Since the 1950s, when the first cases of myxomatosis were reported in wild rabbits in Europe and Australia, myxoma virus (MYXV) has been circulating constantly in wild and farmed populations of lagomorphs, causing outbreaks of the disease (5). Although the traditional antigen-based serological assays used in viral diagnostics have been gradually replaced by molecular methods, they are still often used in laboratories due to their low costs of analysis and simplicity of execution. Among them there is the agar gel immunodiffusion (AGID) assay, still listed in the OIE manual as a recommended method for MYXV detection (14). This assay has been routinely used for virus detection over many years, although its diagnostic performance is not wholly known. The aim of this study was to estimate the diagnostic sensitivity (DSe) and specificity (DSp) of AGID and assess the assay’s diagnostic usefulness in comparison to the PCR-based methods currently used for MYXV detection.
The diagnosis of the classic nodular form of myxomatosis generally is not difficult and is based on clinical investigation and the presence of the characteristic skin lesions in affected animals. Nevertheless, in some cases, laboratory testing is required to confirm MYXV infection. The methods used in viral diagnostics should be validated to allow confidence of obtaining the most reliable results. Apart from the detection limit, the most significant validation parameters of each method are the DSe and DSp (13). In particular, the DSe value is important for a qualitative method such as AGID because it determines the assay’s ability to detect a pathogen in the sample.
In this study, the results obtained for samples tested by AGID assay and IAC-PCR depended on the virus concentration in the sample. When samples of rabbit skin collected from myxomatosis outbreak victims were analysed, 18 out of 23 samples gave positive results in AGID (78%, CI95%: 56–93). The presence of MYXV DNA was shown in all samples tested by IAC-PCR (Table 1). Full concordance of the results obtained in AGID and PCR-based methods (OIE-PCR and OIE real-time PCR) was observed for control reference materials. In addition, full result concordance (100%, CI95%: 83–100) was also demonstrated for tissue homogenates of rabbit skin containing at least 3,125 PCRU of MYXV DNA. Only samples containing 1 PCRU of MYXV DNA gave false negative results in AGID (0%, CI95%: 0–17). The assay’s DSe and DSp were estimated based on the results obtained for all samples tested in AGID. The AGID DSe was 0.65 (0.53–0.76) and DSp 1.00 (0.40– 1.00); AC of 0.67 (0.55–0.76) was also attained. In the case of the IAC-PCR method, these parameters were 0.98 (0.91–1.00) as the DSe and 0.95 (0.84–0.99) as the DSp (10). Direct comparison of method parameters showed that AGID gave only half the DSe of the molecular methods for samples containing low virus concentration. It is highly probable that the DSp of the OIE-PCR and OIE real-time PCR methods is comparable to that of the AGID assay, however, due to the low number of negative samples it could not be accurately estimated, as indicated by the wide confidence interval (0.40–1.00) for the AGID DSp.
Results of samples tested using AGID and molecular methods with confidence intervals (CI) determined by the Clopper–Pearson method
Molecular methods/ type of samples | Number of tested and PCR-positive/negative samples | AGID-confirmed results | Assessed results Median values obtained from proportions of confirmed results in AGID to the total number of samples for which test results agreed positively and negatively |
References |
---|---|---|---|---|
IAC-PCR (3,125 PCRU DNA of MYXV/sample) | 20 | 20 | 1.00 (0.83–1.00) | 10 |
IAC-PCR (1 PCRU DNA of MYXV/sample) | 20 | 0 | 0.00 (0.00–0.17) | |
IAC-PCR (rabbit skin homogenates) | 23 | 18 | 0.78 (0.56–0.93) | This study |
OIE PCR and OIE real-time PCR (control reference material) | 8 | 8 | 1.00 (0.63–1.00) | |
OIE PCR and OIE real-time PCR (control reference material without MYXV) | 4 | 4 | 1.00 (0.40–1.00) | |
All positive (with MYXV) | 71 | 46 | 0.65 (0.53–0.76) | |
All samples in this study (accuracy) | 75 | 50 | 0.67 (0.55–0.76) |
Analysing ĸ values for the AGID and IAC-PCR, the highest result concordance between methods was obtained for samples containing at least 3,125 PCRU of the virus (ĸ = 1.00) (10). Only IAC-PCR showed 100% sensitivity for samples with low (1 PCRU) virus concentration. None of the tested samples from this pool was positive in AGID. However, when the results of samples containing low and high virus levels were considered together, the concordance between the methods was fair (ĸ = 0.40). The reliability of the results was confirmed by a P-value (χ2 < 0.0001) indicating that the number of samples used for comparison of the methods was sufficient. The observed difference in the sensitivity of the AGID and IAC-PCR methods was only 3,125 PCRU. This finding could explain why AGID is still included in the panel of OIE methods used for disease detection. It is noteworthy that AGID is also widely employed in the diagnosis of other viral diseases of animals mainly as a screening tool (1, 8, 9). It appeared to be a sensitive assay in detection of Marek’s disease virus (MDV) in clinically affected and dead birds from disease outbreak flocks, in the testing of which no significant difference between PCR and AGID in the detection of MDV was observed (8).
The probability of getting the correct results with the particular assay used is indicated by NPVs, which are related to the method’s DSe. When AGID NPVs are analysed, it could be assumed that the number of false negative results obtained will be higher for samples with different virus concentrations (low and high viral load). With the increase in the number of true positive samples tested in AGID, especially those with levels of MYXV lower than 3,125 PCRU, the percentage of false negative results will also increase. For example, assuming that 40% of tested samples will contain the virus (the probability of infection), the probability of getting a false negative result using AGID will be 20% (Fig. 1). Using the IAC-PCR method to test these samples, no false negative results will be obtained, and all virus positive samples will be correctly identified. Due to a lack of samples originating from rabbits with the atypical (amyxomatous) form of the disease, it was not possible to assess the diagnostic value of AGID for rabbit tissues other than skin samples. As in the typical nodular form of the disease, in the atypical form the virus was also frequently recovered from the eyelids besides the lungs and liver (12). The skin lesions characteristic for myxomatosis were missing; however, oedematous swelling of the eyelids, scrotum, and anorectal area was observed (4). Because the highest concentration of the virus mainly occurs in rabbit skin with myxomas, testing of other tissues using an assay with a lower sensitivity than molecular methods may give false negative results.
In conclusion, the data from this study suggest that AGID can be used as a screening tool for detection of MYXV infections. Its simplified procedure could be particularly useful in poorly equipped laboratories. However, due to the low DSe value compared to molecular methods, its application for virus detection in samples collected from animals with mildly manifested symptoms of the disease may produce false negative results.