Doris Stünzner

Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B

BACKGROUND The Amplicor 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 HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor HBV Monoitor assay was compared to the Digene Hybrid Capture 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 PCR assay compared to 10(6) to 10(7) copies/ml with the Digene hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor 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 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.

Investigation of Anaplasma phagocytophila Infections in Ixodes ricinus and Dermacentor reticulatus Ticks in Austria

The Ixodes ricinus tick is the main vector of the tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in European countries. 1 It is also the most important human-biting tick although several other species of ticks (for example, Dermacentor reticulatus , Haemaphysalis conncina , etc.) are present in Austria as well. 2 Recently, studies in neighboring countries have demonstrated the presence of Anaplasma phagocytophila DNA in nymphs and adult ticks. 3,4 One human case of ehrlichiosis has been reported in Austria and the patient apparently acquired the disease while camping in Slovenia. 5 However, the causative agent was not identified in detail and a specific genetic sequence was not deposited in GeneBank.

Isolation of Borrelia afzelii from overwintering Culex pipiens biotype molestus mosquitoes

SummaryDuring the years 1995–1996, a total of 1,743 overwintering Culex pipiens biotype molestus female mosquitoes were tested for the presence of spirochetes in several localities in South Moravia, Czech Republic. The spirochetes were observed in 5% of the mosquitoes investigated. One of the five isolated strains of spirochetes (BR-84) was identified as Borrelia afzelii. The potential role of mosquitoes in the ecology and epidemiology of Lyme disease (LD) borreliae should be further investigated.

Prevalence of Borrelia burgdorferi s.l. in Ixodes ricinus ticks from Styria (Austria) and species identification by PCR-RFLP analysis

A total of 1163 I. ricinus ticks were collected in 3 different regions (15 localities) in Styria (Austria) in June 1997 and examined for the presence of spirochetes by dark field microscopy. The mean infection rate was 20.8%. Among 310 adults, 24.2% were positive and among 853 nymphs, 19.6% were positive. All 15 collection areas were shown to harbour infected nymphs with a positivity rate ranging from 5.8% (3/52) to 32.1% (18/56). Isolation attempts in BSKII medium resulted in 29 isolates. Species identification by PCR-RFLP analysis revealed 16 strains of B. garinii, 10 strains of B. afzelii and 2 strains of B. burgdorferi s. s. One isolate showed a mixed population of B. garinii and B. afzelii. In two collection areas, all three major Borrelia species were shown to be present in the tick population.

Prevalence of Borrelia burgdorferi sensu lato in the tick Ixodes ricinus in the Styrian mountains of Austria

SummaryA total of 691 Ixodes ricinus (22 male, 39 female, 501 nymphs and 129 larvae), the tick vector of Lyme borreliosis, were collected by flagging from vegetation in 11 areas at altitudes between 789 m and 1350 m above sea level in mixed woodland with pasture land (cattle) in the province of Styria in Austria. The ticks were individually examined for presence of Borrelia burgdorferi sensu lato by dark-field microscopy and 107 of them by real-time PCR. Attempts to cultivate borreliae were made in BSK-H medium. The overall positivity rate of all collected ticks (excepting larvae) was 10.9%: 9.1% in males, 17.9% in females and 10.4% in nymphs. The 129 larvae examined showed no presence of B. burgdorferi s.l. The mean infection rate of I. ricinus collected at the highest altitude in this study, Gaberl at 1350 m a.s.l. – and at the same time the highest one reported in Europe – was 6.4%: 1/9 males, 2/18 females and 6/114 (5.3%) nymphs were positive. Culture attempts were positive in 12 cases and species identification showed eight isolates were B. afzelii and four B. garinii. Three additional positive results found by PCR (negative by culture) were identified twice as B. afzelii and once as B. garinii. This study shows that the risk of acquiring Lyme borreliosis in habitats at higher altitudes is limited, because of the lower density of I. ricinus and its lesser infection rate than at lower altitudes in central Europe, but nevertheless the risk does exist.

Performance of the automated COBAS AMPLICOR™ system for the detection of hepatitis C virus RNA

BACKGROUND The COBAS AMPLICOR (CA) instrument for the amplification and detection steps of the AMPLICOR molecular diagnostic assays has recently been introduced. The system contains a single thermal cycler with two independently controlled heating/cooling blocks, a pipettor, a magnetic particle washer, a photometer and an incubator. OBJECTIVE The performance of the CA instrument was evaluated in a routine diagnostic laboratory for the detection of hepatitis C virus (HCV) RNA. The new system was compared with the corresponding microwell plate assay (AMPLICOR HCV Test). STUDY DESIGN Routine clinical sera (350) from hemodialysis patients and patients with chronic HCV infection and interferon therapy were studied. If discrepant results were obtained, both assays were repeated (specimen preparation, amplification and detection); in addition, the HCV copy number was determined with the AMPLICOR HCV MONITOR Test. RESULTS There was a correlation between the CA HCV Test and the AMPLICOR HCV Test in 341 of 350 specimens (97%). After resolution of 9 discrepant results, the CA HCV Test gave a sensitivity of 97.8% and a specificity of 99.4%. The most common reason for discrepant results was a low HCV RNA copy number. CONCLUSION The CA system was found to be a labor-saving, fast and reliable instrument for the amplification and detection steps of a RT-PCR molecular assay for detection of HCV RNA.

