Evaluation of histopathology, PCR, and qPCR to detect Mikrocytos mackini in oysters Crassostrea gigas using Bayesian latent class analysis.

Latent class analysis (LCA) is a common method to evaluate the diagnostic sensitivity (DSe) and specificity (DSp) for pathogen detection assays in the absence of a perfect reference standard. Here we used LCA to evaluate the diagnostic accuracy of 3 tests for the detection of Mikrocytos mackini in Pacific oysters Crassostrea gigas: conventional polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), and histopathology. A total of 802 Pacific oysters collected over 12 sampling events from 9 locations were assessed. Preliminary investigations indicated that standard LCA assumptions of test independence and constant detection accuracy across locations were likely unrealistic. This was mitigated by restructuring the LCA in a Bayesian framework to include test-derived knowledge about pathogen prevalence and load for categorizing populations into 2 classes of infection severity (low or high) and assessing separate DSe and DSp estimates for each class. Median DSp estimates were high (>96%) for all 3 tests in both population classes. DSe estimates varied between tests and population classes but were consistently highest for qPCR (87-99%) and lowest for histopathology (21-51%). Acknowledging that detection of M. mackini may be fitted to multiple diagnostic and management purposes, qPCR had the highest DSe while maintaining similar DSp to both conventional PCR and histopathology and thus is generally well-suited to most applications.

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.

Molecular detection of Mikrocytos mackini in Pacific oysters using quantitative PCR.

Mikrocytos mackini is an internationally regulated pathogen and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Recent phylogenetic breakthroughs have placed this parasite within a highly divergent and globally distributed eukaryotic lineage that has been designated a new taxonomic order, Mikrocytida. The discovery of this new radiation of parasites is accompanied by a heightened awareness of the many knowledge gaps that exist with respect to the general biology, epizootiology, and potential impact of mikrocytid parasites on hosts, ecosystems, and commercial fisheries. It has also highlighted current shortcomings regarding our ability to detect these organisms. In this study, we developed a species-specific, sensitive, and quantitative method for detecting M. mackini DNA from host tissues using probe-based real-time qPCR technology. A limit of sensitivity between 2 and 5 genome copy equivalents was achieved in a reaction matrix containing ≥ 40 ng/µL host gDNA without inhibition. This detection proved superior to existing methods based on conventional PCR, histology or gross pathology and is the first species-specific diagnostic test for M. mackini. Quantitative assessment of parasite DNA using this assay remained accurate to between 10 and 50 copies identifying that during infection, M. mackini DNA was significantly more prevalent in hemolymph, labial palp, and mid-body cross-sections compared to mantle or adductor muscle. DNA extracted from a mid-body cross-section also provided the highest likelihood for detection during diagnostic screening of infected oysters. Taken together, these findings provide strong analytical evidence for the adoption of qPCR as the new reference standard for detecting M. mackini and give preliminary insight into the distribution of the parasite within host tissues. Standardised operating methodologies for sample collection and qPCR testing are provided to aid in the international regulatory diagnosis of M. mackini and serve as a useful platform for the future development of multiplexed or alternate mikrocytid species detection.

Molecular taxonomy of Mikrocytos boweri sp. nov. from Olympia oysters Ostrea lurida in British Columbia, Canada.

Mikrocytos mackini is a microcell parasite that usually infects Crassostrea gigas distributed along the Pacific Northwest coast of North America. For many years, M. mackini was the only known species in the genus, but there have been multiple recent findings of genetically divergent forms of Mikrocytos in different hosts and in distantly located geographic locations. This note describes M. boweri sp. nov. found in Olympia oysters Ostrea lurida collected from and native to British Columbia, Canada, primarily using a molecular taxonomic approach.

Rediscovery of the Yesso scallop pathogen Perkinsus qugwadi in Canada, and development of PCR tests.

Perkinsus qugwadi, a pathogenic protozoan parasite of Yesso scallops Patinopecten yessoensis, is found only in cultured populations in British Columbia, Canada. This pathogen was first identified in 1988 and caused significant mortalities at some locations during the early 1990s. Prevalence of infection decreased dramatically following 1995, and the disease was last reported in 1997, leading to speculation that the Yesso scallop stocks in Canada had developed resistance to the disease, or that P. qugwadi had disappeared. However, the present study revealed that infection with P. qugwadi and associated mortality is still occurring in scallops from at least one location in British Columbia. One of the PCR tests developed for P. qugwadi detected the parasite in a 105-fold dilution of DNA extracted from a heavily infected sample and detected 52% more positive scallops than histology; however, the assay also cross-reacted with P. honshuensis and P. olseni. The other PCR test was less sensitive and detected 34% more positives, but did not react to any of the other Perkinsus species tested, suggesting that these PCR tests are powerful tools for screening for the presence of P. qugwadi. Phylogenetic analysis of 1796 bp of SSU rRNA gene sequence clearly indicated that P. qugwadi is positioned basally to other Perkinsus species.

