Toxin-Producing Klebsiella oxytoca in Healthy Infants: Commensal or Pathobiont?

OBJECTIVES: Klebsiella oxytoca is a gastrointestinal pathobiont with the potential to produce the toxins tilivalline and tilimycin, which cause antibiotic-associated hemorrhagic colitis. Overgrowth of toxigenic K oxytoca has recently been implicated in necrotizing enterocolitis. K oxytoca colonizes 2-9% of healthy adults, however, there is no systematic data on colonization in healthy children. We investigated K oxytoca colonization and its toxigenic properties in healthy infants. METHODS: We sampled stool of healthy infants and determined K oxytoca colonization using stool culture and PCR (pehX). Toxin in stool was measured with HPLC/high-resolution mass spectrometry. K oxytoca isolates were typed using multi-locus sequence typing (MLST) and K oxytoca toxin PCR (npsA/B). Cytotoxin production of isolates was analyzed by MTT assay. RESULTS: K oxytoca was detected in 30 of 61 infants (49%) using stool culture and in 45 of 61 (73%) using PCR (pehX). Toxin marker PCR (npsA/B) was positive in 66% of stool samples positive for K oxytoca PCR. Stool toxin levels were too low for quantitation but traces of tilivalline were detected. Contrarily, 49% of K oxytoca isolates demonstrated toxicity in the MTT assay. MLST revealed 36 distinct sequence types affiliated with all known K oxytoca sequence type clusters (A, B1 and B2). CONCLUSIONS: More than 70% of healthy infants were colonized with K oxytoca. Toxin quantities in stool of colonized healthy infants were below detection level, yet half of the isolates produced toxin in vitro demonstrating their pathobiont potential. The high occurrence of toxigenic K oxytoca in healthy infants has to be considered for future disease association studies.

Improved diagnosis of antibiotic-associated haemorrhagic colitis (AAHC) in faecal specimens by a new qualitative real-time PCR assay detecting relevant toxin genes of Klebsiella oxytoca sensu lato.

OBJECTIVE: Toxin-producing Klebsiella oxytoca causes antibiotic-associated haemorrhagic colitis (AAHC). The disease-relevant cytotoxins tilivalline and tilimycine produced by certain K. oxytoca isolates are encoded by the non-ribosomal peptide synthetase genes A (npsA) and B (npsB). In this study, the new LightMix® Modular kit for the detection of relevant K. oxytoca sensu lato (s.l.) toxin genes was evaluated. METHODS: DNA was extracted on the automated EMAG® platform. Amplification was done on the Light Cycler® 480 II instrument. In total, 130 residual faecal specimens collected from patients with antibiotic-associated diarrhoea were studied to determine the clinical sensitivity and specificity. Toxigenic culture served as reference method. RESULTS: With the new kit, the limit of detection was 15 CFU/mL for all targets. For the pehX target specific to K. oxytoca s.l., 65 of 130 clinical specimens were positive, while toxin-specific targets (npsA/npsB) were positive in 47 of 130. The npsA/npsB PCR targets showed a clinical sensitivity of 100% (95%CI 80.5-100%) and a specificity of 73.5% (95%CI 64.3-81.3%) with a positive predictive value of 16.5% (95%CI 12.7-21.2%) and a negative predictive value of 100%. CONCLUSION: Compared with culture, additional clinical specimens positive for K. oxytoca s.l. were detected with real-time PCR. The specificity of the toxin targets appears moderate due to the inferior sensitivity of the culture-based reference method. Since the developed assay is highly sensitive, it may be used as first-line method to improve the diagnosis of AAHC.

Low prevalence of Clostridium difficile colonization in patients in long-term care facilities in Graz, Austria: A point-prevalence study.

