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A proportion of individuals vaccinated with live attenuated Oka varicella-zoster virus (VZV) vaccine subsequently develop attenuated chicken pox and/or herpes zoster. To determine whether postvaccination varicella infections are caused by vaccine or wild-type virus, a simple method for distinguishing the vaccine strain from wild-type virus is required. We have developed a TaqMan real-time PCR assay to detect and differentiate wild-type virus from Oka vaccine strains of VZV. The assay utilized two fluorogenic, minor groove binding probes targeted to a single nucleotide polymorphism in open reading frame 62 that distinguishes the Oka vaccine from wild-type strains. VZV DNA could be genotyped and quantified within minutes of thermocycling completion due to real-time monitoring of PCR product formation and allelic discrimination analysis. The allelic discrimination assay was performed in parallel with two standard PCR-restriction fragment length polymorphism (RFLP) methods on 136 clinical and laboratory VZV strains from Canada, Australia, and Japan. The TaqMan assay exhibited a genotyping accuracy of 100% and, when compared to both PCR-RFLP methods, was 100 times more sensitive. In addition, the method was technically simpler and more rapid. The TaqMan assay also allows for high-throughput genotyping, making it ideal for epidemiologic study of the live attenuated varicella vaccine.

Context: Meningococcal infection has a high public profile because of its dramatic presentation, high fatality rate, and propensity to occur in outbreaks and clusters of cases. Use of a diagnostic polymerase chain reaction (PCR) assay could enhance laboratory confirmation of cases and guide the public health response in North America., Objective: To assess the performance of a PCR assay for the diagnosis of meningococcal disease after its implementation in a North American setting and to evaluate sensitivity and specificity of the assay for the detection of prevalent bacterial isolates., Design: Laboratory evaluation of the sensitivity and specificity of a PCR assay for Neisseria meningitidis and observational study of a series of cases comparing molecular diagnosis against the criterion standard of conventional laboratory diagnostic tests., Setting: A Canadian province with a population of 4 million people., Patients: Children and adults presenting with suspected meningococcal disease in British Columbia., Main Outcome Measures: The sensitivity and specificity of the PCR assay when compared against standard laboratory methods., Results: The PCR assay correctly identified all of 38 Canadian isolates of Neisseria meningitidis and correctly assigned the serogroup to each isolate. None of 57 other gram-positive or gram-negative bacteria or yeasts were detected by the PCR assay. In a clinical evaluation, for diagnosis of meningococcal disease, the PCR assay had a sensitivity and specificity of 91% and 76%, respectively, against conventional methods of diagnosis. Use of the PCR assay increased the laboratory confirmation of clinically suspected cases by 36%. During an outbreak, the PCR assay allowed serogroup determination in 3 of 7 cases, aiding in the public health decision to launch an immunization campaign., Conclusions: The PCR assay is more sensitive than conventional methods for the diagnosis of meningococcal disease, and enhanced surveillance may help direct the public health response to the changing epidemiology of disease in North America.

The etiology of central nervous system (CNS) infections such as meningitis and encephalitis remains unknown in a large proportion of cases partly because the diversity of pathogens that may cause CNS infections greatly outnumber available test methods. We developed a metagenomic next generation sequencing (mNGS)-based approach for broad-range detection of pathogens associated with CNS infections suitable for application in the acute care hospital setting. The analytical sensitivity of mNGS performed on an Illumina MiSeq was assessed using simulated cerebrospinal fluid (CSF) specimens (n = 9). mNGS data were then used as a training dataset to optimize a bioinformatics workflow based on the IDseq pipeline. For clinical validation, residual CSF specimens (n = 74) from patients with suspected CNS infections previously tested by culture and/or PCR, were analyzed by mNGS. In simulated specimens, the NGS reads aligned to pathogen genomes in IDseq were correlated to qPCR CT values for the respective pathogens (R = 0.96; p < 0.0001), and the results were highly specific for the spiked pathogens. In clinical samples, the diagnostic accuracy, sensitivity and specificity of the mNGS with reference to conventional methods were 100%, 95% and 96%, respectively. The clinical application of mNGS holds promise to benefit patients with CNS infections of unknown etiology. [ABSTRACT FROM AUTHOR]

