Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne infectious disease found in Europe, the United States, and other regions where Ixodes ticks and Lyme disease are endemic. HGE typically presents with myalgia, fever and nausea. Only a few single cases of HGE predominantly manifesting with neurological symptoms and signs have been described to date. The prevalence of this ,,neuro-ehrlichiosis" (NE) is not known and a systematic search for NE in patients with neurological deficits has not yet been performed. As both mode of transmission and clinical neurological presentations of HGE are similar to those of neuroborreliosis (NB) we analyzed 47 inpatients of a neurological tertiary care facility in South-eastern Austria in which NB was suspected but not confirmed by stringent diagnostic criteria. In five patients - two patients with radiculoneuritis, two with polyradiculoneuritis (one of which with Guillain Barre syndrome) and one with peripheral facial nerve palsy - antibodies to Ehrlichia equi related agents (HGE-agents) were detected by means of indirect immunofluorescent antibody assay and in four this was confirmed by Western blot. We suspect that in tick endemic areas of Central Europe HGE may be responsible for neurological deficits but further large scale prospective studies of seroprevalence in neurological patients are needed to make it an established differential diagnosis of NB.

Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 10(4) copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2x10(3) to 5x10(3) HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.

The use of AMPLILINK version 1.0 software was evaluated for the operation and control of one COBAS AMPLICOR instrument and for two COBAS AMPLICOR instruments run simultaneously to perform and detect nucleic acid amplification reactions. A total of 3,384 results were analyzed. The initial accuracy of the results was 99.91%. Three errors of omission of transfer of data from the COBAS AMPLICOR to the AMPLILINK system were observed. Two of these errors were from a single specimen, where both the analyte and internal control results were not transmitted. These errors did not interfere with the correctness of any other data. There were no interruptions of runs, and no data were mixed. AMPLILINK increased convenience, saved labor, and was found to be a very useful addition for clinical laboratories performing molecular-diagnostic procedures with the COBAS AMPLICOR system.

Background: The Amplicor(TM) HBV Monitor Test for quantitative determination of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates.Objective: The performance of the Amplicor(TM) HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor(TM) HBV Monoitor assay was compared to the Digene Hybrid Capturer(TM) System HBV DNA assay for the quantitation of HBV in patient sera.Study design: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays.Results: The detection limit was found to be 10(3) copies/ml with the Amplicor(TM) PCR assay compared to 10(6) to 10(7) copies/ml with the Digene(TM) hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor(TM) PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10(3) and 10(7) copies/ml and all of them tested below the detection limit with the hybridization assay.Conclusion: The Amplicor(TM) HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products. (C) 1998 Elsevier Science B.V. All rights reserved.

Background: Little is known about the role of bell peppers in food allergy. We collected sera from 11 patients with food allergy to bell peppers to analyze bell pepper extracts for allergen composition. Methods: Proteins of mature fruits of eight horticultural strains of bell peppers were extracted and tested with patients’ sera for IgE binding and with monoclonal and polyclonal antibodies in immunoblot. Results: Profilin was detected in bell pepper extracts by an anti-celery profilin antibody. It showed high IgE binding activity in all extracts, which could be inhibited by recombinant birch pollen profilin. Anti-birch pollen monoclonal antibody BIP3, directed against birch pollen proteins between 30 and 69 kD, bound to bell pepper antigens of comparable molecular weights. A homologue of the major birch pollen allergen Bet v 1 was detected in four of eight horticultural strains of bell peppers, and was shown to bind IgE in 1 of the 11 patients. A 23-kD allergen of bell peppers was shown to correspond to the 23-kD major paprika allergen by IgE absorption experiments. Its N-terminal sequence showed 100% identity to P23 from tomatoes. Conclusion: The appearance of profilin in all and Bet v 1 in 50% of the tested horticultural strains indicates that bell peppers have to be considered potentially dangerous for Bet v 1- and profilin-sensitized patients. Moreover, in 4 of 8 horticultural strains of bell peppers a homologue of the osmotin-like protein P23 from tomatoes is responsible for substantial IgE binding. Contact with Bet v 1 and P23 homologues in bell peppers can therefore be minimized by avoidance of the respective horticultural strains.