BACKGROUND: We aimed to determine the prevalence of asymptomatic colonization by C. difficile in stool of residents in four long-term care facilities (LTCFs) in Graz, Austria and to identify factors associated with colonization. METHODS: We conducted a point-prevalence study in March 2018. Stool samples were examined by GDH enzyme immunoassay and when positive a toxin A/B-enzyme immunoassay was carried out. Additionally, all samples were tested by toxin A and B PCR and were plated manually as well as in automated fashion onto selective C. difficile agar. RESULTS: In 4/144 (2.8%) residents the GDH assay was positive. Each resident was colonized by a different C. difficile ribotype. C. difficile was not detected in any of the environmental samples. Significantly more colonized residents (60%) had stayed at a hospital in the 3 months previous to the study compared to 10% of non-colonized patients (p=0.01). CONCLUSIONS: The prevalence of colonization by toxigenic C. difficile was 2.8% in patients in LTCFs in Graz, Austria.

Evaluation of a new extraction platform in combination with molecular assays useful for monitoring immunosuppressed patients.

BACKGROUND: In the immunosuppressed, detection of viral reactivation at the earliest convenience and molecular monitoring are of paramount importance. Nucleic acid extraction has a major impact on the reliability of results obtained from molecular assays. OBJECTIVES: The aim of this study was to investigate the accuracy of the new EMAG® nucleic acid extraction platform and to compare the performance of the new platform to that of the standard NucliSENS® easyMAG® instrument in the routine clinical laboratory. STUDY DESIGN: For accuracy testing, reference material and for comparison studies, clinical specimens were used. In addition, a lab-flow analysis including estimation of hands-on time and that for automated extraction was performed. RESULTS: When accuracy was tested, all 89 results obtained were found to be concordant with the results expected. When 648 clinical results were compared, 85.7% were found to be within ±0.5 log10 unit, 9.5% between ±0.5 and ±1.0 log10 unit, and 4.8% more than ±1.0 log10 unit. The overall time required for nucleic acid extraction of 8 samples in parallel was 94 min for the fully automated extraction mode and 82 min for the partly automated mode with the new platform, and 73 min with the standard instrument. Hands-on time was found to be shorter with the new platform. CONCLUSIONS: The extraction performance of both platforms was found to be similar for EDTA whole blood, BAL, and urine specimens. The total turn-around time for nucleic acid extraction was found to be longer with the EMAG® platform, whereas hands-on time was reduced.

Analytical and clinical performance of a new molecular assay for Epstein-Barr virus DNA quantitation.

Quantitation of EBV DNA has been shown to be a useful tool to identify and monitor patients with immunosuppression and high risk for EBV-associated disease. In this study, the analytical and clinical performance of the new Realquality RS-EBV Kit (AB Analitica, Padova, Italy) was investigated. The clinical performance was compared to that of the EBV R-gene (bioMerieux, Varilhes, France) assay. When the accuracy of the new assay was tested, all results except of one were found to be within ±0.5log10 unit of the expected panel results. Determination of linearity showed a quasilinear curve, the between day imprecision ranged from 18% to 88% and the within run imprecision from 16% to 53%. When 96 clinical EDTA whole blood samples were tested, 77 concordant and 19 discordant results were obtained. When the results for the 69 samples quantifiable with both assays were compared, the new assay revealed a mean 0.31log10 unit higher measurement. The new assay proved to be suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory. The variation between quantitative results obtained by the assays used in this study reinforces the use of calibrators traceable to the existing international WHO standard making different assays better comparable.

Extraction of viral nucleic acids: comparison of five automated nucleic acid extraction platforms.

BACKGROUND: Nucleic acid extraction has a major impact on the reliability of results in routine molecular diagnostics. Optimal isolation of nucleic acids and removal of inhibitors are essential. OBJECTIVES: This study compares five different automated extraction platforms for the extraction of norovirus RNA from stool and cytomegalovirus (CMV) DNA from plasma samples. STUDY DESIGN: Norovirus positive stool samples and CMV positive plasma samples were aliquoted and analyzed using five different automated platforms (easyMAG, bioMerieux; m2000sp, Abbott; MagNA Pure LC 2.0, Roche; QiaSymphony, Qiagen; sample preparation module of the VERSANT kPCR Molecular System, Siemens). Similar sample input and output volumes, the identical real-time PCR cycler, and the identical assays for amplification and detection for norovirus RNA and CMV DNA, respectively, were chosen. RESULTS: Of 39 stool samples, 36 tested positive for norovirus RNA with all extraction platforms. The three discrepant samples showed inhibition after extraction with at least one platform. Only with the VERSANT platform all samples tested positive for both the target RNA and the internal controls. Of 42 plasma samples, 27 gave quantifiable results for CMV DNA with all extraction platforms. There was significant variance between viral concentrations when different extraction platforms were compared. The majority of the 15 discrepant samples showed low viral concentrations. The internal control of the CMV assay gave positive results for all samples tested below the limit of quantification. CONCLUSIONS: The five automated extraction platforms yielded comparable results. However, the extraction performance was found to be impaired by inhibitory substances in stool samples.

