[Effective acquisition of basic surgical techniques through blended learning]. / Effizienter Erwerb chirurgischer Basistechniken durch "blended learning"

BACKGROUND: Large student numbers and heterogeneous teaching pools hamper standardized teaching and impede objective assessment of surgical skills. This article presents the advantages of new teaching media in a "blended learning" concept for training surgical skills to medical students at the Basel University Medical School in Switzerland. MATERIAL AND METHODS: The surgical skills course (suture course) for medical students was redesigned according to a blended learning concept consisting of an introduction with a multimedia CD-ROM, a practical course, and a skills lab. The learning targets of the course were evaluated through an objective structured clinical examination (OSCE) at the end of each study year. The students' own course evaluations were compared with the OSCE results before and after introduction of the new blended learning. RESULTS: The students' evaluations with regard to teaching material, subjective practical achievement, prospective value for the practical year, and overall course evaluation were significantly higher than in the old course format. The proportion of passed OSCEs was 10% higher after the redesign of the course. CONCLUSION: Blended learning can improve cognition and performance as well as the training efficiency and duration required for mentoring. Thus human resources can be saved indirectly. Surgical procedures may be presented more clearly.

Evaluation of AMPLILINK software for the COBAS AMPLICOR system.

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.

Is IgM of diagnostic value in case of delayed intrathecal production of IgG antibodies?

The neurological manifestations of Lyme borreliosis comprise a wide range of clinical signs. However, these symptoms might have other aetiologies. Therefore detection of intrathecal production of specific antibodies is necessary to confirm the clinical assumption of neuroborreliosis (NB). In case of delayed intrathecal production of specific IgG antibodies, detection of IgM could play a role in the early diagnosis of NB. To clarify whether IgM is of diagnostic value in such cases, paired CSF serum samples from 176 patients with suspected NB admitted to the department of Neurology, Karl Franzens University, Graz, Austria, were tested. Testing was performed with the IDEA Neuroborreliosis Kit (Dako, Denmark) and Enzygnost Borreliosis (Behring, Germany) and results of both methods were compared. According to well defined criteria 63 of the 176 patients had defined NB and 113 were regarded as possible NB. Twelve out of 63 patients with defined NB had delayed intrathecal IgG production. Only one patient with delayed IgG production had an intrathecal IgM production prior to IgG. In all patients with possible NB no intrathecal production of IgM was detected. At the time of the first lumbar puncture IgG intrathecal production could be detected with the IDEA seven times more often than with the Enzygnost Borreliosis. The determination of intrathecal production of IgM does not appear to be of diagnostic value in patients with delayed IgG antibody production. Therefore a consecutive lumbar puncture is more likely to confirm clinical assumption if there is strong clinical evidence of NB.

Species identification of Borrelia burgdorferi sensu lato from tick and human isolates in Styria (Austria) by PCR-RFLP analysis.

Seventy-one isolates of Borrelia burgdorferi sensu lato (B.b.s.l.) derived from Ixodes ricinus ticks (50 strains) and patients (21 strains) were characterised by PCR-RLFP analysis. In four cases the human isolates were obtained from the cerebrospinal fluid (CSF) of patients with clinical symptoms of neuroborreliosis and in 17 cases from skin biopsies of patients with dermatological manifestation of Lyme borreliosis. Ixodes ricinus isolates originated from 14 localities in three regions (Mur valley, eastern and western Styria) in Styria. Thirty six strains of B.b.s.l. were isolated from nymphal ticks, nine strains from female and five strains from male ticks. Species identification of human isolates revealed three B. garinii and one B. afzelii isolates in CSF. In the PCR-RFLP analysis of 17 skin specimens a pattern for B. afzelii was found in ten cases, while six could be identified as B. garinii and one as a mixed infection of B. afzelii and B. garinii. Genetic characterisation of tick isolates resulted in 24 strains of B. afzelii (48%), 11 strains of B. garinii (40%) and 5 strains of B. burgdorferi s.st. (10%); one isolate showed a mixed infection of B. afzelii and B. garinii. Our findings indicate that B. afzelii and B. garinii predominate over B. burgdorferi s.str. in Ixodes ricinus ticks from Styria, which is similar to findings in neighbouring countries. This also reflects the occurrence of different pathogenic Borrelia strains in human samples.

Automation of polymerase chain reaction-based systems for detection of hepatitis C virus RNA.

