to detect early immunoglobulin G antibodies to Toxoplasma gondii.

Two ELISA procedures, one using sonicated antigen coated with carbonate buffer and the other formalin fixed trophozoites with dry coating, differ in their ability to detect early antibodies in toxoplasmosis. In order to identify factors responsible for this difference, seven ELISA systems differing from each other in antigen used and/or coating procedure were compared. Both fixation of the trophozoites with formalin and air-drying of the antigen in the microtiterplate were important factors determining the ability of the assay to detect IgG antibodies in the early stage of infection. Differences in the results of the two ELISA procedures can be used to distinguish between the acute and chronic stages of infection.

T lymphocyte function in hairy cell leukaemia.

The high incidence of infections characteristic of impaired cell-mediated immunity in patients with hairy cell leukemia led us to study T lymphocyte function in sixteen patients with the lymphocyte transformation test. All patients showed imparied responses to mitogens, attributable to one or more of the following causes: dilution of responsive T cells by inert hairy cells, shortage of monocytes to give adequate interaction with the T cells and a significant decrease in the number of T cells with Fcmu receptors proportional to the percentage of hairy cells in the peripheral blood. The response to antigens was severely depressed; PPD was one of the few antigens that induced positive reactions in half the cases. We conclude that in patients with hairy cell leukaemia, T lymphocyte function, as tested in a proliferative assay, is severely impaired and that this may contribute to the deficient resistance to infection.

LDH analysis of human thymocytes and thymocyte subsets.

The lactate dehydrogenase (LDH) isoenzyme pattern (expressed as the B:A subunit ratio) and two enzymes of the purine metabolism adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) (expressed as the ADA/PNP ratio) were studied in human prenatal thymocytes, in subsets of human infant thymocytes, and in peripheral T lymphocytes. Prothymocytes were enriched by E rosette depletion, cortical thymocytes were enriched with a monoclonal antibody rosette technique using OKT6, and medullary thymocytes were enriched either with a monoclonal antibody rosette technique using OKT3 or with complement-mediated cytolysis using normal fresh rabbit serum. Peripheral T lymphocytes were isolated from normal adult peripheral blood by E rosette sedimentation. Prenatal thymocytes had the lowest B:A ratio. In the infant thymuses, prothymocytes had a lower B:A ratio (0.99 +/- 0.10) than the cortical thymocytes (1.04 +/- 0.08). The medullary thymocytes obtained either by OKT3 selection or normal rabbit serum cytotoxic treatment had higher B:A ratios (1.30 +/- 0.15 and 1.42 +/- 0.17, respectively). The highest B:A ratio is found in peripheral T lymphocytes (2.07 +/- 0.28) together with the lowest ADA/PNP ratio (0.50 +/- 0.07). The B:A ratios are paralleled by a progressive decrease in the ADA/PNP ratio. These findings indicate that during intrathymic T cell development, important changes occur not only in the activities of the enzymes of the purine metabolism but also in the distribution of the LDH isoenzymes.

NE1: a new neutrophil specific antigen.

The sera of three children with chronic benign neutropenia, due to anti-neutrophil antibodies, were studied with respect to their antibody specificity. This was done by screening the sera against a panel of leukocyte donors in the EDTA micro-agglutination test and in the indirect fluorescence test. Two of the sera contained antibodies against the known neutrophil-specific antigen NA2. The third serum was directed against a new neutrophil-specific antigen. Genetic analysis showed no correlation between this antigen and the already known neutrophil-specific antigens: 9A, NA1, NA2, NB1, and NC1. In the Dutch population the frequency of the new antigen, tentatively called NE1, is 23%, which gives a gene frequency of 0.12.

Cell-marker studies in CLL with monoclonal OKT antisera and lactic dehydrogenase isoenzyme analysis.

In 16 patients with B-type CLL, the T and B cell compartment was analysed using the monoclonal anti-T-cell antisera OKT1, 3, 4 and 8 and the lactic dehydrogenase enzyme marker. Pertinent findings were: the presence on the CLL cells in 15 out of 16 patients of the antigen determined by the OKT1 antiserum, and, in all patients, a lower total LDH content on a per cell basis combined with a higher percentage-activity in the LDH-3 band as compared to the normal B-cell. Furthermore, the T-cell compartment was also disturbed in these patients, as the ratio of OKT4+ to OKT8+ cells was significantly depressed accompanied by a significant decrease of the LDH-1 percentage-activity in favour of LDH-3 and 4. These findings argue for the B-cell being immature and confirm the recent evidence that the T-cell compartment is changed in B-CLL.

Hepatitis C virus infection in African patients with liver cirrhosis or primary hepatocellular carcinoma.

We have studied the prevalence of hepatitis C virus (HCV) infection in Rwandan patients with histologically proven liver cirrhosis (LC) or primary hepatocellular carcinoma (HCC). Anti-HCV antibodies were determined by using a second-generation test, with a line immunoassay for structural and non-structural antigens as confirmation. Seventy-nine patients with LC, 26 with HCC, and 54 voluntary blood donors as controls were evaluated. Anti-HCV antibodies were more prevalent in LC patients (48%) and in HCC patients (38%) than in the controls (17%; difference, p = 0.0001 and p = 0.03, respectively). Eighty-four per cent of LC patients and 54% of HCC patients were HBsAg-negative. The prevalence of anti-HCV antibodies was significantly higher for LC and HCC patients who had been in contact with HBV but who had no persistent HBV infection (p < 0.05). We conclude that HCV infection is common in Rwanda and is linked to LC and HCC.

Urinary tract infections with Aerococcus urinae in the south of The Netherlands.

Aerococcus urinae is an uncommon urinary tract pathogen that causes infections predominantly in elderly persons with local or general predisposing conditions. During a one-year study, the clinical features of Aerococcus urinae urinary tract infections (> or = 10(5) cfu/ml) were investigated in two large medical microbiology laboratories in the Netherlands. The incidence of Aerococcus urinae urinary tract infections ranged between 0.31 and 0.44% for the two laboratories. The median age (range 35-95 years) of patients with this infection was 82.5 years for women and 77.5 for men. Men had significantly (p < 0.01) more local predisposing conditions than did women. Underlying systemic diseases such as diabetes mellitus, malignancy, and dementia were found in 67.5% of patients. Most patients (97.5%) had the classic signs of a urinary tract infection, but none of them developed serious symptoms. All isolates tested were susceptible to penicillin, amoxicillin, and nitrofurantoin, 78.3% were susceptible to norfloxacin, and all were resistant to sulfonamides. The majority of patients were treated with amoxicillin, amoxicillin with clavulanic acid, or norfloxacin.

Clinical spectrum of infections due to the newly described Actinomyces species A. turicensis, A. radingae, and A. europaeus.

Over a 7-year period, we isolated 294 Actinomyces-like organisms (ALOs) which were not clearly identifiable. Using well-defined probes coding for sequences specific for recently described Actinomyces species (A. turicensis, A. radingae, and A. europaeus), we were able to identify 128 strains. The majority belonged to the A. turicensis species. A. radingae was found only in patients with skin-related pathologies. A. europaeus was also detected in patients with urinary tract infections. The main sources of A. turicensis were genital infections, followed by skin-related and urinary tract infections. Additional clinical pictures were appendicitis, cholecystitis, ear, nose, and throat infections, and bacteremia. In a small number of patients these ALOs were found as the only pathogen. Strains of the three species were tested by two widely used biochemical identification methods. A. turicensis was easily identifiable by both these methods. We conclude that these ALOs are not infrequent pathogens and are found in a wide range of human infections. At least A. turicensis is easily identifiable by clinical diagnostic laboratories.