Objectives: Chronic hepatitis C virus (HCV) infection is common in patients who receive hemodialysis (HD). The aim of this study was to determine the natural history of hepatitis C viremia and the clinical utility of quantitation and genotyping of HCV in this population of patients. Methods: Consecutive sera from two groups of HD patients who were HCV RNA positive, a group of 33 patients treated with interferon alfa (5 MU, three times a week for 4 months) and a group of 31 untreated patients, were analyzed by qualitative polymerase chain reaction, quantitative polymerase chain reaction, and a line probe assay for genotyping. Results: Serum HCV RNA was detected continuously in 20 of 31 untreated patients (65%), and 11 patients (35%) showed a fluctuating pattern of viremia with virus-free intervals of up to 4 wk. Twenty-five of 33 patients (76%) treated with interferon alfa became HCV RNA negative during therapy; eight of these 25 patients had a breakthrough, which was transient in seven patients and persistent in one. Of the remaining 24 end-of-treatment responders, 17 relapsed after completion of therapy, and seven (21%) had a sustained response with undetectable serum HCV RNA for 1 yr of follow-up. Initial serum HCV RNA levels in HD patients were generally low (median, 1 x 10(5) genome eq/ml). Sustained responders had significantly lower median levels of viremia (4 x 10(4) eq/ml) than relapsers and nonresponders (9 x 10(4) and 1.8 x 10(5) eq/ml, respectively). Genotyping revealed a predominance of genotype 1a (33%) and 1b (48%). Conclusions: This study documents that fluctuating hepatitis C viremia with periods of undetectable HCV RNA is common and that low viral load predicts a sustained response to interferon therapy in HD patients. Diagnosis of chronic hepatitis C and monitoring of interferon therapy in HD patients should include initial HCV RNA quantitation and repeated qualitative measurements of HCV RNA.

The AMPLICOR Enterovirus Test was evaluated with 103 cerebrospinal fluid (CSF) specimens. Twenty-seven CSF specimens were culture positive. With the AMPLICOR test, enterovirus RNA was detected in 34 specimens. Compared with culture, the AMPLICOR test gave a sensitivity of 96.3% and a specificity of 100%. The sensitivity of culture was 79.4% in comparison with the AMPLICOR test.

Background: Demonstration of the hepatitis C virus (HCV) genome is usually done with combined reverse transcription and polymerase chain reaction (RT-PCR) employing nested primer sets. Recently, a commercial PCR assay (Amplicor PCR assay), based on a simplified sample preparation procedure, a single, combined reverse transcription and polymerase chain reaction (RT-PCR), and a microwell plate capture and detection, has been developed.Objective: The aim of the present study was to compare the new Amplicor assay with an 'in-house' PCR. Additional testing included a third-generation enzyme immunoassay for anti-HCV antibodies, the Wellcozyme HCV Western Blot, which is equivalent to a third-generation recombinant immunoblot assay. Furthermore, HCV genotypes were classified.Study design: Sera from a total of 127 patients were studied. After screening with a third-generation enzyme immunoassay (EIA), the Wellcozyme HCV Western Blot, was performed as well as the conventional RT-PCR and the Amplicor PCR. Specimens, which were found positive by testing with the Amplicor kit, were subjected to storage at room temperature for 96 h.Results: A total of 52 patients were found to be positive for anti-HCV by the third-generation ELA. With the Amplicor assay, the HCV genome was detected in 38 patients. In comparison with the 'in-house' assay, two discrepant results were found. Resolution of discrepant samples increased the total number of true positives to 39. A good correlation was found between a positive anti-HCV test result and the presence of HCV-RNA by RT-PCR. No significant reduction in the amount of amplification product was observed by retesting of suboptimally stored samples with the Amplicor assay.Conclusion: Because of the rapidity and the improved ease of handling, the Amplicor assay was found to be a good contribution for detection of HCV in serum.

A molecular assay for the detection of herpes simplex virus (HSV), including a novel, nonradioactive hybridization technique, was evaluated with a total of 123 cerebrospinal fluid specimens. After DNA extraction, specific HSV DNA sequences were amplified with digoxigenin-labeled primers derived from the DNA polymerase gene-coding region from HSV. Amplified products were detected by the Enzymun-Test DNA detection assay (Boehringer, Mannheim, Federal Republic of Germany), which uses biotinylated probes. Amplification with nonlabeled primers and then Southern blotting and nonradioactive detection of hybrids by the digoxigenin technique was the reference system. The sensitivities of the molecular assays were determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene obtained from the HSV type 1 strain Angelotti. The Enzymun assay was able to detect all of the 16 positive samples, giving 100% agreement with the Southern blot hybridization results. Optical density values were widely separated for the positive and negative groups of specimens. Ten copies of plasmid pS4 per microliter could be distinctly detected by the Enzymun assay. The cutoff was determined for the hybridization assay, and an equivocal zone was defined. The whole molecular assay including the Enzymun-Test DNA detection proved to be sensitive and easy to use. It may contribute to the rapid and safe detection of HSV DNA in cerebrospinal fluid.