Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3)

Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/μl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity.


Introduction
In the past few decades, aquaculture has evolved as an important business segment worldwide. No global food production sector is growing faster. In 2016, over 80 million tons of food fish have been produced with a value of 231.6 billion US Dollar [1]. Among the finfish, the common carp (Cyprinus carpio carpio) accounted for the third most produced species worldwide [1]. Koi carp (Cyprinus carpio var. koi) the ornamental variant of common carp has a high breeding and economic value and belongs to the most popular ornamental pond fish worldwide [2].
Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass death in captive and free-ranging common carp and koi carp [3,4]. The causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), genus Cyprinivirus, Alloherpesviridae [5]. First descriptions of the disease originated from Israel, the USA, and Germany [6,7]. Clinical signs include gill necrosis, discolored patches on the skin, erratic swimming and enophthalmos [8]. Water temperatures between 22˚C and 26˚C favor KHVD outbreaks [9]. CyHV-3 causes latent infection which can be reactivated someday after the initial exposure. Healthy but latently infected carrier-fish constitute a considerable risk for the spread of CyHV-3 during trade and fish farming procedures [9][10][11][12][13]. KHVD outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain [9]. Because of its epizootic appearance, KHVD is listed as a notifiable disease in Germany [13,14] and by the World Organization for Animal Health (OIE) (https://www.oie. int/en/animal-health-in-the-world/oie-listed-diseases-2019/) [15].
A safe and effective vaccine is currently not widely available. Hence, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures [16]. An early diagnosis of the disease is crucial to prevent its spread and minimize economic losses. There are several methods for targeted surveillance and diagnosis of KHV but PCR is the recommended technique for reasons of availability, ease-of-use, diagnostic specificity and sensitivity [16]. A rapid and reliable pond-side test would shorten the time required for routine diagnostic. Lateral flow immunoassays (LFA) are widely used in human and veterinary medical diagnostic investigations [17] and can be used for primary screening at the point of care (POC) need.
The aim of this study was to develop and evaluate an easy-to-handle, sensitive, specific and reliable test prototype for a POC detection of KHV. The overall assay performance should be comparable to approved reference methods. Therefore, we chose a combination of DNAamplification via multiplex-endpoint-PCR followed by a specific probe hybridization step, then visualized with a simple and universal lateral flow immunoassay (PCR-LFA).
For PCR-LFA, three different ready-to-use solutions (A, B and H) were developed. Solution A (Milenia Biotec GmbH) consisted of two KHV-specific primers (KHV 86 F, KHV 163R FITC) and a "carp-independent" internal amplification control (IAC). The latter was composed of digoxigenin (DIG)-and fluorescein isothiocyanate (FITC)-labeled primers and the IAC template (Milenia Biotec GmbH, Gießen, Germany). It was highly compatible to the KHV-PCR of Gilas et al. (2004) [18] without decreasing assay sensitivity (data not shown).
Solution B (Milenia Biotec GmbH) contained 2x polymerase-mix with hot-start DNA-polymerase, dNTP's, and PCR-buffer. Solution H was used for post-PCR hybridization. It consisted of 2.9x saline-sodium citrate (SSC) buffer, 18.2 nM biotinylated KHV probe (KHV 109P (rc) BIO), and 181.8 nM KHV-Helper 2. PCR was carried out in a 25 μl-mixture of Solution A (7.5 μl), Solution B (12.5 μl), and 5 μl template in a Sensoquest Labcycler 48s (Sensoquest GmbH, Göttingen, Germany) under the following conditions: initial denaturation for 240 seconds at 95˚C followed by 40 cycles of 15 seconds at 95˚C, 20 seconds at 60˚C, 10 seconds at 72˚C. The PCR protocol was stopped at 8˚C after a final elongation of 10 seconds at 72˚C. Subsequently, 55 μl of Solution H were added to each PCR-tube, gently mixed, and the hybridization step was started (60 seconds of denaturation and 5 min of incubation at 50˚C). Thereafter, a LFA-dipstick (HybriDetect 2T, Milenia Biotec GmbH) was placed in each PCR-tube whilst maintaining cycler temperature at 50˚C for 10 min. Dipsticks were removed after 10 min of incubation and results documented photographically. Results were evaluated manually by naked eye. The HybriDetect 2T dipstick is a universal lateral flow dipstick for simultaneous detection of two different analytes. Each dipstick comprised three lines: immunoassay control line (C-line), IAC-test line, and KHV-test line. Appearance of the C-line indicated functionality of the LFA. Appropriate DNA amplification was confirmed by the IAC-test line. This feature is crucial for discrimination between a valid negative result and an inhibited PCR. The KHV-test line indicated the presence of a KHV-specific PCR product. The detection principle of the PCR-LFA is given in Fig 1. Viruses KHV isolates 'Israel' (HP 951) and 'Taiwan 832' were grown in common carp brain cells (CCLV-RIE 816) as described elsewhere [19,20]. Spring viremia of carp virus (SVCV; isolate RC 56/70, RVB-405), pike fry sprivivirus (PFRV; isolate PFR42, RVB-0431), and viral hemorrhagic septicemia virus (VHSV; isolate Fi13, RVB-0417) were grown in fat head minnow caudal trunk cells (FMH; CCLV-RIE 57) maintained in Minimal Essential Medium Hanks' Balanced Salts (MEM HBSS; Life Technologies GmbH, Darmstadt, Germany) adjusted to 850 mg/l NaHCO 3 (Life Technologies GmbH), 10% fetal bovine serum (FBS; Sigma Aldrich Chemie GmbH, Schnelldorf, Germany) without CO 2 at 20˚C. Fat head minnow cells (EPC; CCLV-RIE 173) grown in a 1:1-mixture of MEM HBSS and MEM Earle's BSS (Life Technologies GmbH) containing 1.5 g/l NaHCO 3 , 1x non-essential amino acids (Life Technologies GmbH), 120 mg/l sodium pyruvate (Life Technologies GmbH), and 10% FBS (Sigma Aldrich Chemie GmbH) were used to culture infectious haematopoietic necrosis virus (IHNV; isolate DF 11/95, RVB-0086) at 20˚C without CO 2 . Cells and viruses were obtained from the Collection of Cell Lines in Veterinary Medicine, Friedrich-Löffler-Institute, Greifswald-Insel Riems,