WITHDRAWN: Primary prevention for alcohol misuse in young people.

BACKGROUND: Alcohol misuse is a cause of concern for health services, policy makers, prevention workers, the criminal justice system, youth workers, teachers and parents. OBJECTIVES: 1. To identify and summarize rigorous evaluations of psychosocial and educational interventions aimed at the primary prevention of alcohol misuse by young people. 2. To assess the effectiveness of primary prevention interventions over the longer-term (> 3 years). SEARCH STRATEGY: Databases searched (no time limits): Project CORK, BIDS, PSYCLIT, ERIC, ASSIA, MEDLINE, FAMILY-RESOURCES-DATABASE, HEALTH-PERIODICALS-DATABASE, EMBASE, BIDS, Dissertation-Abstracts, SIGLE, DRUG-INFO, SOMED, Social-Work-Abstracts, National-Clearinghouse-on-Alcohol-and-Drug-Information, Mental-Health-Abstracts, DRUG-database, ETOH (all searched Feb-June 2002). SELECTION CRITERIA: 1. randomised controlled and non-randomised controlled and interrupted time series designs. 2. educational and psychosocial primary prevention interventions for young people up to 25 years old. 3. alcohol-specific or generic (drugs; lifestyle) interventions providing alcohol outcomes reported. 4. alcohol outcomes: alcohol use, age of alcohol initiation, drinking 5+ drinks on any one occasion, drunkeness, alcohol related violence, alcohol related crime, alcohol related risky behaviour. DATA COLLECTION AND ANALYSIS: Stage 1: All papers screened by one reviewer against inclusion criteria. Stage 2: For those papers that passed Stage 1, key information was extracted from each paper by 2-3 reviewers. MAIN RESULTS: 20 of the 56 studies included showed evidence of ineffectiveness. No firm conclusions about the effectiveness of prevention interventions in the short- and medium-term were possible. Over the longer-term, the Strengthening Families Program (SFP) showed promise as an effective prevention intervention. The Number Needed to Treat (NNT) for the SFP over 4 years for three alcohol initiation behaviours (alcohol use, alcohol use without permission and first drunkeness) was 9 (for all three behaviours). One study also highlighted the potential value of culturally focused skills training over the longer-term (NNT=17 over three-and-a-half years for 4+ drinks in the last week). AUTHORS' CONCLUSIONS: 1. Research into important outcome variables needs to be undertaken. 2. Methodology of evaluations needs to be improved. 3. The Strengthening Families Programme needs to be evaluated on a larger scale and in different settings. 4. Culturally-focused interventions require further development and rigorous evaluation. 5. An international register of alcohol and drug misuse prevention interventions should be established and criteria agreed for rating prevention intervention in terms of safety, efficacy and effectiveness.

Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location.

Mikrocytos mackini is a microcell parasite of Pacific oysters only known to occur on the Pacific coast of North America. It is the only described species in the genus, although a genetically divergent Mikrocytos sp. organism has been reported once in both the Atlantic Ocean and China. We developed methods for sequencing the internal transcribed spacer (ITS) of rDNA for the purpose of characterizing extant diversity within M. mackini throughout its known geographic range, and surveying for other evidence of Mikrocytos sp. organisms. Our specific aims were to examine relatedness of M. mackini among sites to make inferences about its recent evolutionary history, and to provide baseline data for future development of a species-specific molecular detection method. We found a total lack of genetic variation within M. mackini across the complete ITS1-5.8S-ITS2 array in over 70 samples collected throughout its range. We hypothesize that this could be a result of a founder effect if the parasite had been introduced into its known range alongside its host, which was imported from Asia beginning around 1914 to about 1961. We detected a single divergent sequence at a short stretch of 18S that was identical to the Mikrocytos sp. detected elsewhere, which adds to the recent and growing body of evidence that Mikrocytos is much more broadly distributed than the limited range of M. mackini suggests. A 1903 bp section of rDNA from Mikrocytos sp. was generated that contained regions of high divergence from M. mackini (in ITS1 and ITS2) that could be exploited for molecular diagnostics.

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini.

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n=40), all of the subsequent V. philippinarum (n=62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n=100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n=60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n=63) using this technique.

Poetry writing and secretory immunoglobulin A.

17 healthy students provided saliva samples for Immunoglobulin A (s-IgA) assay before and after sessions of either writing poetry or reading magazines (control). Levels of s-IgA increased after the poetry-writing sessions but not after reading.