Evaluation of Diagnostic Accuracy of Eight Commercial Assays for the Detection of Rubella Virus-Specific IgM Antibodies.

Rubella and congenital rubella syndrome are caused by the rubella virus and are preventable through vaccination, making disease eradication possible. Monitoring of progress toward global eradication and local elimination requires high-quality, sensitive disease surveillance that includes laboratory confirmation of cases. Previous evaluations of anti-rubella IgM detection methods resulted in the broad adoption of the Enzygnost (most recently manufactured by Siemens) enzyme-linked immunosorbent assay (ELISA) kits within WHO's global measles and rubella laboratory network, but they have been discontinued. This study evaluated seven comparable ELISAs from six manufacturers (Trinity Biotech, Euroimmun, Clin-Tech, NovaTec and Virion\Serion) as well as one automated chemiluminescent assay (CLIA) from DiaSorin. These assays include three IgM capture assays and five indirect ELISAs. A panel of 238 sera was used for the evaluation that included 38 archival rubella IgM-positive sera and 200 sera collected from patients with symptomatically similar diseases, such as measles, dengue, parvovirus B19 infection, and roseola. With this panel of sera, the sensitivity of the methods ranged from 63.2% to 100% and the specificity from 80.0% to 99.5%. No single method had both sensitivity and specificity of >90%, unless sera with equivocal results were considered presumptively positive. Some assays, particularly the Serion ELISA, had a large number of false positives with parvovirus B19 IgM-positive sera as well as sera from confirmed measles cases. The performance characteristics identified in this evaluation serve as a reminder to not rely solely on rubella IgM results for case confirmation in elimination settings.

Development and validation of a multiplex reverse transcriptase-PCR assay for simultaneous testing of influenza A, influenza B and SARS-CoV-2.

In the current pandemic of coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the co-circulation of SARS-CoV-2 and other respiratory viruses during the upcoming fall and winter seasons may present an unprecedented burden of respiratory disease in the population. Important respiratory viruses that will need to be closely monitored during this time include SARS-CoV-2, influenza A and influenza B. The epidemiology of these viruses is very similar in terms of susceptible populations, mode of transmission, and the clinical syndromes, thus the etiological agent will be difficult to differentiate without target specific assays. The availability of a sensitive and specific multiplex assay that can simultaneously detect all these targets will be valuable. Here we report the validation of a real-time reverse transciptase-PCR assay for the simultaneous detection of SARS-CoV-2, influenza A and influenza B. This multiplex assay is comparable to its singleplex counterparts with a limit-of-detection being less than 5 copies/reaction, 100 % specificity, over seven logs of dynamic range, less than 1 % coefficientof variation showing high precision, and equivalent accuracy using patient samples. It also offers the added benefits of savings in reagents and technologist time while improving testing efficiency and turn-around-times in order to respond effectively to the ongoing pandemic.

Evaluation of Diagnostic Accuracy of Eight Commercial Assays for the Detection of Measles Virus-Specific IgM Antibodies.

The World Health Organization (WHO) has targeted measles for global eradication through mass immunization. For effective monitoring of eradication targets, high-quality surveillance is needed. The detection of IgM antibodies, specific to the measles virus, with the use of commercial enzyme-linked immunosorbent assays (ELISA or EIA) is broadly used within the WHO global measles and rubella laboratory network for laboratory confirmation, and in particular, ELISA kits manufactured by Siemens (Enzygnost kits) have been primarily used. Spurred by the discontinuation of these kits, this study aims to report on the clinical sensitivity and specificity of comparable commercial ELISA kits and one automated chemiluminescent immunoassay (CLIA) method. A panel of 239 serum samples was assembled that included sera from confirmed measles cases (n = 50) and probable post-MMR vaccine response (n = 2). Measles-negative sera (n = 187) were collected from individuals presenting with other fever and rash illnesses. A total of 7 ELISA kits (Euroimmun native antigens and recombinant nucleoprotein, IBL, Clin-Tech Microimmune, NovaTec NovaLisa, Serion, and Siemens Enzygnost) and one CLIA method (DiaSorin LIAISON XL) were evaluated. The ELISA kits included two IgM capture methods and five indirect methods. Calculated sensitivities and specificities ranged from 75.0% to 98.1% and 86.6% to 99.5%, respectively. The parvovirus B19 IgM positive sera were noted to cause false-positive results, particularly for the ELISA kits from Serion and NovaLisa; specificities for this subset of samples ranged from 51.4% to 100.0%. The capture IgM ELISA methods provided the best combination of sensitivity and specificity.