Carp tissue
Gill and kidney samples were obtained from KHV-negative and experimentally KHV-infected common carp (K2). All carp were part of another study which was approved by the Animal Welfare Officer of the Veterinary Faculty at Leipzig University and it was performed with permission of the Landesdirektion Sachsen (TVV A09/13, TVV A09/14). Experimental infection was carried out using KHV isolate 'Israel'. The study was published in 2016 elsewhere [19].

Reproducibility (inter-assay variance)
In order to test for variation in results between runs [23] the samples used for intra-assay variance were tested as single-preparations on two consecutive days.

Diagnostic specificity and sensitivity
Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA (S1 Table). Samples were tested previously with the qPCR using the protocol published by Gaede  In addition, the usability of the PCR-LFA for the detection of CyHV-3-DNA in gill swabs taken from 20 deceased common carp during a KHVD-outbreak in a commercial carp stock was examined. Dry cotton swabs were used for sampling. Swabs (S1 Table) were transported cooled (+4˚C) to the laboratory and PCR-LFA was performed at the same day. Each swab was placed in 250 μl sterile phosphate-buffered saline (pH 7.4) for 10 min, rinsed, and the eluate was submitted to PCR-LFA without further processing.
All PCR-LFA results were compared to the gold standard reference qPCR [18,20]. Specificity, sensitivity, positive predictive value (probability that a test-positive fish is actually positive), and negative predictive value (probability that a test-negative fish is a true-negative) of the PCR-LFA were calculated according to Thrusfield (2018) [24].

