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Rapid pathogen identification is essential for the targeted treatment and effective infection control in horses with suspected infectious diseases. The prompt availability of diagnostic results enables clinicians to initiate appropriate antimicrobial or antiviral therapy, implement isolation measures and prevent further spread within a yard or clinic. In most clinical settings, the polymerase chain reaction (PCR) is the gold standard for pathogen detection; however, samples must be submitted to accredited external laboratories, and the transport and processing time typically exceeds 24 hours. The loop-mediated isothermal amplification (LAMP) method represents a promising point-of-care alternative, offering rapid on-site results without the need for a thermocycler. Unlike PCR, the LAMP reaction proceeds under strictly isothermal conditions, utilising the strand-displacement activity of a Bst DNA polymerase, and employs a six-primer configuration including two inner primers (FIP and BIP), two outer primers (F3 and B3) and two loop primers (LF and LB), which together enable highly specific and sensitive nucleic acid amplification within 30 minutes or less (Fig. 1–4). The LAMP method is already in clinical use in human medicine for the rapid detection of Zika virus, Mycobacterium tuberculosis and SARS-CoV-2, and is particularly well suited to resource-limited or field settings.

The aim of the current study was to evaluate the diagnostic performance of the EPONA test device (Enalees, France), a LAMP-based rapid diagnostic system for equine infectious diseases, by comparing its results with PCR analysis as the gold standard, performed in accredited reference laboratories (Laboklin, Bad Kissingen, Germany; Labor Dr. Böse GmbH, Harsum, Germany; Idexx, Kornwestheim, Germany; Swedish Veterinary Agency, Uppsala, Sweden). A secondary objective was to assess the practical field applicability of the system under routine clinical conditions.

A total of 206 samples were collected from sick horses of all ages, breeds and sexes across eight veterinary clinics in Germany, Sweden and Denmark: Tierärztliches Kompetenzzentrum Kirchheim, Pferdeklinik am Kirchberg, Pferdeklinik Destedt, Tierklinik Domäne Karthaus, Pferdeklinik der Tierärztlichen Hochschule Hannover, Pferdeklinik Ludwigshafen, Højgård Hestehospital and Mälaren Hästklinik. Horses presenting with clinical signs suggestive  viral respiratory disease, including fever, coughing and nasal discharge, underwent sampling of the upper respiratory tract. Of the 206 samples, 122 nasopharyngeal swabs or guttural pouch lavages were tested for equine herpesvirus 1 (EHV 1) and equine herpesvirus 4 (EHV 4), and 107 of these were additionally tested for equine influenza virus (EIV) using a combined LAMP assay (Fig. 5). A further 71 nasopharyngeal swab or guttural pouch lavage samples were collected from horses presenting with enlarged lymph nodes, fever or purulent nasal discharge and tested for Streptococcus equi ssp. equi (Strep. equi). Additionally, 13 blood samples were obtained by jugular venepuncture from horses exhibiting high or recurrent fever, inappetence or reduced performance, and examined for the tick-borne pathogens Anaplasma phagocytophilum, Babesia caballi and Theileria equi (Tab. 1–3).

Diagnostic sensitivity and specificity were calculated for each pathogen using standard formulae based on the number of true positive, true negative, false positive and false negative results, with PCR serving as the reference method. The 95 % confidence interval for each estimate was determined using the Wilson score interval method. Where sensitivity or specificity equalled 1, confidence intervals could not be meaningfully calculated and are not reported.

Complete concordance between LAMP and PCR was achieved for EIV, with a sensitivity and specificity of 100 %, thus meeting or exceeding the manufacturer's validated values of 92.0 % and 97.3 %, respectively (nasopharyngeal swabs) (8). For EHV 4, one false negative result was recorded, corresponding to a sensitivity of 87.5 % and a specificity of 100 %, compared to the manufacturer's reference values of 95.3 % (nasopharyngeal swabs) and 94.2 % (guttural pouch lavages) for sensitivity, and 98.6 % and 100 % for specificity, respectively (Tab. 1). For EHV 1, three false positive and five false negative results were observed, yielding a sensitivity of 54.5 % and a specificity of 97.3 %, which falls notably below the manufacturer's validated sensitivity of 94.0 % (nasopharyngeal swabs) and 93.8 % (guttural pouch lavages) (Tab. 1). For Strep. equi, four false negative and no false positive results were recorded, corresponding to a sensitivity of 73.3 % and a specificity of 100 %, versus manufacturer values of 92.3 % and 94.2 % for sensitivity (nasopharyngeal swabs and guttural pouch lavages, respectively) and 90.3 % and 92.9 % for specificity, respectively (Tab. 2). Direct comparability between the present study and the manufacturer's validation data remains limited, as the manufacturer reports sensitivity and specificity values separately by sample type, whereas the present study pooled results across nasopharyngeal swabs and guttural pouch lavages. Furthermore, the manufacturer's validation data should be interpreted with caution, as they originate from non-peer-reviewed technical documentation (8).

Regarding the tick-borne pathogens, complete concordance was achieved for Theileria equi, with the single PCR-positive sample and all 12 PCR-negative samples correctly identified by the LAMP method, yielding a sensitivity and specificity of 100 % (Tab. 3). For Babesia caballi, one false positive and one false negative result were observed, corresponding to a sensitivity of 50.0 % and a specificity of 90.9 %; the false negative is attributed to a co-infection with Theileria equi, in which the higher pathogen load may have competitively inhibited detection of Babesia caballi. For Anaplasma phagocytophilum, two false positive and no false negative results were recorded, yielding a sensitivity of 100 % and a specificity of 81.8 %, compared to manufacturer reference values of 92.5 %, 91.4 % and 91.9 % for sensitivity and 96.0 %, 96.6 % and 93.1 % for specificity for Theileria equi, Babesia caballi and Anaplasma phagocytophilum, respectively (Tab. 3). Due to the very limited number of PCR-positive samples for the tick-borne pathogens, all calculated values should be regarded as preliminary estimates only, and the findings require confirmation in larger field studies.

Several factors may account for the observed discrepancies between LAMP and PCR results. Differing cut-off values between methods represent a key consideration, as PCR laboratories define their own clinical decision thresholds based on clinical studies, meaning that a negative LAMP result at low pathogen concentrations is not necessarily equivalent to a false result. Low pathogen load is a known challenge for LAMP-based detection, and sensitivity can be significantly improved through professional DNA extraction or heat treatment prior to testing. Samples from the respiratory tract may additionally contain inhibitors that interfere with DNA polymerase activity or interact with sample DNA during extension. False positive results may be attributed to primer dimerisation, non-specific amplification or cross-contamination, the latter of which can be minimised by keeping reaction tubes closed throughout the procedure. The limited number of PCR-positive samples, particularly for EHV 1 and the tick-borne pathogens, reduces the statistical reliability of the calculated sensitivity values and results in wide confidence intervals, a challenge inherent to field studies where the number of naturally positive cases cannot be controlled.

The LAMP-based EPONA diagnostic system demonstrated generally good diagnostic performance for equine respiratory pathogens, with particularly reliable results for EIV and EHV 4. Performance was more variable for EHV 1 and Strep. equi, likely influenced by the limited number of PCR-positive samples and differences in sample handling. The results for tick-borne pathogens require validation in larger studies. Overall, the LAMP method represents a rapid, field-applicable alternative to laboratory-based PCR that supports timely clinical decision-making in equine practice. However, repeated testing or confirmation with laboratory-based PCR may be advisable in clinically suspicious cases with an initially negative LAMP result.