Inter-Laboratory Ring Trial to Evaluate Reverse Transcription Polymerase Chain Reaction Methods Used for Dolphin Morbillivirus Detection in Italy

Dolphin Morbillivirus (DMV) is one of the most frequently detected pathogens in stranded cetacean specimens worldwide as well as in Italy. Due to the persistence of DMV in the Mediterranean Sea and to the lack of information about the efficiency of the available diagnostic techniques, the Italian National Reference Centre for diagnostic activities on dead stranded marine mamHow to cite this paper: Zoccola, R. , Pautasso, A. , Grattarola, C., Monnier, M. , Miceli, I., Ingravalle, F., Crescio, I., Giorda F., Masoero L., Zaccaria, G., Purpari, G., Cersini, A., Viscardi, M., Serracca, L., Padalino, I., Puggioni, G., Canonico, C., Toffan, A., Centelleghe, C., Di Francesco, C.E., Mazzariol, S., Di Guardo, G., Mignone, W., Casalone, C.


Introduction
Dolphin Morbillivirus (DMV), a single negative stranded RNA virus within the genus Morbillivirus, subfamily Paramyxovirinae, family Paramyxoviridae [1], is included in the cluster of Cetacean Morbillivirus (CeMV) [2]. DMV infection, in a similar manner to many other Morbillivirus genus members, affects mainly the upper respiratory tract as well as the central nervous system and the immune system of marine mammals [3] [4], having been associated with high mortality rates and stranding of cetaceans in different regions of the world [5]. In the Mediterranean Sea, two well documented DMV outbreaks occurred between 1990 and 1992 [6] as well as between 2006 and 2008 [7]. Moreover, DMV was deemed as the most likely cause of three cetacean unusual mortality events (UMEs) oc- curred since 2011 along Spanish [8] and Italian coastlines [9] [10]. Viral isolation and subsequent PCR identification are usually considered the "gold standard" for the definitive diagnosis of morbilliviral infections [4]. Nevertheless, this is often a challenging issue given the poor preservation of virus-targeted tissues when dealing with stranded cetacean carcasses; therefore, direct detection in tissues by means of reverse transcription-PCR (RT-PCR) followed by sequencing represents the first rapid, sensitive and specific tool for DMV detection. In the last few years, many conventional and Real-time RT-PCR methods have been developed to detect the presence of DMV worldwide [11] [12] [13].
In Italy, the National Reference Centre for diagnostic activities on dead stranded marine mammals (C.Re.Di.Ma) coordinates post mortem investigations on stranded cetaceans along the entire Italian peninsula, promoting the application of standardized guidelines regarding sampling and diagnostic techniques and the sharing of diagnostic results. In regard to direct DMV detection in tissues, C.Re.Di.Ma developed an RT-PCR restriction fragment length polymorphism (RFLP) technique, based upon the use of an RT-PCR with degenerate primers targeting a 287 bp fragment of the nucleoprotein (N) gene, followed by MseI RFLP analysis [14]. This work is aimed at assessing the performances of different diagnostic methods routinely applied in Italy for DMV infection's diagnosis. In this respect, identical panels of ad hoc samples were analyzed by the whole Italian dead stranded marine mammals' diagnostic network. More in detail, the accuracy (Se = sensitivity and Sp = specificity) and precision (reproducibility) of the method described by Verna et al. 2017 [14] were verified and, at the same time, they were also compared with the accuracy of other 7 biomolecular techniques routinely applied for DMV detection in Italy [11] [12] [15]- [20].

Material and Methods
Twelve Public Diagnostic Laboratories belonging to the Italian diagnostic network on stranded cetaceans, listed in Table 1, took part in the ring trial. They constantly remained anonymous, thereby respecting privacy requirements and were named as L1 to L12. All the participating Laboratories were asked to apply the method proposed by C.Re.Di.Ma [14] on the first panel of samples. That method [14] was developed to detect a 287 bp sequence of a highly conserved region of Canine Distemper Virus (CDV) nucleoprotein (NP). Considering the relevant polymorphism of the target region, primers developed by Frisk et al.. 1999 [16] were redesigned through an in silico analysis. C.Re.Di.Ma, as the ring trial organizer, and in order to ensure the most consistent homogeneity of results, provided all of the participating Laboratories with detailed instructions and reagents to carry out the protocol described by Verna [20] whose essential details are reported in Table 2.

Samples Preparation
Each laboratory was provided with a panel composed of 40 samples to be analyzed with the method proposed by C.Re.Di.Ma (Panel 1: Samples from 1 to 40). Each panel consisted of 27 positive samples and 13 negative ones. C.Re.Di.Ma expected that its own method was able to reach values of Se ≥ 80% and Sp ≥ 90%, therefore the number of positive and negative samples included in the study had to be sufficient to ensure that performance, with a limit lower than the 95% confidence interval for both indices was set at over 60%. Laboratories that routinely employed other detection methods were also provided with a second panel containing the same number of samples, with distinguishable header and numbering (Panel 2: Samples from 51 to 90). The positive samples consisted in viral suspensions at different concentrations obtained from a cell culture infected with a given DMV isolate [21], recognized as the "gold standard". (Gen-Bank Acc. No. MF589987). The virus was propagated on Vero/dogSLAM cell line [21] and after 6 days, when the cytopathic effect (CPE) affected 80% -90% of the cells, supernatants were collected and clarified by centrifugation at 3500 rpm.  Semi Nested RT-PCR [19] Gene P-300 bp Real time RT-PCR sybrgreen [16] Gene N-287 bp P-1 (CDV) 5'-ACAGGATTGCTGAGGACCTAT-3' P-2 (CDV) 5'-CAAGATAACCATGTACGGTGC-3' 9 Nested PCR [11] Gene H-612 bp-nested 200 bp in aliquots of 250 μl, identified individually with numerical code and stored at −80˚C.

