Charles Caraguel

Selection of a cutoff value for real-time polymerase chain reaction results to fit a diagnostic purpose: analytical and epidemiologic approaches

Diagnostic laboratories frequently select a subjective cutoff value for real-time amplification assays, above which a threshold cycle (Ct) value is deemed false. Commonly, higher Ct values are interpreted as amplification or fluorescence artifacts, or cross contaminations. Although the implementation of Ct cutoff might be reasonable, its justification and selection should be based on evidence. The current article reviewed evidence-based strategies to select Ct cutoffs grouped in analytical and epidemiologic approaches. Analytical strategies use criteria gathered during the assay development and include fluorescence threshold, reaction end-cycle, limit of detection, and artifact investigation. Variability in amplification efficacy across test runs may induce some instability in an intended Ct cutoff and requires some standardization or normalization procedures. Epidemiologic strategies use criteria based on either the probability or the cost of a false test result associated with a specified cutoff. Cutoffs, depending on the intended purpose of the test, can be selected graphically to minimize the probability of either false-positive or false-negative results by using two-graph receiver operating characteristics curves. The assays diagnostic sensitivity and specificity may vary with the tested population, thus, the estimated two-graph receiver operating characteristics curve is population dependent and should be established for the targeted population. Although the selection of a cutoff based on misclassification cost depends on infection prevalence, the selection based on predictive values does not. To optimize the test average diagnostic performance, the Ct cutoff should be selected when diagnostic odds ratio is maximal. Epidemiologic approaches were illustrated by selecting Ct cutoffs for a real-time assay for Infectious salmon anemia virus.

The two-step Fagan's nomogram: ad hoc interpretation of a diagnostic test result without calculation

In 1975, Fagan published a nomogram to help practitioners determine, without the use of a calculator or computer, the probability of a patient truly having a condition of interest given a particular test result. Nomograms are very useful for bedside interpretations of test results, as no test is perfect. However, the practicality of Fagans nomogram is limited by its use of the likelihood ratio (LR), a parameter not commonly reported in the evaluation studies of diagnostic tests. The LR reflects the direction and strength of evidence provided by a test result and can be computed from the conventional diagnostic sensitivity (DSe) and specificity (DSp) of the test. This initial computation is absent in Fagans nomogram, making it impractical for routine use. We have seamlessly integrated the initial step to compute the LR and the resulting two-step nomogram allows the user to quickly interpret the outcome of a test. With the addition of the DSe and DSp, the nomogram, for the purposes of interpreting a dichotomous test result, is now complete. This tool is more accessible and flexible than the original, which will facilitate its use in routine evidence-based practice. The nomogram can be downloaded at: www.adelaide.edu.au/vetsci/research/pub_pop/2step-nomogram/.

Association between Indoor Environmental Contamination by Salmonella enterica and Contamination of Eggs on Layer Farms

ABSTRACT This study involves longitudinal and point-in-time surveys of Salmonella carriage and environmental contamination on two commercial cage layer farms positive for Salmonella enterica subsp. enterica serovar Typhimurium (flock A age, 32 weeks; flock B age, 34 weeks). Salmonella-positive fecal, egg belt, and dust samples were all unconditionally associated with eggshells testing positive for Salmonella. The odds of an eggshell testing positive for Salmonella were 91.8, 61.5, and 18.2 times higher when fecal, egg belt, and dust samples, respectively, tested positive for Salmonella. The agreement between the culture-based methods and real-time PCR on preenriched broths for detecting Salmonella was almost perfect for eggshell (observed agreement, 99.19%; kappa coefficient, 0.94) and egg belt samples (observed agreement, 95%; kappa coefficient, 0.88), and it was substantial for fecal (observed agreement, 87.14%; kappa coefficient, 0.47) and floor dust samples (observed agreement, 80.61%; kappa coefficient, 0.58). A 1-log increase in the load of Salmonella detected in the fecal, egg belt, and floor dust samples resulted in 35%, 43%, and 45% increases, respectively (P < 0.001), in the odds of an eggshell testing positive for Salmonella. The multilocus variable-number tandem-repeat analysis (MLVA) patterns of the S. Typhimurium strains isolated from flock A were distinct from those of flock B. S. Typhimurium strains detected from human food poisoning cases exhibited an MLVA pattern similar to those of the strains isolated from flocks A and B.