Development and validation of RT-PCR assays for testing for SARS-CoV-2.

Background: The recent emergence and rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrates the urgent need for laboratory-developed assays for clinical diagnosis and public health interventions in the absence of commercial assays. Methods: We outline the progression of reverse-transcriptase polymerase chain reaction (RT-PCR) assays that were developed and validated at the Alberta Precision Laboratories, Public Health Laboratory, Alberta, Canada, to respond to this pandemic. Initially, testing was performed using SARS-CoV-2-specific and pan-coronavirus gel-based assays that were soon superseded by real-time RT-PCR assays targeting the envelope and RNA-dependent RNA polymerase genes to accommodate the high anticipated volumes of samples. Throughput was further enhanced by multiplexing the different targets together with the co-detection of an internal extraction control. Results: These assays are comparable in sensitivity and specificity to the assays recommended by the World Health Organization and the US Centers for Disease Control and Prevention. Conclusions: The availability of real-time RT-PCR assays early in the pandemic was essential to provide valuable time to local health authorities to contain transmission and prepare for appropriate response strategies.

Historique: La récente émergence et la propagation mondiale rapide du coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2) a démontré l'urgence de créer des dosages en laboratoire pour poser un diagnostic clinique et adopter des interventions sanitaires en l'absence de dosages commerciaux. Méthodologie: Les chercheurs exposent la progression des dosages d'amplification en chaîne par polymérase couplée à la transcriptase inverse (RT-PCR) mis au point et validés par les Alberta Precision Laboratories du Laboratoire de santé publique de l'Alberta, au Canada, pour répondre à cette pandémie. Les tests ont d'abord été effectués au moyen de dosages sur gel spécifiques au SARS-CoV-2 ou décelant tous les coronavirus, mais ont vite été remplacés par des dosages RT-PCR en temps réel ciblant l'enveloppe et les gènes d'ARN polymérase sous la dépendance d'ARN pour répondre au fort volume anticipé d'échantillons. Le criblage a également été renforcé par le multiplexage conjoint des différentes cibles et la codétection d'un contrôle d'extraction interne. Résultats: Ces dosages ont une sensibilité et une spécificité comparables à ceux recommandés par l'Organisation mondiale de la Santé et les Centers for Disease Control and Prevention des États-Unis. Conclusions: Il était essentiel de disposer de dosages RT-PCR au début de la pandémie pour que les autorités sanitaires locales puissent profiter de temps précieux pour contenir la transmission et préparer les stratégies de réponse appropriées.

A Public Health Laboratory Response to the Pandemic.

An outbreak of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) began in Wuhan, Hubei, China, in December 2019 and spread rapidly worldwide. The response by the Alberta Precision Laboratories, Public Health Laboratory (ProvLab), AB, Canada, included the development and implementation of nucleic acid detection-based assays and dynamic changes in testing protocols for the identification of cases as the epidemic curve increased exponentially. This rapid response was essential to slow down and contain transmission and provide valuable time to the local health authorities to prepare appropriate response strategies. As of May 24, 2020, 236,077 specimens were tested, with 6,475 (2.74%) positives detected in the province of Alberta, Canada. Several commercial assays are now available; however, the response from commercial vendors to develop and market validated tests is a time-consuming process. In addition, the massive global demand made it difficult to secure a reliable commercial supply of testing kits and reagents. A public health laboratory serves a unique and important role in the delivery of health care. One of its functions is to anticipate and prepare for novel emerging pathogens with a plan for pandemic preparedness. Here, we outline the response that involved the development and deployment of testing methodologies that evolved as SARS-CoV-2 spread worldwide, the challenges encountered, and mitigation strategies. We also provide insight into the organizational structure of how a public health response is coordinated in Alberta, Canada, and its benefits.

Emergence of a Novel Recombinant Norovirus GII.P16-GII.12 Strain Causing Gastroenteritis, Alberta, Canada.

We identified a novel recombinant GII.P16-GII.12 norovirus associated with epidemic and endemic gastroenteritis during March 1, 2018-February 12, 2019, in Alberta, Canada. GII.12 viruses have not been detected in Alberta since 2000. Comparing the full genome of this strain to previously published sequences revealed this virus to be a novel recombinant strain.