Homogeneity and Stability Tests on Ring Trial Samples
Homogeneity in sample preparation was evaluated by analyzing with the method Verna et al. 2017 [14] five replicates for each level of positivity under the same conditions planned for the execution of ring trial analyses ( Figure 1). Extractions were carried out using the QIAamp viral RNA mini kit QIAGEN and retrotranscription and amplification kit OneStep RT-PCR Qiagen. Evaluation of homogeneity was performed at the end of the preparation of all samples and prior to their shipment to all laboratories involved in the ring trial. Five aliquots for each virus concentration were randomly selected from every group of samples ready for distribution. In order to eliminate possible confounding factors, tests were carried out under strict standard conditions; therefore they were performed by the same operator and in the same analytical session. Acceptability criteria for homogeneity tests were set up at reproduction, on each selected aliquot, of the same amplicon yield, corresponding to their respective reference samples for each viral dilution, including negative ones. With reference to stability tests, aliquots for each level of virus concentration were used to perform two different trials. In order to simulate the effect of potential temperature failure  occurrences during shipment on virus samples, a stability test was designed to evaluate the resistance of samples to stress temperature for each level of positivity, including the negative ones and it was carried out before the shipment. Analyses were performed on three replicates for each virus concentration maintained at room temperature at: t 0 = corresponding to homogeneity test day; t 1 = day1; t 2 = day2; t 3 = day3 (Figure 2). A second stability test was carried out to verify the sample stability at the storage conditions (−80˚C), during the entire period of execution of the ring trial. In this case, three replicates for every single virus concentration (including negative controls) were analyzed, every 15 days, from the day of shipment until the defined closing time for results submission. Acceptability criteria for stability tests were also set up at reproduction, during all ring-testing period under all storage and stress conditions, on each selected aliquot, of the same amplicon yield, corresponding to their respective reference samples for each viral dilution, including negative ones.

Statistical Analysis
Performances of each laboratory in the application of the method described by Verna et al. were evaluated in terms of both accuracy and precision (reproducibility). Conversely, for each of the other 7 methods routinely applied throughout the Country, accuracy was evaluated. In order to assess the accuracy, Sensitivity (Se) and Specificity (Sp), positive (PPV) and negative (NPV) predictive values were estimated, as well as their 95% confidence intervals (95% CI). To assess precision Cohen's kappa was pairwise estimated for each participant towards everyone, as well as for each participant with respect to the majority judgment (MJ, i.e. the outcome given by most of participants for each sample examined).  Moreover, the k-combined was calculated [22] to evaluate the agreement between all the participants in the trial. Statistical analysis was carried out using the software STATA 15 software (StataCorp, College Station, Texas, USA).

Samples Homogeneity and Stability
Outcomes satisfied the acceptability requirements, thereby confirming expectations, as summarized in Figure 1 and Figure 2. All dilution samples gave back the expected results. Pictures show: in Figure  1(a) five replicates of negative samples (lanes 1 to 5); five replicates of DMV at 1:500 dilution (lanes 6 to 10); five replicates of DMV at 1:100 dilution (lanes 11 to 15); in Figure 1 For stability test at storage conditions, replicates of each viral dilution, stored at −80˚C, were examined after 0, 15 and 30 days to cover all testing period until the deadline for results' submission. Presence of viral RNA was confirmed in all replicates containing DMV at each time interval.

PCR Results
This study allowed us to verify the performances of most of molecular detection  Figure 3 and

Discussion
As previously stated, DMV represents one of the most relevant threats to free-ranging cetaceans. Different RT-PCR assays have been used for DMV and,  more in general, for CeMV detection from stranded cetaceans worldwide.
Within such context, the consistent advances in molecular biology have allowed a progressively faster, easier and more reliable development of biomolecular protocols for DMV infection's laboratory diagnosis [4]. The main goal of the herein reported interlaboratory ring trial was to assess the diagnostic performances of molecular assays to detect DMV used by 12  preservation conditions. In this respect, the good stability of viral genomes, observed under unsuitable storage conditions, leads us to suppose that the "morbillivirus-related samples" preservation degree may not be "dramatically" affected during transfer between different laboratories for confirmatory analyses and comparison of results.

Conclusion
Based upon the results of the herein described ring trial, we can conclude that most of the biomolecular techniques used in our Country for DMV infection's diagnosis show a satisfactory reliability and reproducibility, while an adequate level of expertise appears to be also present in all of the participating Laboratories regarding the application of the method by Verna et al. 2017, which could be recommended as a reliable RT-PCR protocol to be used for the routine laboratory diagnosis of DMV infection in Italy.