PCR-LFA development PCR adaption and cross primer-dimer analysis.
In order to prevent any nonspecific interaction between primers and probes, every labeled component of the detection system was tested. Approximately seven pmol of each labeled oligonucleotide were co-incubated under hybridization conditions and analyzed by LFA. No cross primer or probe interaction was observed for all the combinations tested (Fig 2). This confirmed compatibility of the primers and probes developed by Gilad et al. (2004) [18] with the PCR-LFA test principle.
Optimization of hybridization conditions. Physicochemical optimization of hybridization parameters was carried out, i.e. adjustment of ionic strength, time for denaturation, hybridization temperature and duration of the hybridization reaction (data not shown). The addition of helper oligonucleotides to the hybridization solution considerably increased KHVspecific signal intensity on the dipstick (Fig 3). These helpers were designed to match the FITC labeled DNA-strand in regions adjacent to the probe binding site to minimize potential secondary structures of the 78 bp target strand. The usage of the KHV-Helper 2, which was complementary to the 5' end of the FITC labeled strand, led to at least 200% more intense KHVspecific signals on the test strip. The effect of unlabeled helpers is not a new phenomenon and has been described for rRNA based hybridization techniques elsewhere [25][26][27].
Optimal hybridization conditions were defined as follows: 55 μl of solution H (2.9x SSCsolution containing 10 pmol helper and 1 pmol KHV 109P(rc) Bio per hybridization reaction) were added to 25 μl of PCR amplicons (i.e. final SSC concentration 2x). Denaturation was carried out at 50˚C for 1 min followed by 5 min of hybridization at 50˚C in the PCR cycler. Thereafter, dipsticks were placed into the PCR tubes and LFA was carried out in the thermal cycler while constantly heating the mix at 50˚C. Representative dipsticks showing signal intensity during optimization can be seen in Fig 3A. Signal intensities were additionally quantified via ImageJ (https://imagej.nih.gov/ij/) and results are displayed in Fig 3B. Comprehensive results are given in S1 Fig. Development of a PCR-LFA protocol. A well working PCR-LFA protocol was developed for screening at the POC need. The protocol was based on the HybriDetect 2T (Milenia Biotech GmbH), which is a ready-to-use, simple and universal lateral flow device to develop quick test methods. Its test principle is based on lateral flow technology using gold particles. The developed reagents were ready-to-use and therefore easy to handle which constitutes a prerequisite for POC analyses. Hereby, the test became less laborious and less prone to handling errors. The simple 3-step PCR protocol ensures assay transfer to various benchtop or portable PCR machines. The PCR-LFA protocol allowed a sensitive detection of KHV-DNA in approximately 1 h analysis time with a hands-on time less than 10 min (without sample preparation). Preliminary experiments showed a satisfying performance in terms of sensitivity and specificity. The overall functionality of the KHV PCR-LFA protocol is presented in Fig 4.

PCR-LFA validation
Analytical sensitivity of PCR-LFA. The concentrations of the plasmid standard cTL-21 and the results obtained for analytical sensitivity are given in Table 2. Based on the number of positive tests per total number of tests (i.e. n = 5) the LOD was calculated. The LOD (95% probability) determined for the PCR-LFA was 9 copies/μl (Fig 5), i.e. 45 copies/5 μl.
Analytical specificity of PCR-LFA. No false positive signals were observed with any of the virus-cell culture samples, viral, bacterial, and fish DNA or the alga (Fig 6). Hence, test strips only show the IAC-line and the C-line indicating a valid test performance. This was also seen for the no template control. In addition to these lines, the KHV-specific test line was present in the KHV positive-control.
Intra-and inter-assay variability. There was no variation seen in PCR-LFA results obtained for positive and negative samples within and between runs. All KHV-positive and -negative samples revealed either positive or negative results when tested in triplicate within the same PCR-LFA run (intra-assay variability) as well as when tested in PCR-LFAs at two consecutive days (inter-assay variability). Results are given in Table 3.
Diagnostic sensitivity and specificity. Diagnostic sensitivity and specificity were tested using DNA from a total of 100 tissue samples from common carp with known disease status: i.e. 43 KHV-negative samples from KHV-negative carp and 57 KHV-positive samples from experimentally CyHV-3-infected carp. In addition, 20 gill swab samples from deceased common carp of a confirmed KHVD outbreak were tested. All samples were examined in a single test run. PCR-LFA accurately detected no KHV-DNA in samples from KHV-negative carp assuming a diagnostic specificity of 100%. Of 77 KHV-positive samples, 55/57 tissue specimen and 18/20 gill swab eluates were precisely identified as positive (diagnostic sensitivity = 94.81%).  The two false-negative tissue samples (gill UI-55, kidney UI-55; S2 Table) were examined again each in three replicates. The gill sample was then determined as positive in 1 out of 3 replicates whereas the kidney sample remained negative in all three replicates. When tested in five replicates, the latter reacted positive in 1 out of 5 replicates. Of the two gill swabs (no. 10 and no. 11) negative by PCR-LFA, swab eluate no. 10 remained negative after repeated testing. The eluate of swab no. 11 completely inhibited the multiplex-endpoint-PCR which became obvious by a missing IAC-line. The IAC was also affected by another five swab eluates but the PCR-LFA revealed a KHV-positive result. When eluates were tested at a dilution of 1:10 or using extracted DNA, inhibition disappeared and the 20 samples were accurately identified as