Simultaneous Detection and Differentiation between Wild-Type and Vaccine Measles Viruses by a Multiplex Real-Time Reverse Transcription-PCR Assay.

Measles is one of the most contagious viral respiratory infections and was declared to be eliminated from Canada in 1998; however, measles cases and outbreaks still occur every year through reintroduction from other parts of the world. Laboratory confirmation of measles virus (MV) RNA by real-time PCR provides a definitive diagnosis, and molecular analysis to determine the genotype is the only way to distinguish between wild-type and vaccine strains. This distinction is important since live attenuated vaccine strains are able to replicate in the patient and can be associated with rash and fever but are poorly transmissible, if at all. Prompt reporting of measles cases to local authorities, including differentiation between wild-type and vaccine strains, allows for optimal management and contact tracing. The development and validation of a multiplex real-time reverse transcription-PCR (rtRT-PCR) assay for the simultaneous detection and differentiation of the Moraten and Schwarz vaccine strains from presumptive wild-type MV in a format that can be easily implemented for high-throughput testing of patient samples are reported here. This assay is sensitive, specific, reproducible, and 100% accurate in comparison with the gold standard comparator assay.

Simultaneous detection of Zika, Chikungunya and Dengue viruses by a multiplex real-time RT-PCR assay.

BACKGROUND: In the recent past, arboviruses such as Chikungunya (CHIKV) and Zika (ZIKV) have increased their area of endemicity and presented as an emerging global public health threat. OBJECTIVES: To design an assay for the simultaneous detection of ZIKV, CHIKV and Dengue (DENV) 1-4 from patients with symptoms of arboviral infection. This would be advantageous because of the similar clinical presentation typically encountered with these viruses and their co-circulation in endemic areas. STUDY DESIGN: In this study we have developed and validated a triplex real time reverse transcription PCR assay using hydrolysis probes targeting the non-structural 5 (NS5) region of ZIKV, non-structural protein 4 (nsP4) from CHIKV and 3' untranslated region (3'UTR) of DENV 1-4. RESULTS AND CONCLUSIONS: The 95% LOD by the triplex assay was 15 copies/reaction for DENV-1 and less than 10 copies/reaction for all other viruses. The triplex assay was 100% specific and did not amplify any of the other viruses tested. The assay was reproducible and adaptable to testing different specimen types including serum, plasma, urine, placental tissue, brain tissue and amniotic fluid. This assay can be easily implemented for diagnostic testing of patient samples, even in a high throughput laboratory.

Determining rubella immunity in pregnant Alberta women 2009-2012.

Rubella IgG levels for 157,763 pregnant women residing in Alberta between 2009 and 2012 were analyzed. As there have been no reported cases of indigenous rubella infection in Canada since 2005, there has been a lack of naturally acquired immunity, and the current prenatal population depends almost entirely on vaccine induced immunity for protection. Rubella antibody levels are significantly lower in younger maternal cohorts with 16.8% of those born prior to universal vaccination programs (1971-1980), and 33.8% of those born after (1981-1990) having IgG levels that are not considered protective (<15 IU/mL). Analysis across pregnancies showed only 35.0% of women responded with a 4-fold increase in antibody levels following post-natal vaccination. Additionally, 41.2% of women with antibody levels <15 IU/mL had previously received 2 doses of rubella containing vaccine. These discordant interpretations generate a great deal of confusion for laboratorians and physicians alike, and result in significant patient follow-up by Public Health teams. To assess the current antibody levels in the prenatal population, latent class modeling was employed to generate a two class fit model representing women with an antibody response to rubella, and women without an antibody response. The declining level of vaccine-induced antibodies in our population is disconcerting, and a combined approach from the laboratory and Public Health may be required to provide appropriate follow up for women who are truly susceptible to rubella infection.

Full-genome analysis of avian influenza A(H5N1) virus from a human, North America, 2013.

Full-genome analysis was conducted on the first isolate of a highly pathogenic avian influenza A(H5N1) virus from a human in North America. The virus has a hemagglutinin gene of clade 2.3.2.1c and is a reassortant with an H9N2 subtype lineage polymerase basic 2 gene. No mutations conferring resistance to adamantanes or neuraminidase inhibitors were found.

Bioinformatics of varicella-zoster virus: single nucleotide polymorphisms define clades and attenuated vaccine genotypes.