Polymerase chain reaction-based molecular assays are gaining increasing importance in the diagnosis and monitoring of infectious diseases. Over the past several years, the development and application of these techniques has initiated a revolution in the diagnosis and monitoring of hepatitis C infection. Presently, molecular assays are exclusively done in especially dedicated laboratories. Advances in automation will bring these technologies into routine diagnostic laboratories and will make them widely used.

Quantitative detection of hepatitis B virus DNA with a new PCR assay.

The Amplicor HBV Monitor Test for quantitative detection 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. Evaluation in a routine diagnostic laboratory showed excellent sensitivity and adequate reproducibility; however, a more automated format would be desirable. The Amplicor HBV Monitor Test is useful for recognizing those patients who might benefit from antiviral treatment and for evaluation of the efficacy of anti-hepatitis B virus treatment.

A simple and robust method for the complete dissociation of HIV-1 p24 and other antigens from immune complexes in serum and plasma samples.

Accuracy of antigen determination in human plasma samples is often adversely affected by immune complex formation between antigens (e.g., HIV-1 p24 protein) and specific antibodies. In this study we describe an optimized method for complete immune complex dissociation (ICD) in plasma. This method is based on heat denaturation of antibodies and utilizes a defined solution of sodium dodecyl sulfate (SDS) and diethylenetriaminepentaacetic acid (DTPA) as diluent. The efficiency of this procedure for ICD was compared with those of published methods, employing heat denaturation alone and acidification. Plasma samples from patients participating in anti-retroviral treatments and samples reconstituted in vitro were treated and analyzed in parallel. HIV-1 p24 antigen was determined by quantitative enzyme-linked immunosorbent assay (ELISA). In 312 samples from 97 patients, antigenemia was found in 44.9% when measured directly and in 87.2% after this treatment. In a subset of 56 samples, 21.4% tested positive prior to treatment, while after either novel treatment, heat denaturation or acidification, these samples tested positive in 80.4%, 62.5% and 60.7%, respectively. In 94% of cases viral RNA was detected. This improved procedure for ICD provides a reliable and convenient method for complete and accurate p24 antigen detection in human plasma and is applicable to commercially available test kits.

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.

Evaluation of molecular parameters for routine assessment of viremia in patients with chronic hepatitis C who are undergoing antiviral therapy.

OBJECTIVE: To define the usefulness of molecular parameters in patients with chronic hepatitis C who are undergoing antiviral therapy. Anti-hepatitis C virus (HCV) treatment was monitored by determination of serum HCV load and by presence of HCV RNA in peripheral blood mononuclear cells (PBMCs). STUDY DESIGN/METHODS: Fifty-one patients with chronic hepatitis C undergoing antiviral therapy with interferon-alpha plus ribavirin were studied. Serum HCV RNA load was tested with a quantitative assay (Amplicor HCV Monitor Test) before, during, and up to 12 months after end of treatment. If HCV RNA was not detectable, serum samples were subsequently tested with a qualitative assay (Cobas Amplicor HCV Test) and corresponding ethylenediaminetetraacetic acid (EDTA)-treated blood was checked for presence of HCV RNA in peripheral blood mononuclear cells (PBMCs). Sustained virologic response was defined by loss of HCV RNA 12 months after the end of treatment. RESULTS: Four patients (7.8%) were found to be sustained virologic responders, 17 (33.3%) were transient virologic responders, and 30 (58.8%) were virologic nonresponders. No significant difference was found in the median pretreatment serum HCV RNA load between sustained virologic responders, transient virologic responders, and virologic nonresponders. At 1 month after start of therapy, HCV RNA was not detectable with both the serum and the PBMC assay in 12 (23.5%) of 51 patients. Four remained HCV RNA-negative until 12 months after the end of treatment. In 14 of 17 transient virologic responders, reappearance of HCV RNA was detected earlier in PBMCs than in serum. CONCLUSIONS: Based on these results in 51 patients, quantitation of baseline serum HCV RNA does not appear to be a decisive factor to the management of the individual patient. Early assessment of serum HCV RNA level after start of anti-viral treatment seems to be of major importance to identify virologic nonresponders. Reappearance of HCV RNA may be demonstrated earlier in PBMCs than in serum.

Prevalence of Borrelia burgdorferi s.I. 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.

Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format.

