Direct detection and characterization of foot‐and‐mouth disease virus in East Africa using a field‐ready real‐time PCR platform

Summary Effective control and monitoring of foot‐and‐mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype‐specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD‐endemic countries have motivated the development of simple diagnostic platforms to support local decision‐making. Using a portable thermocycler, the T‐COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan‐serotype‐specific real‐time RT‐PCR (rRT‐PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa‐specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan‐serotype‐specific lyophilized assay were comparable to that of an OIE‐recommended laboratory‐based rRT‐PCR (determined using a panel of 57 FMDV‐positive samples and six non‐FMDV vesicular disease samples for differential diagnosis). The FMDV‐typing assay was able to correctly identify the serotype of 33/36 FMDV‐positive samples (no cross‐reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal–pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre‐clinical, clinical and clinically recovered cattle. These data support the use of field‐ready rRT‐PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.


| INTRODUCTION
Early detection of foot-and-mouth disease virus (FMDV), a highly infectious picornavirus, is essential to minimize the impacts of footand-mouth disease (FMD) in susceptible populations. FMD is endemic in many countries throughout Africa, Asia and parts of South America, existing as seven distinct FMDV serotypes (A, O, C, Asia 1, Southern African Territories [SAT] 1, 2 and 3), which are distributed unevenly worldwide within seven geographically defined pools (Paton, Sumption, & Charleston, 2009). Laboratory-based diagnostic tests play an essential role in FMD control and eradication by providing accurate confirmation of clinical signs. Real-time reverse-transcription polymerase chain reaction (rRT-PCR) has been widely adopted by FMD reference laboratories as a principal tool for FMDV detection, offering high analytical sensitivity and rapid sample throughput Shaw et al., 2007). Pan-serotype-specific rRT-PCR assays, targeting conserved regions of the FMDV RNA genome (Callahan et al., 2002;Reid et al., 2002), were used during the UK 2007 FMDV outbreak to test 99.1% of 3,246 diagnostic samples submitted to the UK National Reference Laboratory for FMD (The Pirbright Institute, UK) .
Although laboratory-based tests provide rapid and accurate results, sample transportation to the laboratory can negatively affect the quality of the specimens (if shipped incorrectly) and delay/or even hinder the processes of immediate critical decision-making and disease control. Furthermore, many diagnostic procedures require sophisticated and expensive equipment, limiting their application in low-and middle-income countries (LMICs), which lack the infrastructure and financial resources for veterinary diagnostics and surveillance of endemic diseases such as FMD (Namatovu et al., 2013;Paton et al., 2009;Vosloo, Bastos, Sangare, Hargreaves, & Thomson, 2002). The control of FMD has been recognized by the Food and Agricultural Organization of the United Nations (FAO) and The World Organisation for Animal Health (OIE) as a global priority (Sumption, Domenech, & Ferrari, 2012). However, with effective control strategies requiring knowledge of FMD distribution and epidemiology, the deployment of simple point-of-care test (POCT) platforms for active FMDV detection, monitoring and characterization remains an ongoing research effort.
The compatibility of an OIE-recommended rRT-PCR assay with POCT platforms has already been demonstrated (Hearps, Zhang, & Alexandersen, 2002;Madi et al., 2012;Monwina, Clavijo, Li, Collignon, & Kitching, 2007;Paixão et al., 2008). For instance, portable rRT-PCR has been deployed in field settings for accurate detection of FMDV in serum, epithelial suspensions and oesophageal-pharyngeal fluid (OP) fluid (Howson et al., 2015). However, current assay formats and platforms are currently limited by low sample throughput, the requirement for RNA extraction (and thus methods and equipment to perform this) or are commercially unavailable. Furthermore, evaluation of POCT rRT-PCR platforms has only been performed using FMDV pan-serotype-specific assay formats. With methods of FMD control in endemic settings relying upon rapid and accurate identification of the particular FMDV serotype circulating in the field (e.g. targeted vaccination) (Sumption et al., 2012), there have been efforts to design serotype-specific assays (tailored to the region) that target variable capsid-coding regions of the FMDV genome (Ahmed et al., 2012;Bachanek-Bankowska et al., 2016;Giridharan, Hemadri, Tosh, Sanyal, & Bandyopadhyay, 2005;Jamal & Belsham, 2015;Knowles et al., 2016;Reid et al., 2014). Samples comprised of serum (n = 11) and mouth swabs (n = 11) taken daily from two animals from the day of challenge and epithelium (n = 4) and OP fluid (n = 2) collected from the same animals post-mortem (carried out on the day of culling).

