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

Abstract

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.

Keywords: diagnostics; disease control; foot-and-mouth disease; foot-and-mouth disease virus; lyophilized; rRT-PCR.

Figure 1

Field locations in East Africa. The bottom right map depicts the location of the antigenically defined foot‐and‐mouth disease virus pools within Africa (Paton et al., 2009). Field sampling locations are representative of foot‐and‐mouth disease virus pool 4

Figure 2

Limit of detection analysis for lyophilized pan‐serotype‐specific reagents compared to the reference real‐time reverse transcription PCR (rRT‐PCR) across four different serotypes (O/TAN/39/2012; A/TAN/6/2013; Southern African Territories [SAT] 1/KEN/72/2010; SAT 2/KEN/2/2008). (●) reference rRT‐PCR performed on a benchtop thermocycler; (○) pan‐serotype‐specific lyophilized reagents performed on the T‐COR™ 8. Points represent the mean of two replicates; crossed points indicate that of the identical replicates, one was positive and the other negative. The error bars indicate the range

Figure 3

Comparison between lyophilized pan‐serotype‐specific reagents and reference real‐time reverse transcription PCR. (a) Pan‐serotype‐specific rRT‐PCR compared to the reference rRT‐PCR across a panel of 57 foot‐and‐mouth disease virus (FMDV)‐positive clinical samples. (b) Serotype‐specific rRT‐PCR compared to the reference rRT‐PCR across a panel of 36 FMDV‐positive clinical samples from East Africa. The colour of points indicates serotype: blue (A), red (O), yellow (Southern African Territories [SAT] 1) and purple (SAT 2). For both graphs, points represent the mean of two replicates

Figure 4

Determination of clinical detection window for the pan‐serotype‐specific lyophilized real‐time reverse transcription PCR reagents. Assays performed on extracted RNA on the T‐COR™ 8 included the following sample types: (●) serum; (■) mouth swabs; (▲) foot epithelium; (▼) mouth epithelium; (♦) oesophageal–pharyngeal (OP) fluid. Assays performed directly (one in 10 dilution) on samples using the T‐COR™ 8 included the following sample types: (○) serum; (□) mouth swabs; (▵) foot epithelium; (▽) mouth epithelium; (♢) OP fluid. Points represent the mean of two replicates and crossed points represent when one replicate was positive and the other negative

Figure 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 (10⁻¹ to 10⁻⁹). Elution from antigen‐detection lateral‐flow devices (Ag‐LFD) was tested for epithelial suspensions only: ± 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)

Figure 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™ 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 (serotype A), red (serotype O) and white (serotype not detected). The 22/46 FMDV‐positive samples where the serotype was not detected had pan‐serotype‐specific C _T values of > 29 (using lyophilized reagents)

Figure 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