Varicella zoster virus (VZV) is one of the human herpesviruses. To date, over 40 complete VZV genomes have been sequenced and analyzed. The VZV genome contains around 125,000 base pairs including 70 open reading frames (ORFs). Enumeration of single nucleotide polymorphisms (SNPs) has determined that the following ORFs are the most variable (in descending order): 62, 22, 29, 28, 37, 21, 54, 31, 1 and 55. ORF 62 is the major immediate early regulatory VZV gene. Further SNP analysis across the entire genome has led to the observation that VZV strains can be broadly grouped into clades within a phylogenetic tree. VZV strains collected in Singapore provided important sequence data for construction of the phylogenetic tree. Currently five VZV clades are recognized; they have been designated clades 1 through 5. Clades 1 and 3 include European/North American strains; clade 2 includes Asian strains, especially from Japan; and clade 5 includes strains from India. Clade 4 includes some strains from Europe, but its geographic origins need further documentation. Within clade 1, five variant viruses have been isolated with a missense mutation in the gE (ORF 68) glycoprotein; these strains have an altered increased cell spread phenotype. Bioinformatics analyses of the attenuated vaccine strains have also been performed, with a subsequent discovery of a stop-codon SNP in ORFO as a likely attenuation determinant. Taken together, these VZV bioinformatics analyses have provided enormous insights into VZV phylogenetics as well as VZV SNPs associated with attenuation.

Rubella diagnostic issues in Canada.

With the success of the rubella vaccination program, a goal for the elimination of rubella and congenital rubella syndrome (CRS) by 2010 has been established. To monitor the progress toward elimination, surveillance is critical. The laboratory plays an important role in both diagnostics and surveillance for rubella and CRS. In the elimination phase, there are particular issues and challenges that are important to consider when undertaking rubella diagnostics and surveillance activities. Although immunoglobulin (Ig) M serological testing is the primary diagnostic test used to confirm acute rubella infection, additional tests, such as paired IgG serological testing, molecular detection of rubella virus, and rubella IgG avidity testing need to be considered for confirming cases, depending on the clinical and epidemiologic context of a particular suspected rubella case.

Detection of mumps virus RNA by real-time one-step reverse transcriptase PCR using the LightCycler platform.

A real-time reverse transcriptase PCR (RT-PCR) assay that targeted both the mumps virus F gene and human RNase P in clinical samples was adapted for use with the LightCycler platform. LightCycler RT-PCR is as sensitive as conventional nested RT-PCR and significantly less expensive and labor-intensive, making it ideal for mumps diagnosis during outbreaks.

Foscarnet salvage therapy for acyclovir-resistant varicella zoster: report of a novel thymidine kinase mutation and review of the literature.

The authors describe an acyclovir-resistant varicella zoster virus infection in a pediatric patient after hematopoietic stem cell transplant, the use of foscarnet as salvage therapy, and review the literature to clarify the pediatric experience with foscarnet in this setting. A novel thymidine kinase mutation is described, along with a new phenotypic assay for characterizing acyclovir resistance in varicella zoster virus.

A prospective assessment of cytomegalovirus immune evasion gene transcription profiles in transplant patients with cytomegalovirus infection.

BACKGROUND: The cytomegalovirus (CMV) immune evasion genes US3, US6, and US11 may disrupt the host immune response via downregulation of major histocompatibility complex molecules. Transplant recipients with CMV infection were prospectively assessed for immune evasion gene expression. METHODS: Seventy solid organ transplant patients with CMV infection who were given antiviral therapy were enrolled. Quantitative mRNA levels of US3, US6, and US11 were assessed using real-time polymerase chain reaction assays from peripheral blood mononuclear cells at regular time-points after starting therapy. RESULTS: High immune evasion mRNA levels were detectable at start-of-therapy (median US3-4.5 log10 copies; US6- 3.7 log10 copies, and US11-3.3 log10 copies/10 cells). With therapy, immune evasion mRNA levels declined exponentially. For example, median calculated US3 half-life was 1.59 days (range 0.74-12.5 days). By day7, US3 mRNA was detectable in 55.7%, US6 in 38.6%, and US11 in 41.4% of patients. Early phase kinetics correlated with outcomes. When adjusted for baseline DNA level, there was a trend to higher mRNA levels in patients who relapsed. Also, detectable mRNA at day 14 after start of therapy was associated with virologic relapse after initial treatment (P

Evaluation of commercial rubella immunoglobulin G avidity assays.