Performance of new enzyme-linked fluorescent assays for detection of Epstein-Barr virus specific antibodies in routine diagnostics.

BACKGROUND AND AIMS: Indirect fluorescent antibody assays are still the gold standard for the detection of Epstein-Barr-virus-specific antibodies; however, this technique requires several manual steps and an experienced technician. This retrospective study investigated the performance of the new VIDAS(®) enzyme-linked fluorescent assays for automated qualitative detection of EBV VCA IgM, VCA/EA IgG, and EBNA IgG antibodies in routine diagnostics. METHODS: 174 serum samples were tested first with the gold standard. In context with the clinical status, 60 samples IFA IgG/IgM positive and with clinical symptoms compatible with infectious mononucleosis, 26 samples IFA IgG/IgM negative and missing any clinical symptoms and 88 samples with varying IFA status and without any clinical information were defined. In a second step all samples were retested with the new assays. RESULTS: In the overall agreement between VIDAS(®) and IFA for evaluable results, almost perfect agreement was observed (kappa = 0.91; 95% confidence interval (CI), 0.86-0.97). Estimating all indeterminate VIDAS(®) results as discordant or concordant the observed kappa values were 0.71 (CI 0.63-0.79) and 0.93 (CI 0.88-0.98), respectively. With the new assays 45, 22, and 70 identical results were obtained, respectively. Western blot analysis of the discrepant samples showed a quasi similar performance of both assays. CONCLUSIONS: The new VIDAS(®) assays can be an alternative to IFA testing especially in high-throughput laboratories. Full automation of EBV serological diagnostis by the new VIDAS assays is of major importance for routine diagnostic laboratories.

Evaluation of four molecular assays for detection of pandemic influenza A (H1N1) 2009 virus in the routine diagnostic laboratory.

BACKGROUND: Detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA have gained significance because of their widespread community transmission. OBJECTIVE: To study the accuracy and the performance of four molecular assays for the detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA in the routine diagnostic laboratory. STUDY DESIGN: The accuracy of the molecular assays was determined with reference material. For evaluation of the performance, 104 clinical specimens were studied. Sample preparation was done on a fully automated extraction instrument. For amplification and detection of influenza RNA, all molecular assays evaluated were based on real-time PCR. RESULTS: When the accuracy was tested, the majority of assays yielded results as expected. When clinical samples were analyzed, 94 samples gave concordant results with all assays. One of the assays showed one false-negative result and another assay 10 false-negatives. CONCLUSION: The majority of assays evaluated in this study proved suitable for the detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA. All assays are easy to handle and provide results rapidly.

Rapid quantitation of cytomegalovirus DNA in whole blood by a new molecular assay based on automated sample preparation and real-time PCR.

Quantitation of cytomegalovirus (CMV) DNA in whole blood samples has gained significance in the routine diagnostic laboratory. In this study, the analytical performance of the artus™ CMV RG PCR kit in conjunction with automated sample preparation on the new QIAsymphony™ SP instrument was evaluated. Clinically referred samples were tested and results compared to those obtained with the routinely used molecular test system. Accuracy testing showed results to be within ±0.2 log(10) unit of the expected panel results. The assay was linear (R = 0.9972) from a lower quantification limit of 148 copies/ml to 1.3 × 10(7) copies/ml. The between-day imprecision CV was 8-63%, and the within-run imprecision CV was 16-61%. When 100 clinically referred samples were tested, results obtained with the new test system showed an acceptable concordance with those obtained with the routinely used easyMAG™ sample preparation and CMV HHV6,7,8 R-gene™ test system. Discrepant results were found with low-titer samples containing CMV DNA concentrations under the lower limit of quantification or within half a log unit above. The time to results was comparable for both test systems. The QIAsymphony™ sample preparation and artus™ CMV RG PCR test system allows for a rapid, sensitive, precise, and accurate high-throughput quantitation of CMV DNA in whole blood in the routine diagnostic laboratory.

