Diagnosing herpesvirus infections by real-time amplification and rapid culture

Procedures using real-time technique were developed to demonstrate the presence of herpes simplex virus type 1 (HSV-1) and HSV-2, varicella zoster virus (VZV), and cytomegalovirus (CMV) in miscellaneous clinical specimens. The assays were compared to rapid culture using centrifugation followed by detection with monoclonal antibodies. A total of 711 consecutive samples were collected from different patient groups. Throat swabs were obtained from transplant patients; dermal or oral specimens were collected from patients suspected for VZV or HSV infection. Genital specimens were taken from patients who attended the Clinic for Sexually Transmitted Diseases at the Dijkzigt Hospital Rotterdam presenting with symptoms of a primary genital ulcer. Nucleic acid extraction was carried out using a MagnaPure LC instrument. The amplification steps were performed on the ABI Prism 7700 sequence detection system. To monitor the process of extraction and amplification, a universal control consisting of seal herpesvirus type 1 (PhHV-1

Students’ experiences of learning in the operating theatre

Human parechoviruses (HPeVs) are members of the large and growing family of Picornaviridae. Although 16 types have been described on the basis of the phylogenetic analyses of the VP1 encoding region, the majority of published reports relate to the HPeV types 1-8. In pediatrics, HPeV1, HPeV2 and HPeV4-8 mainly cause mild gastrointestinal or respiratory illness; only occasionally more serious diseases have been reported, including myocarditis, encephalitis, pneumonia, meningitis, flaccid paralysis, Reye syndrome and fatal neonatal infection. In contrast, HPeV3 causes severe illness in young infants, including sepsis and conditions involving the central nervous system. Currently, the most sensitive method for detecting HPeV is real-time polymerase chain reaction assays on stools, respiratory swabs, blood and cerebrospinal fluid. However, although it is known that HPeVs play a significant role in various severe pediatric infectious diseases, diagnostic assays are not routinely available in clinical practice and the involvement of HPeV is therefore substantially underestimated. Despite long-term efforts, the development of antiviral therapy against HPeVs is limited; no antiviral medication is available and the use of monoclonal antibodies is still being evaluated. More research is therefore needed to clarify the specific characteristics of this relevant group of viruses and to develop appropriate treatment strategies

Improved performance of PACE 2 with modified collection system in combination with probe competition assay for detecti

The Gen-Probe PACE 2 assay (GP) in combination with a modified collection system was compared with cell culture (CC) for the detection of Chlamydia trachomatis in urethral specimens from males. Analysis of discordant results was performed by PCR. The modifications, i.e., application of a more rigid swab type and a 50% reduction in the amount of transport medium, were made to improve the sensitivity of the assay. By using the modified GP on 302 urethral specimens from males, a sensitivity of 89.5% and a specificity of 100% were determined. In addition, performance of a probe competition assay on all GP samples with a result > 0.6 and < 1.0 times the cutoff factor (gray zone) detected three more true-positive samples. The sensitivity of GP in combination with the probe competition assay increased to 94.9%, with a specificity of 100%. This was identical to the performance of CC. The modified GP offers a very sensitive and specific alternative to CC

Identification of a new variant in the YMDD motif of the hepatitis B virus polymerase gene selected during lamivudine therapy

A new hepatitis B virus variant selected during lamivudine treatment was detected, in which the methionine (rtM204) in the so-called YMDD motif in the C

Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

A highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B viru

Epitopes on the peplomer protein of infectious bronchitis virus strain M41 as defined by monoclonal antibodies.

Sixteen monoclonal antibodies (Mcabs) were prepared against infectious bronchitis virus strain M41, all of them reacting with the peplomer protein. One of them, Mcab 13, was able to neutralize the virus and to inhibit hemagglutination. Competition binding assays allowed the definition of five epitopes, designated as A, B, C, D, and E, of which epitopes A and B are overlapping. Furthermore, the binding of Mcab 13 (epitope E) could be enhanced by the addition of Mcabs from group B, C, and D. A dot immunoblot assay was used to analyze the effect of denaturation on antibody recognition

Comparison of two enzyme-linked immunosorbent assays and one rapid immunoblot assay for detection of herpes simplex virus type 2-specific antibodies in serum

The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull Laboratories and Centocor), were compared by testing a panel of 1,250 serum samples from individuals attending an outpatient clinic for sexually transmitted diseases. A qualitative agreement among the three assays was observed with 1,080 serum samples (86.4%); 291 of the serum samples (23.3%) were positive, 789 samples (63.1%) were negative, and 170 serum samples (13.6%) gave a discordant result. Results were considered conclusive when a concordant result was obtained with two of three assays. The sensitivities and specificities of the RIBA, the Gull EIA, and the Centocor EIA proved to be 99.2, 99.7, and 89.9% and 97.1, 96.7, and 99.3%, respectively. These results indicate that the Chiron RIBA and the Gull EIA are especially useful and reliable fo

Lamivudine plasma levels in chronic hepatitis B patients

Lamivudine has recently been registered for the treatment of chronic hepatitis B patients. The main therapeutic outcome in the studies on which the registration was based was a drop of HBV DNA below 10(7) genome equivalents/ml, the level of detection of the insensitive Abbott Genostics assay. However, as we have reported previously, with the use of sensitive PCR-based assays, individual differences in virological response to lamivudine can be detected. As a first step in analysing the chain of events after oral intake of lamivudine we modified and validated a high-pressure liquid chromatography (HPLC) method to evaluate lamivudine plasma levels. Lamivudine levels in chronic hepatitis B patients who participated in a study on the efficacy of lamivudine were comparable to our reference curve, which was derived from eight chronic hepatitis B patients. From the reference curve, a mean area under the curve (AUC) of 4994 mcg/l.h (SD 1524), a mean t(max) of 42 minutes (SD 11), and a mean C(max) of 1.9 mg/l (SD 0.70) were calculated. Lamivudine exerts its action as the active triphosphate inside the hepatocyte after extensive handling. Therefore, additional steps in the pharmacokinetic process should be evaluated to explore the potential mechanisms that are responsible for the diversity in quantitative HBV DNA response to lamivudine

Molecular epidemiology of gibbon hepatitis B virus transmission

Although transmission of human hepatitis B virus (HBV) variants to nonhuman primates is well documented, it remains to be elucidated whether nonhuman primate HBV is transmissible to humans. The prevalence and transmission routes of gibbon HBV were analysed in 101 captive gibbons in Thailand. Approximately 40 % of these animals showed at least one marker of HBV infection; 19 animals were chronic HBV carriers, characterized by elevated levels of alanine amino transferase and the presence of HBV DNA. Some of the chronic animals were found to be anti-HBc (HBV core antigen) negative (4 of 19), while precore promoter point mutations (nt 1762 or 1764) were determined in four animals by RFLP analysis. Phylogenetic tree analysis of the complete surface gene sequences revealed that gibbon viruses clustered separately from hepadnaviruses of other hosts. Evidence for horizontal and vertical transmission in captive gibbons was obtained. HBV DNA was also detected in the saliva of HBV carrier gibbons. Although some of the animal caretakers at the Krabok Koo Wildlife Breeding Centre were found to be chronic HBV carriers, genotype and sequence analysis did not reveal any evidence for zoonotic disease transmission