We compared the performances of five commercial rubella virus immunoglobulin G (IgG) avidity assays. The Adaltis (kappa = 0.28) and Diesse (kappa = 0.33) assays showed poor correlation, the Behring assay (kappa = 0.68) showed good correlation, and the Euroimmun (kappa = 0.95) and Radim (kappa = 0.94) assays showed excellent correlation with a well-established in-house rubella virus IgG avidity assay. The Euroimmun and Radim assays were statistically significantly better than the other commercial assays (P < 0.01).

A full-genome phylogenetic analysis of varicella-zoster virus reveals a novel origin of replication-based genotyping scheme and evidence of recombination between major circulating clades.

Varicella-zoster virus (VZV) is a remarkably stable virus that until recently was thought to exhibit near-universal genetic homogeneity among circulating wild-type strains. In recent years, the expanding knowledge of VZV genetics has led to a number of groups proposing sequence-based typing schemes, but no study has yet examined the relationships between VZV genotypes at a full-genome level. A central hypothesis of this study is that VZV has coevolved with humankind. In this study, 11 additional full VZV genomic sequences are presented, bringing the current number of complete genomic sequences publicly available to 18. The full-genome alignment contained strains representing four distinct clades, but the possibility exists that a fifth clade comprised of African and Asian-like isolates was not represented. A consolidated VZV genotyping scheme employing the origin-associated region between reiteration region R4 and open reading frames (ORFs) 63 and 70 is described, one which accurately categorizes strains into one of four clades related to the geographic origin of the isolates. The full-genome alignment also provided evidence for recombination having occurred between the major circulating VZV clades. One Canadian clinical isolate was primarily Asian-like in origin, with most of the genome showing strong sequence identity to the Japanese-like clade B, with the exceptions being two putative recombination regions, located in ORFs 14 to 17 and ORFs 22 to 26, which showed clear similarity to the European/North American clade A. The very low rate of single-nucleotide polymorphisms scattered across the genome made full-genome sequencing the only definitive method for identifying specific VZV recombination events.

Prevention of congenital rubella syndrome--what makes sense in 2006?

This review summarizes the practical aspects of rubella immunization programs in both developed and developing countries. Routine use of rubella vaccine is gradually resulting in the elimination of endemic rubella and congenital rubella syndrome (CRS) in the developed world, and routine use of vaccine in young children is now being implemented in many developing countries. However, such programs must achieve high immunization rates or be supplemented by the immunization of seronegative women of childbearing age to prevent a paradoxical increase in CRS as the burden of illness is shifted to an older age group. There are many successful prenatal screening programs for rubella immunity in developed countries, but screening prior to pregnancy could theoretically prevent even more cases of CRS. Enzyme-linked immunosorbent assay is the most commonly used laboratory test for screening, but the protective titer remains to be established. The need for reimmunization of women who serorevert or who remain seronegative following rubella vaccine has not been established. Surveillance for rubella cases and for CRS is vital in assessment of the ongoing success of rubella immunization programs.

Complete DNA sequence analyses of the first two varicella-zoster virus glycoprotein E (D150N) mutant viruses found in North America: evolution of genotypes with an accelerated cell spread phenotype.

Varicella-zoster virus (VZV) is considered to be one of the most genetically stable of all the herpesviruses. Yet two VZV strains with a D150N missense mutation within the gE glycoprotein were isolated in North America in 1998 and 2002. The mutant strains have an accelerated cell spread phenotype, which distinguishes them from all wild-type and laboratory viruses. Since the VZV genome contains 70 additional open reading frames (ORFs), the possibility existed that the phenotypic change was actually due to an as-yet-undiscovered mutation or deletion elsewhere in the genome. To exclude this hypothesis, the entire genomes of the two mutant viruses were sequenced and found to contain 124,883 (VZV-MSP) and 125,459 (VZV-BC) nucleotides. Coding single-nucleotide polymorphisms (SNPs) were identified in 14 ORFs. One missense mutation was discovered in gH, but none was found in gB, gI, gL, or gK. There were no coding SNPs in the major regulatory protein ORF 62. One polymorphism was discovered which could never have been anticipated based on current knowledge of herpesvirus genomics, namely, the origins of replication differed from those in the prototype strain but not in a manner expected to affect cell spread. When the two complete mutant VZV sequences were surveyed in their entirety, the most reasonable conclusion was that the increased cell spread phenotype was dependent substantially or solely on the single D150N polymorphism in glycoprotein gE. The genomic results also expanded the evolutionary database by identifying which VZV ORFs were more likely to mutate over time.