Diagnostic pipeline and large-scale monitoring: the experience in the Apulian outbreak

In Apulia (southern Italy) since 2016 one of the largest monitoring campaign in EU is ongoing, with an average number of 100.000 samples tested per year in the framework of the official monitoring program for Xylella fastidiosa, in the demarcated and Xylella-free areas.  More than 85% of the samples were from olive trees, the remaining from almond, oleander and other host plants. Before the promulgation of the EU Regulation 2020/1201, the diagnostic program was organized with two levels of checks. Samples were first screened in 4 regional laboratories by ELISA, with each laboratories processing 300-600 samples/day. Samples yielding positive or doubtful ELISA-results along with 3-5% of the negative samples, were re-tested by qPCR as confirmation tests and for verifying the lab performance for the serological tests.  An average of 8.000 sample/year were re-tested by qPCR. Using this large dataset of samples (double tested by ELISA and qPCR) we determined the diagnostic specificity and sensitivity of the test used, and assessed the influence of other factors (period of sampling, origin of the samples).

Overall, less than 0.03% of the ELISA-tested samples could not be assigned as negative/positive (i.e. undetermined) and thus re-tested by qPCR, yielding in the majority of the cases (>85%) negative qPCR-results. The diagnostic sensitivity and specificity calculated on the panel of ELISA-negative/positive samples re-tested in qPCR, approx. 26.000 samples, were 94% and 97%, respectively.  These values did not vary significantly when the results were analyzed based on the period of sampling or location of the sampled trees. In conclusion, under the specific scenario characterizing the  Apulian epidemics (olive predominant host plants, climatic conditions) this diagnostic workflow had allowed to screen with good diagnostic accuracy a relevant number of samples/year.

Given the need to replace ELISA with qPCR tests, the capability of the laboratories to process the samples is now reduced (approx. half samples/day) and other possibilities will be consider for the implementation of the upcoming monitoring campaign (i.e testing pooled samples) to ensure testing the same amount of samples.