Shedding of Salmonella in single age caged commercial layer flock at an early stage of lay

The shedding of Salmonella in a single age commercial egg layer flock was investigated at the onset of lay (18weeks) followed by two longitudinal samplings at 24 and 30weeks. At the age of 18weeks, when the first sampling was performed, the prevalence of Salmonella in faeces was 82.14% whereas all egg belt and dust samples were Salmonella positive by culture method. In later samplings, at the age of 24 and 30weeks, the prevalence of Salmonella in faeces was significantly reduced (p<0.001) to 38.88% and 12.95% respectively, however all egg belt and dust samples remained positive by culture method. The prevalence of Salmonella in faeces collected from the low tier cages was significantly higher (p=0.009) as compared with samples from the high tier cages. In all types of samples processed by culture method, S. Mbandaka was the most frequently (54.40%) isolated serovar followed by S. Worthington (37.60%), S. Anatum (0.8%), and S. Infantis (0.8%). All samples were also tested by real-time PCR method. The observed agreement between culture method and real-time PCR in detecting Salmonella-positive dust and egg belt samples was 100%. There was almost perfect agreement (observed agreement=99.21%) for the detection of Salmonella-positive eggshells. Observed agreement between culture method and real-time PCR for detecting Salmonella-positive shoe cover and faecal samples was, however, moderate (80%) and low (54.27%) respectively. Real-time PCR results showed that there was a significant increase in the load of Salmonella on egg belt, dust and shoe cover samples at the 24 and 30weeks of lay as compared to the 18weeks of lay. Real-time PCR provided a more rapid and reliable method of detection of Salmonella on all dry sample types whereas the traditional culture method proved much more reliable when trying to detect Salmonella in wet faecal samples.

Microheterogeneity and Coevolution: An Examination of rDNA Sequence Characteristics in Neoparamoeba pemaquidensis and Its Prokinetoplastid Endosymbiont

ABSTRACT. Neoparamoeba pemaquidensis, the etiological agent of amoebic gill disease, has shown surprising sequence variability among different copies of the 18S ribosomal RNA gene within an isolate. This intra‐genomic microheterogeneity was confirmed and extended to an analysis of the internal transcribed spacer (ITS) region. High levels of intra‐genomic nucleotide diversity (Pi=0.0201–0.0313) were found among sequenced ITS regions from individual host amoeba isolates. In contrast, the ITS region of its endosymbiont revealed significantly lower levels of intra‐genomic nucleotide diversity (Pi=0.0028–0.0056) compared with the host N. pemaquidensis. Phylogenetic and ParaFit coevolution analyses involving N. pemaquidensis isolates and their respective endosymbionts confirmed a significant coevolutionary relationship between the two protists. The observation of non‐shared microheterogeneity and coevolution emphasizes the complexity of the interactions between N. pemaquidensis and its obligate endosymbiont.

The Canadian harp seal hunt: observations on the effectiveness of procedures to avoid poor animal welfare outcomes

The Canadian harp seal (Pagophilus groenlandicus) hunt has, for several decades, raised public concerns related to animal welfare. The field conditions under which this hunt is carried out do not lend themselves easily to detailed observations and analyses of its killing practices. This article reports observations carried out over several seasons that aimed at obtaining more specific information about the conditions under which seals are killed, in order to assess potential welfare issues and explore avenues for possible improvements in its practice. A standardised three-step process for killing seals (ie stunning, checking by palpation of the skull, and bleeding) was recently implemented to maximise the proportion of animals that are killed rapidly with minimum pain. Based on field observations, the rifle and the hakapik, when used properly, appeared to be efficient tools for stunning and/or killing young harp seals. All carcases of seals observed to be killed with a rifle, either on the ice or in the water, could be recovered. However, shooting seals in water rather than on ice carried a higher risk of poor welfare outcome because of the limited opportunities to shoot the animals again if not stunned with the first shot. Based on current practices, there is no reliable evidence that the Canadian harp seal hunt differs from other forms of exploitation of wildlife resources from the perspective of animal welfare. Although opportunistic field observations may be less amenable to generalisation than structured studies, we believe that they reflect the reality of the hunt and provide valuable information to direct the evolution of its practice.

Use of a third class in latent class modelling for the diagnostic evaluation of five infectious salmon anaemia virus detection tests