| Initial laboratory analysis of the lyophilized
East Africa typing rRT-PCR reagents Initial laboratory analysis of the East Africa-specific typing assay was performed on RNA extracted from 36 samples (serotypes O, A, SAT 1 and SAT 2) originating from East Africa (samples from Tanzania and Kenya included in Appendix S1). These samples were characterized by the WRLFMD using an antigen-detection ELISA (Ferris & Dawson, 1988) and sequencing of the VP1 region (Knowles et al., 2016), as stated in the guidelines of the diagnostic manuals of the OIE (OIE, 2012).

| Sample preparation for field evaluation
Loose epithelial tissue of ruptured vesicular lesions from either the mouth or feet was prepared using the SVANODIP â FMDV-Ag Extraction Kit and SVANODIP â FMDV-Ag LFD Kit (Boehringer Ingelheim) as previously described (Howson et al., 2015). Epithelial samples from the feet (inter-digital space/coronary band) were briefly washed in nuclease free water (NFW) prior to processing to remove soil contaminants. The homogenate was left to settle for 1 min, and then, the supernatant was removed and diluted one in 10 in NFW prior to analysis.
Swabs (GenoTube Livestock, Thermo Fisher Scientific) were collected by swabbing the surface of ruptured lesions in the mouth or on the feet (inter-digital space/coronary band). The feet were cleaned in sterile water prior to swabbing in order to remove soil contaminants. Swabs were then agitated by hand in 1 ml NFW, which was used directly in analysis.
OP fluid was collected using a suitably sized probang cup following the guidelines within the diagnostic manuals of the OIE (2012) and diluted one in 10 in NFW prior to analysis.
Blood (10 ml) was collected from either the jugular or tail vein using Vacutainer â Plus Plastic Serum Tubes (BD, Plymouth, UK), or similar. Samples were then transported back to the local laboratory and centrifuged at 3,000 g for 10 min. Serum was removed and then diluted one in 10 in NFW prior to analysis.

| Laboratory-based rRT-PCR
An OIE-recommended pan-serotype-specific one-step rRT-PCR was used as the reference assay. Each sample was tested in duplicate, using primers and probes reported by Callahan et al. (2002)

| rRT-PCR using the T-COR TM 8
The FMDV lyophilized pan-serotype-specific assay was performed in duplicate using FMDV 2.0 reagents with inhibition control (TC-9092-064, Tetracore Inc.). This assay contains proprietary primers and probes to target two areas within the highly conserved 3D RNA was extracted as above using a DynaMag TM -Spin magnet (Thermo Fisher Scientific). To meet biosecurity procedures the sample was added to lysis buffer before the magnetic beads

| Sensitivity and specificity of lyophilized pan-serotype-specific rRT-PCR reagents
The analytical sensitivity of the lyophilized pan-serotype-specific reagents, performed on the T-COR TM 8, was equivalent to the reference rRT-PCR across the four serotypes tested (both consistently detected down to 10 À6 for each serotype) ( Figure 2). Diagnostic sensitivity of the pan-serotype-specific lyophilized assay was 100% across the 57 FMDV-positive samples tested (Appendix S1), with all diagnostic samples tested displaying comparable C T values to the reference rRT-PCR (Figure 3a). The specificity of the pan-serotype-specific lyophilized assay was also 100% (n = 6), in that no crossreactivity was observed with the other vesicular disease viruses tested (Appendix S1). FMDV RNA was detected in serum from 1-4 days post-challenge, mouth swabs from 2 days onwards post-challenge and all epithelium and OP samples collected post-mortem using the lyophilized pan-serotype-specific reagents (Figure 4).