Detection and quantitation of Epstein-Barr virus (EBV) DNA in EDTA whole blood samples using automated sample preparation and real time PCR.

BACKGROUND: Detection and quantitation of Epstein-Barr virus (EBV) DNA in EDTA whole blood samples has gained significance in the routine diagnostic laboratory. METHODS: In this study, the analytical and clinical performance of the artus EBV RG PCR kit in conjunction with automated sample preparation on the QIAsymphony SP instrument was evaluated. RESULTS: When the accuracy of the new test system was tested, all results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity showed a quasilinear curve over 4 log units. The lower limit of detection was determined to be 391 EBV DNA copies/mL in EDTA whole blood. The between day imprecision ranged from 18% to 66%, and the within run imprecision ranged from 11% to 50%. When clinical samples were tested and the results compared with those obtained with the routinely used easyMAG sample preparation and EBV R-gene test system, 60 samples tested positive and 31 samples tested negative by both assays. Nineteen samples were found to be positive using the QIAsymphony sample preparation and artus EBV RG PCR test system only, and no samples tested positive with the routinely used test system only. CONCLUSIONS: The QIAsymphony sample preparation and artus EBV RG PCR test system is suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory.

Detection of CMV DNA: is EDTA whole blood superior to EDTA plasma?

The early diagnosis of human cytomegalovirus (CMV) infection in immunosuppressed patients has been improved by molecular detection of CMV DNA. In this study, corresponding EDTA whole blood and EDTA plasma specimens were obtained from 42 bone marrow transplant recipients. For detection of CMV DNA, two commercially available assays, the CMV HHV6,7,8 R-gene (ARGENE), and the artus CMV LC PCR Kit (QIAGEN), were employed. The linearity of both assays was determined by using a clinical EDTA whole blood sample with high CMV DNA load. With the CMV HHV6,7,8 R-gene test, CMV DNA was detected in 40 EDTA whole blood and in 19 EDTA plasma samples, while the artus CMV LC PCR Kit test detected CMV DNA in 27 EDTA whole blood and in 30 EDTA plasma samples. In conclusion, EDTA whole blood samples were found to be the superior material when using the CMV HHV6,7,8 R-gene test. However, this benefit may not exist when employing alternative assays.

Reliable detection and quantitation of viral nucleic acids in oral fluid: Liquid phase-based sample collection in conjunction with automated and standardized molecular assays.

Oral fluid has been used widely as sample matrix for the detection and quantitation of viral nucleic acids. However, in the vast majority of previous studies, various methods for collection of oral fluid and molecular assays lacking automation and standardization were used. In this study, a new standardized liquid phase-based saliva collection system was employed followed by a fully automated viral nucleic acid extraction and real-time PCR using commercially available in vitro diagnostics (IVD)/Conformité Européene (CE) labeled molecular assays. When the lower limit of detection of herpes simplex virus (HSV)-1/2 DNA, varicella zoster virus (VZV) DNA, and hepatitis C virus (HCV) RNA in spiked oral fluid was tested, the results were found to be comparable to those with defined sample materials recommended by the assay manufacturers. When clinical specimens were investigated, 21 of 25 (84%) oral fluids obtained from patients with clinically apparent herpetic lesions tested positive for HSV DNA, 7 of 10 (70%) oral fluids obtained from patients with Ramsay Hunt Syndrome tested positive for VZV DNA, and 19 of 40 (48%) oral fluids collected from patients with chronic HCV infection tested positive for HCV RNA. The automated extraction instruments completed all extractions without malfunction and no inhibitions were observed throughout the entire study. Liquid phase-based saliva collection in conjunction with automated and standardized commercially available molecular assays allows reliable quantitation of viral nucleic acids in oral fluid samples and may contribute to improved comparable and interpretable test results.