A new molecular assay, based on a rapid DNA extraction protocol, PCR, and hybridization to a specific probe in a nonradioactive microwell plate format was used to detect Mycoplasma pneumoniae in bronchoalveolar fluid specimens. The sensitivity of the assay was determined to be 10 to 100 organisms with M. pneumoniae reference strains. Specificity testing with different bacteria capable of producing pneumonia showed no cross-reactivity. In a prospective study, bronchoalveolar lavage fluids obtained from patients with pneumonia were investigated with the PCR assay and compared to culture. Twelve positive samples were detected with the PCR assay. Seven of them were subsequently confirmed by culture. All patients with positive PCR results seroconverted. Application of the PCR assay described may lead to safe and early diagnosis of M. pneumoniae in patients with pneumonia.

Rapid diagnosis of enterovirus infection by a new one-step reverse transcription-PCR assay.

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.

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.

Detection of antichlamydial antibodies in tears: a diagnostic aid?

OBJECTIVES: The antibody response in sera and tears of 167 patients with suspected chlamydial conjunctivitis was compared with the antibody response in sera and tears of 45 patients with symptoms of urogenital chlamydial infection to discover whether and which type of antichlamydial antibody detected in tears may be of diagnostic help in chlamydial conjunctivitis. METHODS: Diagnosis was based on chlamydial antigen detection from the conjunctiva and urogenital tract, done by a direct immunofluorescence assay, McCoy cell culture, and polymerase chain reaction. Additionally, antichlamydial immunoglobulin A (IgA) and immunoglobulin G (IgG) were determined in sera and tears of all patients by an immunoperoxidase assay. RESULTS: Two hundred twelve patients were examined--167 with conjunctivitis, 45 with symptoms of urogenital chlamydial infection. Cell culture, direct immunofluorescence assay, and polymerase chain reaction brought identical results. Conjunctival specimens taken from 33 (20%) of the patients with conjunctivitis were Chlamydia antigen positive; specimens taken from 134 (80%) were negative. Antichlamydial antibodies were found in tears of 29 (88%) of the patients with conjunctivitis whose specimens were Chlamydia antigen positive. Fifty-four (40%) of the patients with conjunctivitis whose specimens were Chlamydia antigen negative had antichlamydial antibodies in their tears. Twenty-five patients with urethritis (56%) were Chlamydia antigen positive in urethral swabs; 20 (44%) were negative. Antichlamydial antibodies were found in the tears of eight (32%) of the Chlamydia antigen-positive and two (10%) of the Chlamydia antigen-negative patients with urethritis. In contrast to patients with conjunctivitis, findings for patients with urethritis always were negative for antichlamydial IgG in the tears. CONCLUSION: Antichlamydial antibodies in tears were seen significantly more often in patients with conjunctivitis than in those with urethritis (P < or = 0.05). Antichlamydial IgG was found only in tears of patients with conjunctivitis. Therefore, the authors conclude that the detection of antichlamydial IgG in the tears might be helpful for diagnosis in patients with suspected chlamydial conjunctivitis who have antigen-negative conjunctival swabs.

Evaluation of two nonculture antigen tests and three serotests for detection of anti-chlamydial antibodies in the diagnosis of ocular chlamydial infections.

BACKGROUND: Diagnosis of chlamydial conjuctivitis is difficult in chronic diseases because chlamydial elementary bodies are mostly undetectable in conjunctival scrapings by cell culture. We therefore compared two nonculture antigen tests and three different serotests for anti-chlamydial antibodies with McCoy cell culture, the "gold standard" of chlamydial diagnosis. Conjunctival scrapings and serum samples of 93 patients attending the outpatient eye clinic in Graz because of chronic follicular conjunctivitis were tested. METHODS: A total of 558 conjunctival scrapings and 93 serum samples were investigated. Chlamydial antigen detection was done by McCoy cell culture, polymerase chain reaction (PCR; Amplicor, Roche), and direct immunofluorescence assay (DFA; Microtrak, Syva). Antichlamydial IgA and IgG antibodies in the sera were detected by an immunoperoxidase assay (IPAzyme, Savyon) and two different enzyme-linked immunosorbent assays (SeroELISA, Savyon and rELISA, medac). RESULTS: Cell culture and PCR yielded identical results. The positivity rate for chlamydial conjunctivitis was 8.6% (8 of 93 patients). PCR proved most sensitive and most specific. IPAzyme was 75% sensitive for IgA and 100% for IgG; SeroELISA and rELISA were less sensitive. IPAzyme was 81% specific for IgA and 47.3% for IgG. SeroELISA and rELISA were less specific for IgA, but more specific for IgG. Post-test likelihood of disease was greatest in IPAzyme. CONCLUSIONS: PCR proved to be a good alternative to cell culture; DFA is useful for quick diagnosis. Genus-specific serotests cannot compete with chlamydial antigen detection. They differ in sensitivity and specificity because of the antigen type they present. They are still of only supportive value in cases where chlamydial antigen detection is not possible. Recently introduced species-specific antibody tests should be of greater value.