| Initial laboratory analysis of the lyophilized
East Africa-specific typing rRT-PCR reagents  Figure 5). However, when these same sample preparations were tested with the lyophilized rRT-PCR reagents (e.g., dilution in NFW and the use of a filter), a log 10 reduction in analytical sensitivity compared with the use of extracted RNA. Alternative methods, such as the use of use of Chelex â 100 or elution from Ag-LFDs, did not increase analytical sensitivity for either rRT-PCR assay ( Figure 5).
Although serum could be added directly to both the reference rRT-PCR and lyophilized rRT-PCR, the analytical sensitivity was higher with the lyophilized rRT-PCR. For all other methods tested, the analytical sensitivity for the two rRT-PCR assays was similar, with dilution in NFW and/or the use of Chelex â 100 or a syringe filter required to improve the LOD ( Figure 5). For OP fluid, a log 10 reduction (relative to MagMax TM extraction) was evident for both rRT-PCR reagents when diluted one in 10 in NFW prior to analysis.
The additional use of syringe filters, Chelex â 100 or other dilutions resulted in a further decrease in LOD ( Figure 5).

| Detection of FMDV in situ using lyophilized rRT-PCR in East Africa
The T-COR TM 8 and the newly developed rRT-PCR protocols were tested on 144 samples from 78 cattle across 13 farms in East Africa.
Using the pan-serotype-specific lyophilized rRT-PCR in combination with the T-COR TM 8, FMDV RNA was identified in 5/5 F I G U R E 5 Comparison of sample preparation methods for the reference real-time reverse transcription PCR (rRT-PCR) and lyophilized pan-serotype-specific rRT-PCR. Sample preparation methods were tested for three sample types (epithelial suspensions, sera and oesophageal-pharyngeal [OP] fluid) across a dilution series (10 À1 to 10 À9 ). Elution from antigen-detection lateral-flow devices (Ag-LFD) was tested for epithelial suspensions only: AE represent Ag-LFD results. Black squares represent dilutions where both replicates were positive for FMDV; grey squares represent dilutions where one replicate was positive; white squares represent reactions where both replicates were negative for FMDV. For the reference rRT-PCR, the use of simple sample preparation methods for epithelium resulted in assay inhibition in known positive samples (bar the use of Ag-LFD) epithelial, 1/1 swab and 3/3 sera samples collected from cattle displaying 1-to 3-day-old lesions. FMDV RNA was identified in 13/14 epithelial, 15/19 swab, 3/4 OP fluid and 3/29 sera samples collected from cattle displaying 4-to 7-day-old lesions. Of the 27 cattle that displayed lesions older than 1 week (8+ days post-initial lesion presentation), FMDV RNA was identified in 3/12 OP fluid samples, whilst all sera (n = 25) and swab (n = 10) samples were negative.
Samples considered positive by the lyophilized pan-serotype-specific assay (n = 46), in addition to a selection of samples from cattle considered clinically negative (n = 7), were then subsequently characterized using the East Africa-specific typing assay which has been developed to detect all known FMDV strains relevant to this region . Of these, 24 were identified as F I G U R E 6 In situ real-time reverse transcription PCR (rRT-PCR) results for 144 East African samples using lyophilized rRT-PCR reagents and the T-COR TM 8. Samples were collected from cattle displaying clinical signs of foot-and-mouth disease and cattle with no clinical signs (NCS). Each point represents an average for a single sample (tested in duplicate) on the pan-serotype-specific rRT-PCR (samples that share the same C T will only appear as a single point with individual C T values presented within supplementary data); crossed points indicate that out of the duplicates, one was positive and the other negative. Positive samples were then tested using the typing assay; the colour of the points represents the serotype detected: blue (