Comparison of molecular assays for detection and typing of human papillomavirus.

OBJECTIVE: The objective of the study was to compare the performance of 3 different extraction instruments in conjunction with 4 different amplification and detection kits for detection and typing of human papillomavirus (HPV) deoxyribonucleic acid (DNA). STUDY DESIGN: A total of 42 cervical swabs were investigated. HPV DNA was extracted on the 3 different instruments. Each of the extracts was then amplified, and HPV DNA amplification products were detected with 4 different kits. RESULTS: In 31 samples, HPV DNA was detected by both the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test in conjunction with DNA extraction on the easyMAG instrument. In another 6 samples, only low-risk types were detected with the linear array HPV genotyping test. After extraction on the easyMAG instrument, 32 samples tested positive when the PapilloCheck with the HotStarTaq DNA polymerase was used. CONCLUSION: Together with extraction on the easyMAG instrument, the Amplicor HPV test, the linear array HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid and divergent HPV typing results.

Detection of cytomegalovirus (CMV) DNA in EDTA whole-blood samples: evaluation of the quantitative artus CMV LightCycler PCR kit in conjunction with automated sample preparation.

Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.

Detection and differentiation of Bordetella spp. by real-time PCR.

Molecular detection of Bordetella pertussis DNA is a sensitive and specific method for the rapid diagnosis of pertussis. In this study, a new molecular assay for the detection and differentiation of Bordetella spp. based on automated DNA extraction and real-time PCR was evaluated. The analytical sensitivity of the new assay was determined by Probit analysis of serial dilutions of both cloned PCR products IS481 and IS1001 and cell suspensions of B. pertussis, B. parapertussis, and B. bronchiseptica. The specificity was analyzed by testing a number of pathogens producing respiratory infections. Moreover, a total of 92 clinical samples were investigated. The results were compared to those obtained by an in-house assay based on manual DNA extraction, followed by real-time PCR and detection of IS481. The analytical sensitivity of the new assay for the detection of IS481 and IS1001 was determined to be 2.2 and 1.2 genome equivalents/mul, respectively. The analytical sensitivity for the detection of B. pertussis, B. parapertussis, and B. bronchiseptica was determined to be 1.6, 1.0, and 2.7 genome equivalents/mul, respectively. When clinical specimens were tested with the new assay, 46 of 92 were found to be positive for Bordetella DNA. With the in-house assay, 45 samples tested positive. The new molecular assay proved to be suitable for the rapid diagnosis of pertussis in the routine diagnostic laboratory.

Evaluation of a commercial real-time PCR assay for quantitation of Epstein-Barr virus DNA in different groups of patients.

The aim of this study was to evaluate the performance of a molecular assay for quantitation of Epstein-Barr virus (EBV) DNA based on real-time PCR, and to determine EBV DNA levels in EDTA whole blood samples derived from different groups of patients. Following a manual DNA extraction protocol, real-time PCR was performed using the LightCycler EBV Quantification Kit, which demonstrated sufficient accuracy and linearity. Coefficients of variations were found to be between 6 and 42% and 5 and 34%, respectively, for inter-assay and intra-assay variations. In clinical specimens, EBV DNA was detected in all patients with acute EBV infection (n=34), in one of 25 adults with past EBV infection, in 16 out of 25 (64%) anti-HIV antibody positive persons, in 10 out of 25 (40%) solid organ transplant recipients, and in none of the 23 infants without history of EBV infection. When EBV DNA levels in positive specimens were compared between different groups, statistically significant differences were not found. The LightCycler EBV Quantification Kit was found to be useful for determination of EBV DNA levels in EDTA whole blood.

Rapid diagnosis of adenoviral keratoconjunctivitis by a fully automated molecular assay.

