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

Abstract

SummaryEffective 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.

Foot‐and‐mouth disease virus detection on a handheld real‐time polymerase chain reaction platform

“…Furthermore, serum cannot be tested on LFST whereas two3 can directly detect FMDV in serum. Our results for direct detection of FMDV in a variety of samples are in agreement with other studies (Ambagala et al, ; Howson et al, ). Diagnostic sensitivity was reduced with direct testing of samples on two3, implying that RNA extraction may be required, particularly if a pre‐clinical case is suspected due to epidemiological links.…”

“…With further field validation, the results from two3 could potentially be accepted with the same level of confidence as the reference lab‐based platforms, especially for oral and nasal swabs and specifically vesicular flu acquired during the acute phase of infection. Similar results have been reported for FMDV on other field‐deployable real‐time molecular assays/platforms (Howson et al, ; Howson, Armson, et al, ; Howson, Kurosaki, et al, ; Moniwa et al, ). The fact that two3 detected FMDV in the absence of RNA extraction further potentiates its utility for onsite testing.…”

“…However, M1 technology may not be appropriate for serum samples because they clog the syringe filters used in M1 technology, possibly due to the high protein content in serum. RNA extraction with other sensitive field methods previously described (Ambagala et al, ; Howson et al, ) can also be evaluated for pairing with two3.…”

“…Real‐time reverse transcription polymerase chain reaction (RRT‐PCR) assays for FMDV are highly sensitive (Callahan et al, ; Howson, Armson, et al, ; Moniwa, Clavijo, Li, Collignon, & Kitching, ; Rasmussen, Uttenthal, Stricker, Belak, & Storgaard, ) but are mostly restricted to specialized laboratories. Nevertheless, significant progress has been made on field‐deployable molecular assays for FMDV (Ambagala et al, ; Dukes, King, & Alexandersen, ; Howson et al, ; Howson, Armson, et al, ; Howson, Kurosaki, et al, ; Kasanga et al, ; Waters et al, ). However, a sensitive handheld platform has not been described for FMDV.…”

ELISA, RT-PCR, semi-quantitative RT-PCR and sequencing methods for investigating an epidemic FMD virus serotype O outbreaks

“…The application of reverse transcription quantitative polymerase chain reaction (RT-qPCR) is highly sensitive and is considered as a valuable tool for the detection of viruses to reducing the risk of crosscontamination (Mackay et al, 2002;Mackay, 2004;Howson et al, 2017;Haidar et al, 2018).…”