| DISCUSSION
The requirement for rapid diagnosis of FMD has led to an increase in the development and evaluation of POCT for FMDV detection. In addition to providing a means to rapidly confirm cases of FMD on or close to the farm during outbreaks, these technologies could potentially provide a route for LMICs to establish robust field/laboratory testing capacity. For example, immunological-based assays such as lateral-flow devices (Ag-LFDs) have been developed for rapid viral antigen detection and successfully tested in situ during the UK 2007 FMDV outbreak (Ryan et al., 2008). However, Ag-LFDs have only been validated for use with epithelial suspension and vesicular fluid, and limited analytical sensitivity restricts their usefulness to the acute stage of infection (Ferris et al., 2009(Ferris et al., , 2010. As a consequence, attempts to transfer highly sensitive rRT-PCR assays onto portable detection platforms have been investigated (Hearps et al., 2002;Howson et al., 2015;Madi et al., 2012;Monwina et al., 2007;Paixão et al., 2008), but are either not commercially available or are only suitable for research purposes (i.e., not diagnostic use).
This study evaluates the performance of a commercially available, lyophilized FMDV pan-serotype-specific assay, in combination with a commercially available portable thermocycler, in laboratory and East African field settings. Such tests have the potential to contribute valuable epidemiological information to support country-level and regional control programmes, such as the Progressive Control Pathway for FMD (Sumption et al., 2012). The future success and implementation of such technologies depends upon several factors, including dissemination of information and adoption of these methodologies into current diagnostic strategies.
The provision of reagents in a lyophilized kit format simplifies reagent storage by negating the requirement for a cold chain, whilst minimizing the requirement for skilled personnel and multiple pipetting stages by streamlining assay set up (assays only require addition of a resuspension buffer and sample). The lyophilized pan-serotypespecific reagents, in combination with the T-COR TM 8, maintained comparable diagnostic performance to the reference rRT-PCR when evaluated with extracted RNA. A one to two log 10 reduction in analytical sensitivity for the lyophilized pan-serotype-specific reagents was evident when no extraction method was used; however, performance was still equivalent to the sensitivity of the reference rRT-PCR when a diagnostic cut-off of C T <32 (as recommended by Shaw et al., 2007) was applied to the reference rRT-PCR. As the lyophilized pan-serotype-specific reagents are still able to perform rRT-PCR in the absence of extraction, they offer a potential solution for molecular POCT in field settings, which is not possible using the reference rRT-PCR.
Throughout field validation of the T-COR TM 8 in East Africa, lyophilized reagents generated results consistent with clinical observations, with FMDV detected in samples from the early onset of infection through to delayed viral clearance. Results were gained in less than 1.5 hr from sample collection. High concordance was evident between results gained in the field (T-COR TM 8 rRT-PCR) and local East African laboratories (reference rRT-PCR), with any disagreements associated with samples that had C T values above the diagnostic threshold of C T < 32 (Shaw et al., 2007). Although the provision of simple FMDV-positive/negative results is sufficient for the confirmation of FMD during outbreaks, the value of this information is limited in countries where FMD is endemic. In these situations, it is beneficial to characterize circulating FMDV outbreaks in order to make tailored control programmes a realistic possibility (Namatovu et al., 2013). In support of this, this study also evaluated a lyophilized version of a published FMDV-typing assay specific to East Africa (Bachanek-Bankowska et al., 2016). The typing assay was able to characterize FMDV present in four different sample types collected from seven small holdings (three in Kenya, three in Tanzania and one in Ethiopia) where cattle were presenting two to sevenday-old FMD lesions. Samples which were unable to be typed F I G U R E 7 Comparison between real-time reverse transcription PCR (rRT-PCR) results performed using lyophilised pan-serotype specific rRT-PCR in the field and reference rRT-PCR performed in local laboratories HOWSON ET AL.
| 229 (n = 22) were all considered as weak positives by the lyophilized pan-serotype-specific rRT-PCR (C T > 29) and therefore at the threshold at which characterization information can be routinely obtained by sequencing. The typing assay performance in field settings is therefore consistent with the ability to obtain characterization data within laboratory settings. Two serotypes were detected during the period of testing (O and A), leading to the first reported characterization of FMDV at the pen-side using molecular methods.
In conclusion, this publication describes the laboratory and field evaluation of lyophilized FMDV-specific rRT-PCR assays in combination with a portable thermocycler. The simplicity of the T-COR TM 8 to operate, combined with robust lyophilized reagents, high sensitivity and compatibility with simple sample preparation methods, demonstrates an important transition for FMDV-specific rRT-PCR assays into formats suitable for use on or close to the point-of-care.