Carol A. McClure

Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) > or = 93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.

Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

Evaluation of a Reverse Transcriptase Polymerase Chain Reaction Test and Virus Isolation on Field Samples Collected for the Diagnosis of Infectious Hematopoietic Necrosis Virus in Cultured Atlantic Salmon in British Columbia

Infectious hematopoietic necrosis virus (IHNV) has been found to cause disease in cultured salmon of the Pacific Northwest region of North America. Diagnosis of IHNV by virus isolation (VI) can take over 2 weeks. Recently, a rapid reverse transcriptase (RT) polymerase chain reaction (PCR) test on fish tissues has been used for diagnosis. Test performances of the VI and RT PCR assays were compared using samples collected in the field. The effect of different storage conditions (tissue frozen with or without RNAlater [Ambion, Inc., Austin, Texas] versus fresh tissue) on the diagnostic tests was also evaluated. Based on the limited number of samples tested, the operating characteristics of RT PCR were very similar to those of VI; therefore, this method is likely suitable for testing field samples for IHNV. The ability of the tests to identify a positive fish ranged from 74% to 89%. Freezing samples at -80 degrees C before testing did not negatively affect the performance of RT PCR or VI. However, due to reduced test performance, RNAlater frozen storage is not recommended without further investigation.

Use of emergency departments and primary care visits for asthma related conditions in the 3 years following an asthma education program

Abstract Background: This study examines changes in Primary Care Visits (PCVs) and Emergency Department Visits (EDVs) among 1918 patients with asthma who attended either two visits, one visit or were no-show referrals at the Dr. Patrick Gill Asthma Education Center (AEC) in Charlottetown Prince Edward Island (PEI) between January 1, 2003 and March 31, 2008 compared to 2799 controls selected from a list of PEI asthma patients developed for the Canadian Chronic Disease Surveillance System (CCDSS). Methods: Hurdle regression was used to model counts of PCVs and negative binomial models were used to model counts of EDVs at 12 months prior to AEC contact and 0–1, >1 to 2 and >2 to 3 years after AEC contact. The PEI Research Board approved the project. Results: No-show referrals had a significant increase in pediatric EDVs and PCVs in the first year after referral. The higher rates of PCVs and EDVs prior to contact with the AEC in patients referred to the AEC were reduced after contact with the AEC, although they remained significantly higher than the CCDSS controls. Conclusions: Compared to patients who attended the AEC, referred patients who did not attend the AEC did not achieve similar reductions in pediatric EDVs and PCVs in the first year after referral.

Diagnostic validation of three test methods for detection of cyprinid herpesvirus 3 (CyHV-3)

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of koi herpesvirus disease in koi and common carp. The disease is notifiable to the World Organisation for Animal Health. Three tests-quantitative polymerase chain reaction (qPCR), conventional PCR (cPCR) and virus isolation by cell culture (VI)-were validated to assess their fitness as diagnostic tools for detection of CyHV-3. Test performance metrics of diagnostic accuracy were sensitivity (DSe) and specificity (DSp). Repeatability and reproducibility were measured to assess diagnostic precision. Estimates of test accuracy, in the absence of a gold standard reference test, were generated using latent class models. Test samples originated from wild common carp naturally exposed to CyHV-3 or domesticated koi either virus free or experimentally infected with the virus. Three laboratories in Canada participated in the precision study. Moderate to high repeatability (81 to 99%) and reproducibility (72 to 97%) were observed for the qPCR and cPCR tests. The lack of agreement observed between some of the PCR test pair results was attributed to cross-contamination of samples with CyHV-3 nucleic acid. Accuracy estimates for the PCR tests were 99% for DSe and 93% for DSp. Poor precision was observed for the VI test (4 to 95%). Accuracy estimates for VI/qPCR were 90% for DSe and 88% for DSp. Collectively, the results show that the CyHV-3 qPCR test is a suitable tool for surveillance, presumptive diagnosis and certification of individuals or populations as CyHV-3 free.

Performance characteristics of polymerase chain reaction and histological methods for the detection of Haplosporidium nelsoni in the eastern oyster (Crassostrea virginica).

Fitness for purpose and validation are increasingly becoming a benchmark in the development of test methods for the diagnosis of infectious diseases in aquatic animals. The design of the evaluation and the analysis of data are critical to demonstrate test method performance characteristics and fitness for purpose, as stated in the World Organization for Animal Health pathway for test validation. Three test methods for the detection of the oyster parasite Haplosporidium nelsoni were selected for the validation study described herein: histology, end-point polymerase chain reaction (PCR), and real-time PCR (qPCR). Preliminary work evaluated the analytical sensitivity and specificity of the PCR and qPCR assay in development. The following stage used test results on 100 oysters in 3 different laboratories to assess diagnostic sensitivity (DSe), diagnostic specificity (DSp), repeatability, and reproducibility. Repeatability and reproducibility were within 68–95%. The final part of the project evaluated DSe and DSp using test results on 400 oysters and results from the first 100 oysters tested. In the absence of a 100% gold standard test, latent class modeling methods were explored to characterize the tests (i.e., Bayesian analyses). For both PCR methods, DSe was >90%, and in the 60% range for histology, whereas DSp was >90% for all methods. Based on the results of this validation, a threshold cycle value of 30 for qPCR corresponds to the limit of sensitivity for histology where unreliable detection becomes more frequent, thus providing a threshold helpful in diagnostic settings where both histology and qPCR are used.

Evaluation of external operculum loop tags to individually identify cage-cultured Atlantic halibut Hippoglossus hippoglossus in commercial research trials.

The growth, survival and tag retention of double-tagged [external FT4 lock-on (FT4) and internal passive integrated transponder (PIT)-tagged] Atlantic halibut Hippoglossus hippoglossus were compared to internal PIT-tagged controls in a randomized trial. The objective was to assess the suitability of these tags for monitoring the performance of individual fish in longitudinal trials under commercial cage-culture conditions in the lower Bay of Fundy, New Brunswick, Canada. The FT4 tags were chosen due to their similarity to tags used by investigators to track H. hippoglossus in the wild. A subset of the population randomly received an external FT4 tag inserted through the operculum and were monitored over a 1105 day period. The specific growth rate of FT4-tagged fish was significantly reduced in the first sea summer with no significant difference observed for the remainder of the trial. The differential growth in the first sea summer created a relative size advantage, permitting controls to increase in size significantly faster than FT4 fish in all subsequent periods. The FT4 tags did not significantly influence survival under normal commercial cage-culture conditions. Results, however, suggest that the survival of FT4-tagged H. hippoglossus may be compromised during stressful handling events. Tag retention of FT4 tags was acceptable with 76% of tags remaining at the end of the 1105 day trial. FT4 tags proved to be an effective method to identify individual H. hippoglossus, with the caveat that they seriously bias productivity measures in commercial research trials.