Background Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar ® BKV PCR Kit. Method Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar ® BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. Results The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8 × 10 2 , 3 × 10 3 and 3.5 × 10 2 genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar ® BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar ® BKV PCR assay, were highly specific when tested against a panel of external proficiency specimens containing both BK and JC viruses. All assays, except the VP1MOD assay determined BK viral load in proficiency specimens within the same log values. With reference to results obtained by RealStar ® BKV PCR assay, the sensitivity and specificity of different assays tested in 116 serum specimens submitted for BK viral load assay were 91% and 97% for VP1 assay, 88% and 97% for VP1MOD assay, and 97% and 98% for BKLTA assay, respectively. BK Viral load in positive specimens determined by various assays was highly correlated (R 2 > 0.97), based on linear regression analysis. Conclusions The performance characteristics of the newly designed, BKLTA assay were highly comparable to RealStar ® BKV PCR assay, and can be used for routine detection and viral load monitoring of BKV in a cost-effective manner. [ABSTRACT FROM AUTHOR]

An assessment was made of the utilization and impact of a diagnostic polymerase chain reaction (PCR) assay for the diagnosis of herpes simplex viruses (HSV) 1 and 2 in cerebrospinal fluid of children who attended a Canadian pediatric referral centre. One hundred and three assays were performed on specimens from 103 patients during the period August 1997 to September 1998. Patient ages ranged from newborn to 16 years. Indications for HSV PCR included seizures with or without fever (56.3%), aseptic meningitis (16.5%), and encephalopathy with or without fever (10.7%). Only 2 of 103 (1.9%) assays were positive, including one each for HSV1 and HSV2. Control specimens that were seeded with virus indicated inhibition for 24.3, 8.8, and 6.8% of assays for HSV1, HSV2, and both HSV1 and HSV2, respectively. The mean turn-around time for HSV PCR was 2.5 days, and 90.3% were completed in less than 5 days. Acyclovir was administered to 78.6% of the patients overall; the results of the HSV PCR impacted on the treatment courses for 36 individuals. Nevertheless, 16.5% of patients continued to receive extended courses of antiviral therapy despite negative HSV PCR assays. Although it is desirable to decrease the frequency of PCR inhibitions and to further decrease the interval to assay completion, HSV PCR does have a significant impact on antiviral use in this setting.Key words: herpes simplex, polymerase chain reaction, central nervous system.Nous avons évalué l'utilisation et l'impact d'un test de réaction de polymérase en chaîne (PCR) à des fins de diagnostic du virus herpes simplex (HSV) 1 et 2 dans le liquide céphalo-rachidien d'enfants qui ont fréquenté un centre canadien de pédiatrie. Cent trois tests ont été effectués sur des spécimens provenant de 103 patients pendant la période allant de août 1997 à septembre 1998. L'âge des patient allait du nouveau-né jusqu'à 16 ans. Les indications prescrivant le PCR HSV incluaient des crises avec ou sans fièvre (56,3%), une méningite aseptique (16,5%) et une encéphalopathie avec ou sans fièvre (10,7%). Seulement 2/103 (1,9%) des tests furent positifs, incluant un chacun pour HSV1 et HSV2. Des spécimens témoins ensemencés avec des virus ont démontré une inhibition de respectivement 24,3, 8,8, et 6,8% des tests pour le HSV1, HSV2, et les deux. Le délai moyen pour l'obtention des résultats de PCR HSV était de 2,5 jours, et 90,3% des tests ont été complétés avant 5 jours. De l'acyclovir fut administré à 78,6% du nombre total de patients; les résultats de PCR HSV a eu un impact sur le déroulement des traitements de 36 individus. Néanmoins, 16,5% des patients ont continué de recevoir des traitements prolongés aux antiviraux malgré des test de PCR HSV négatifs. Bien qu'il soit souhaitable de diminuer la fréquence d'inhibitions de PCR et de diminuer davantage le délai de l'accomplissement du test, le PCR HSV a bel et bien un impact important sur l'utilisation d'antiviraux dans ce contexte.Mots clés : herpes simplex, réaction de polymérase en chaîne, système nerveux central.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

Suspected colonies of methicillin-resistant Staphylococcus aureus (MRSA) on chromogenic, MRSA Select (BioRad) medium were confirmed using routine microbiological methods, and a multiplex real-time PCR ( n = 108). Although the specificity of MRSA Select assessed at 24 h of incubation was much higher than that of 48 h (91.4 vs. 60 %), extending the incubation time to 48 h, along with PCR confirmation, increased the total number of true positive samples by 27.8 %. These results provide a cost effective method for sensitive and specific detection of MRSA. [ABSTRACT FROM AUTHOR]