In the absence of a reference standard, a latent class model (LCM) was used in this study to assess diagnostic sensitivity (DSe) and specificity (DSp) of a recently developed reverse-transcription polymerase chain reaction (RT-PCR) for infectious salmon anaemia virus (ISAV). The study included 4 populations of Atlantic salmon, and to ensure the identifiability of the LCM, four additional detection methods were used in parallel including real-time RT-PCR (qRT-PCR), virus isolation (VI), indirect fluorescent antibody test (IFAT), and a lateral flow immunoassay (LFI). While a conventional LCM assumes DSe and DSp to be constant across the populations, Nérette et al. (2008) previously reported concerns about non-constant DSp of RT-PCR, which detects viral RNA from both active and inactive viral particles. It was suspected that some ISAV recovered fish may carry residual RNA and may be more likely to test positive compared to naïve fish. The various mixture distributions of the two sub-classes of non-infected fish would lead to a non-constant combined DSp estimate across populations. Within a Bayesian framework, the conventional two-class LCM was extended to three classes of infection stages (naïve non-infected, recovered non-infected carrying RNA, and infected). The resulting analysis confirmed the existence of three classes of fish with substantially different test performances for ISAV. For infected fish, DSe of RT-PCRs and VI approximated 90%, and antibody based assays were the least sensitive (DSe around 65%). Regardless of the test, the DSp estimates on naïve fish were all above 98% with LFI being in average the most specific. Only RT-PCR and qRT-PCR tested positive with the additional class of recovered fish (DSp around 30%). The true infectious status of this sub-class (i.e. viral RNA carriers) is debatable and requires further knowledge about ISAV infection dynamics at the fish level. Promising applications of multiple class estimates require adjustments of traditional test interpretation and further epidemiological knowledge of the infection dynamics at the population level.

Traditional descriptive analysis and novel visual representation of diagnostic repeatability and reproducibility: application to an infectious salmon anaemia virus RT-PCR assay.

As a component of diagnostic test evaluation, the estimation of repeatability and reproducibility of an assay is necessary to assess the robustness and the transferability of the method among laboratories. Respectively defined as the agreement within and between laboratories, repeatability and reproducibility of a qualitative diagnostic test are traditionally reported using observed proportion of agreement or Kappa values. Applied to a recently designed RT-PCR assay for the detection of infectious salmon anaemia virus, repeatability only within a national reference laboratory and reproducibility with two additional independent regional laboratories were investigated. Homogenization of fish kidney tissue was conducted to potentially provide more uniform submission material, and to assess the effect of homogenization on laboratory comparability. Comparison of agreement between non-homogenized and homogenized tissue samples revealed different patterns of test results and unexpected alterations of agreement due to homogenization. This observation may be explained by cross-contamination of some samples during the homogenization process. One of the laboratories was in clear disagreement with the two others and impacted the overall reproducibility of the assay. Agreement levels were visually described using a novel tree-shape representation inspired from phylogenetic studies. The resulting phylogram illustrated the proximity of test findings between repeated samples within a laboratory and between laboratories, and facilitated the interpretation of the agreement levels.

Chasing Salmonella Typhimurium in free range egg production system.

Free range production systems are becoming a major source of egg production in Australia and worldwide. This study investigated shedding and ecology of Salmonella Typhimurium and Salmonella species in a free range layer flock, wild birds and foxes in the vicinity of the free range farm in different seasons. Shedding of Salmonella was significantly higher in summer. Within the shed, overall, Salmonella prevalence was highest in dust. Corticosterone level in faeces was highest in spring and lowest in winter. There was no direct association between the Salmonella shedding (MPN/gm) and corticosterone levels in faeces. Salmonella Typhimurium MLVA types isolated from fox and wild birds were similar to MLVA types isolated from layer flock and reported during human food borne illness. Wild birds and foxes appear to play an important role in S. Typhimurium ecology and food safety. Environmental factors could play a role in evolution of S. Typhimurium in free range environment.

Recommended reporting standards for test accuracy studies of infectious diseases of finfish, amphibians, molluscs and crustaceans: the STRADAS-aquatic checklist.

Complete and transparent reporting of key elements of diagnostic accuracy studies for infectious diseases in cultured and wild aquatic animals benefits end-users of these tests, enabling the rational design of surveillance programs, the assessment of test results from clinical cases and comparisons of diagnostic test performance. Based on deficiencies in the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines identified in a prior finfish study (Gardner et al. 2014), we adapted the Standards for Reporting of Animal Diagnostic Accuracy Studies-paratuberculosis (STRADAS-paraTB) checklist of 25 reporting items to increase their relevance to finfish, amphibians, molluscs, and crustaceans and provided examples and explanations for each item. The checklist, known as STRADAS-aquatic, was developed and refined by an expert group of 14 transdisciplinary scientists with experience in test evaluation studies using field and experimental samples, in operation of reference laboratories for aquatic animal pathogens, and in development of international aquatic animal health policy. The main changes to the STRADAS-paraTB checklist were to nomenclature related to the species, the addition of guidelines for experimental challenge studies, and the designation of some items as relevant only to experimental studies and ante-mortem tests. We believe that adoption of these guidelines will improve reporting of primary studies of test accuracy for aquatic animal diseases and facilitate assessment of their fitness-for-purpose. Given the importance of diagnostic tests to underpin the Sanitary and Phytosanitary agreement of the World Trade Organization, the principles outlined in this paper should be applied to other World Organisation for Animal Health (OIE)-relevant species.