Prevalence of borreliae in ixodid ticks from a floodplain forest ecosystem

SummaryHost-seeking ixodid ticks were sampled in a floodplain forest ecosystem along the lower reaches of the Thaya (Dyje) river in South Moravia (Czech Republic) and Lower Austria during the period 1989–2002. The ticks were examined by dark-field microscopy for borreliae (Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis), and attempts were made to culture the spirochetes in BSK-H medium from preparations containing their high numbers. Isolated borreliae were identified by PCR-RFLP analysis using probes directed against ribosomal spacer genes. A total of 797 nymphal and 719 adult (391 female, 328 male)Ixodes ricinus were examined: 16.2% of nymphs, 28.6% of females and 29.0% of males were positive.Dermacentor reticulatus (70 females, 30 males) andHaemaphysalis concinna (12 nymphs, 8 females, 2 males) were negative for spirochetes. The overall prevalence rate of borreliae inI. ricinus from the floodplain forest is slightly higher than the mean European data (i.e., 14% for nymphs, 21% for adults). The difference in infection rate between nymphal and adult ticks was significant, including the proportion of heavily infected (with >100 borreliae) nymphs (2.1%) vs. adults (7.6%). Prevalence of borreliae inI. ricinus showed a significant decrease during autumn in this ecosystem. Three strains of spirochetes, all of theBorrelia afzelii genomic group, were isolated from femaleI. ricinus. Moreover,Trypanosoma/Crithidia sp. protozoa andDipetalonema rugosicauda nematodes were detected in 0.4% and 1.0%, respectively, of allI. ricinus.

Visceral manifestation of cat scratch disease in children. A consequence of altered immunological state

SummaryA 12-year-old girl with a 2-month history of fever and abdominal pain was admitted to our hospital. Ultrasound and CT scans of the abdomen showed multiple hypoechoic lesions of liver and spleen. Screening for zoonosis revealed high positive titers to Bartonella henselae. T-cell deficiency was demonstrated and remained almost unchanged during a follow-up of 11 months. A review of the literature shows that disseminated visceral affection is a rare presentation of cat scratch disease (CSD) in childhood and adolescence. Further immunological investigations are needed in more patients with CSD to confirm whether an altered imunological state may be responsible for the atypical visceral manifestation of CSD.

Prevalence of antibodies to Borrelia burgdorferi flagellin in styrian blood donors

Lyme borreliosis and tick-borne encephalitis (TBE) are the most common diseases in Austria caused by tick bites. TBE endemic areas are well defined. It seemed to be of interest to compare prevalence data of antibodies against Borrelia burgdorferi (B.b.) to TBE endemic and non endemic areas. Blood samples (n = 1162) were obtained from healthy blood donors in combination with a standardized questionnaire during 21 excursions to 7 selected regions of Styria, Austria. Serum samples were screened for IgG antibodies against B.b. by a commercial flagellum ELISA. None of the tested persons showed symptoms of active Lyme borreliosis. A higher prevalence of antibodies against B.b. could be found in TBE endemic areas (7.7%) compared to TBE nonendemic areas (3.8%). There was a significant increase in positive antibodies against B.b. with age, exposure and number of tick bites remembered by test persons. The antibody prevalence to B.b. flagellin antigen is significantly higher in TBE endemic areas than in non-endemic comparative regions.

Quantitation and genotyping of hepatitis C virus RNA in sera of hemodialysis and AIDS patients.

BACKGROUND Hepatitis C virus (HCV) infection is highly prevalent in hemodialysis and AIDS patients. Little information exists about the viral load in those patients. OBJECTIVE To characterize HCV infection in hemodialysis and AIDS patients, the viral load in the sera was measured. Results were compared with genotypes, gender of the patients, and biochemical markers of active hepatitis. STUDY DESIGN Sera from a total of 442 patients were screened with a third-generation EIA, and anti-HCV immunoreactivity was confirmed with the Wellcozyme HCV Western Blot. After qualitative PCR with the Amplicor PCR Test, positives were genotyped using a reverse hybridization test. Determination of HCV levels was done with the Amplicor HCV Monitor assay. RESULTS HCV RNA was detected in the sera of 95 (74.8%) EIA-positive patients. HCV RNA levels ranged from 1 x 10(4) to 1.4 x 10(6) molecules of HCV RNA/ml. Median HCV RNA levels of AIDS patients were slightly higher than those of hemodialysis patients. Male patients had higher median HCV RNA levels compared with female patients. No association between HCV RNA levels and both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was found. The most common genotypes were type 1b and type 1a, followed by type 3, type 4, and type 2a. There were no significant differences in HCV RNA levels among patients with genotypes 1a, 1b, and 2a. Patients infected with types 3 and 4, respectively, had significantly lower HCV RNA levels compared with other genotypes. CONCLUSION Because the Amplicor HCV Monitor assay allows quantitation of low-titer viremic patients, HCV RNA levels were distinctly lower compared with previous reports. HCV RNA levels of males did not differ significantly from those of females. ALT and AST are very poor indicators of ongoing HCV infection. Patients with chronic type 3 or type 4 HCV infection tended to have lower HCV RNA levels.