OBJECTIVE: To establish and evaluate a new test system for rapid detection and diagnosis of adenoviral keratoconjunctivitis. DESIGN: After establishment of the molecular assay, 52 conjunctival smears were studied. PARTICIPANTS: Samples were derived from patients with a clinical presentation compatible with keratoconjunctivitis. METHODS: A molecular assay for detection of human adenovirus (HAdV) based on automated nucleic acid extraction and real time polymerase chain reaction was established and evaluated. The new assay included a heterologous internal control. MAIN OUTCOME MEASURES: Statement about the presence or absence of adenoviral DNA in the specimen. RESULTS: The amplification efficiency was found to be 100%. The detection limit was calculated to be 116 copies per LightCycler capillary. When clinical specimens were tested, 15 of 52 conjunctival smears were found to be positive for HAdV DNA. The internal control was detected in all samples. CONCLUSIONS: The new molecular assay proved to be suitable for rapid diagnosis of adenoviral keratoconjunctivitis in the routine diagnostic laboratory.

Evaluation of automated sample preparation and quantitative PCR LCx assay for determination of human immunodeficiency virus type 1 RNA.

Efforts have been made to achieve full automation of molecular assays for quantitative detection of human immunodeficiency virus type 1 (HIV-1). In the present study, the Abbott LCx HIV RNA Quantitative assay was evaluated in conjunction with automated HIV-1 RNA extraction on the MagNA Pure LC instrument and compared to the conventional LCx HIV RNA Quantitative assay, which uses a manual nucleic acid extraction protocol. Accuracy, linearity, and interassay and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was tested with a total of 105 clinical specimens. When the accuracy of the LCx HIV RNA Quantitative assay with the automated sample preparation protocol was tested, all results were found to be within +/- 0.5 log unit of the expected results. Determination of linearity resulted in a quasilinear curve over 3.5 log units. For determination of interassay variation, coefficients of variation were found to be between 21 and 66% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 10 and 69% for the LCx HIV RNA Quantitative assay with manual sample preparation. For determination of intra-assay variation, coefficients of variation were found to be between 7 and 25% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 7 and 19% for the LCx HIV RNA Quantitative assay with manual sample preparation. When clinical samples were tested by the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and the results were compared with those of the LCx HIV RNA Quantitative assay with manual sample preparation, 95% of all positive results were found to be within +/- 0.5 log unit. In conclusion, the assay with automated sample preparation proved to be suitable for use in the routine diagnostic laboratory and required significantly less hands-on time.

Parallel detection of five human herpes virus DNAs by a set of real-time polymerase chain reactions in a single run.

BACKGROUND: Human herpes viruses cause a spectrum of diseases that are usually self-limiting but can be reactive during immuno-suppression and may then lead to severe or even life-threatening diseases. The LightCycler technology allows rapid polymerase chain reaction (PCR) including product analysis within a closed system. This approach has been demonstrated to be suitable for routine diagnostic virus detection. Several LightCycler PCR assays have been established to the detection of human herpes viruses. The assays vary in their detection formats and PCR cycling protocols. So, they cannot be performed within a single LightCycler run. OBJECTIVES: Development of four LightCycler PCR assays for parallel detection of DNA derived from human cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) in a single run. STUDY DESIGN: Primers and hybridization probes were tailored to suit one LightCycler PCR program. LightCycler PCRs were established, detection limits were determined, and clinical samples were evaluated. RESULTS: With quantified herpes virus type specific DNA spiked into cerebrospinal fluid, serum or EDTA plasma the detection limits were found either at 500 or 250 viral DNA copies per ml depending on the virus DNA specific PCR and on the specimen type used. The applicability of the new LightCycler assays for routine molecular testing was evaluated by testing 96 clinical samples. CONCLUSION: The developed set of LightCycler PCRs permits parallel detection of CMV, EBV, HSV-1, HSV-2, and VZV in a single LightCycler run. The new molecular assays can easily be used to the rapid, simple, and convenient detection of herpes virus DNA in cerebrospinal fluid, serum and EDTA plasma in the routine diagnostic laboratory.