Discussion
Sensitive and rapid POC diagnostic tests are of crucial importance for managing and mitigating the spread of infectious diseases. The latter is of paramount importance for global food security [28]. Since its first description in the late 1990s KHVD rapidly spread worldwide. Diagnosis of KHVD in clinically affected fish can currently be achieved by several methods, but some of them are not reliable (e.g. virus isolation in cell culture) or not validated (e.g. immunodiagnostic methods) [16]. Most commonly, PCR-based assays specific for KHV are used for detection of KHV in fish tissues [16]. They require sophisticated equipment and it will take a few days until results are available. There is a rapid (15 min) pondside test available for the detection of KHV-antigen in swab samples from gill tissues (FASTest 1 KOI HV, Megacor Diagnostik GmbH, Hörbranz, Austria) which is based on an immunochromatographic "sandwich principle". Its specificity and sensitivity are given as 100% and 71.7% (compared to the PCR according to Bercovier et al., 2005 [29]), respectively. The detection limit is given as 10 4 −10 6 KHV/ml (Megacor Diagnostik GmbH). The PCR-LFA used in the study at hand was less rapid (approximately 60 min) but with identical specificity, a much lower detection limit (i.e. 9 gene copies/μl), and a higher analytical sensitivity. Although the reference qPCR [20] had a much higher sensitivity (2 copies/μl), most of the KHV-positive samples were also correctly identified by PCR-LFA. When used at pondsite, gill swab samples would be the most appropriate specimen. Our results show that these samples might inhibit the PCR amplification process. This was prevented by diluting the sample at 1:10. Therefore, a dilution at 1:10 might be recommended when testing gill swab samples. The validation of the PCR-LFA further revealed that a single test might result in false-negative findings. With our test-panel, diagnostic sensitivity was increased by testing five replicates per sample. This highlights the necessity for further test series at POC conditions. Our results were similar to other studies using lateral flow assay strips for the rapid detection of KHV [30,31]. The latter based their diagnostic systems on the loop mediated isothermal amplification and the isothermal DNA amplification assay using recombinase polymerase amplification [30,31]. In contrast, we based our assay on a more common amplification technique, the PCR.
Generally, PCR is defined as the first and most accurately described in vitro DNA amplification technique. It is considered as the gold standard for many DNA-based detection methods [32]. Nevertheless, the POC-suitability of this method is often questioned. The expensive equipment, the need for precise temperature protocols and trained staff as well as the operating time were frequently addressed [33][34][35] (Kim & Easley, 2011). However, many of these arguments can be discussed controversially. Today, low-cost thermal cyclers suitable for POC analyses are available, which generally allow PCR-based rapid and "easy-to-handle" field applications [36,37]. Further improvement of the PCR components can simplify PCR processing significantly (e.g. by using a lyophilized ready-to-use mastermix) [38]. In addition, it has already been shown that a PCR-LFA combination can produce results in less than 30 min in a POC setting [39]. In our study, the combination of PCR and LFA was selected as a relatively simple, inexpensive, time-saving and POC-compatible detection platform. The multiplex potential of the PCR was used for the implementation of an internal amplification control, which is a unique characteristic of our diagnostic approach. It enables the user to identify PCR inhibition, which was crucial when investigating gill swab samples from deceased fish without extraction of DNA. Without that control even strongly KHV-positive samples would have been tested false-negative due to inhibiting substances in the sample. In addition, the assay described was developed as the basis for an improved analytic approach. The PCR-based detection strategy allows a stronger multiplexing and the developed post-PCR hybridization enables the detection of multiple amplicons (e.g. further infectious agents of interest) through the use of specific hybridization probes. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving POC-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be tested to confirm the number of replicates and sample processing needed to reveal a 100% diagnostic sensitivity. The assay prototype was tested with the following templates to determine functionality. Lane [1]