Reversal of Borrelia burgdorferi associated dilated cardiomyopathy by antibiotic treatment?

It is suggested that Borrelia burgdorferi infection could be associated with dilated cardiomyopathy (IDC). Stanek et al. were able to cultivate Borrelia burgdorferi from myocardial biopsy tissue of a patient with longstanding dilated cardiomyopathy. Here we present a study in which we examined the effect of standard antibiotic treatment on the left ventricular ejection fraction (LVEF) in patients with dilated cardiomyopathy associated with Borrelia burgdorferi infection. In this study we assessed the serum (IgG, IgM Elisa) and history of 46 IDC patients with specific regard to Borrelia burgdorferi infection (mean LVEF 30.4 +/- 1.3%, measured by cardiac catheterization and echocardiography with the length-area-volume method). All 46 patients received standard treatment for dilated cardiomyopathy: ACE inhibitors, digitalis, and diuretics. Eleven (24%) patients showed positive serology and a history of Borrelia burgdorferi infection; nine of these also had a typical history of tick bite and erythema chronicum migrans (ECM) and/or other organ involvement, and two had no recollection of tick bite or ECM but showed other Borrelia burgdorferi-associated disorders (neuropathy, oligoarthritis). These 11 patients with Borrelia burgdorferi infection received standard antibiotic treatment with intravenous ceftriaxone 2 g bid for 14 days. Six (55%) recovered completely and showed a normal LVEF after 6 months, three (27%) improved their LVEF, and two (18%) did not improve at all. This amounts to nine (82%) patients with recovery/improvement in the Borrelia burgdorferi group. The 35 patients who did not show positive serology or a history of Borrelia burgdorferi infection did not receive antibiotic treatment. In this group without Borrelia burgdorferi infection 12 (26%), showed recovery/improvement following the standard treatment of dilated cardiomyopathy (see earlier). Our results indicate that Borrelia burgdorferi infection could play a decisive role in the development of dilated cardiomyopathy, especially in a geographical region such as Graz, where Borrelia burgdorferi is endemic. While we are aware of the small number of Borrelia burgdorferi patients in this study, we nevertheless conclude that in a remarkable number of patients with signs of Borrelia burgdorferi infection, dilated cardiomyopathy could be reversed and LVEF improved.

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.

Detection of hepatitis C viral sequences in serum by 'nested' polymerase chain reaction (PCR) and a commercial single-round PCR assay.

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 EIA. 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.

Comparison of two hybridization assays for the rapid detection of PCR amplified HSV genome sequences from cerebrospinal fluid.

Rapid diagnosis of herpes simplex encephalitis (HSE) can only be achieved by the polymerase chain reaction (PCR). In order to carry out PCR under routine conditions, it is of great importance to establish an easy DNA extraction protocol and especially a rapid and sensitive DNA detection method. In the present study, two different solid phase hybridization assays (Gen-Eti-K-DNA Enzyme Immunoassay (DEIA), Sorin Biomedica, Italy and Enzymun-Test DNA detection, Boehringer Mannheim, Germany) were compared for detection of PCR amplified HSV DNA polymerase genome region, using standard primers, from cerebrospinal fluid (CSF) samples. 122 CSF samples obtained from patients suffering from encephalitis and hospitalized at the University Clinics of Frankfurt and Graz during the period January 1992 to July 1993 were tested. To ascertain the sensitivity of the hybridization assays, dilution series of a plasmid, encoding the amplified region of the polymerase gene, were investigated. The detection limit of the DEIA assay was one copy of the plasmid/microliter, and the lowest amount of DNA which could be detected by the Enzymun assay as well as Southern blot was 10 copies/microliter. 15 CSF samples obtained from patients with HSE were found positive by the three assays. Concordant results were also obtained with CSF samples from non-HSE patients. The results of this study show that new hybridization systems guarantee a fast and high-sensitive detection of amplified HSV DNA. HSV PCR in CSF can be carried out routinely by the combined use of rapid hybridization and a simple extraction procedure.

Detection of herpes simplex virus DNA from cerebrospinal fluid by PCR and a rapid, nonradioactive hybridization technique.

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.