BackgroundCurrently dengue viruses (DENV) pose an increasing threat to over 2.5 billion people in over 100 tropical and sub-tropical countries worldwide. International air travel is facilitating rapid global movement of DENV, increasing the risk of severe dengue epidemics by introducing different serotypes. Accurate diagnosis is critical for early initiation of preventive measures. Different reverse transcriptase PCR (RT-PCR) methods are available, which should be evaluated and standardized. Epidemiological and laboratory-based surveillance is required to monitor and guide dengue prevention and control programmes, i.e., by mosquito control or possible vaccination (as soon as an effective and safe vaccine becomes available).ObjectiveThe purpose of the external quality assurance (EQA) study described is to assess the efficiency and accuracy of dengue molecular diagnosis methods applied by expert laboratories.Study DesignA panel of 12 human plasma samples was distributed and tested for DENV-specific RNA. The panel comprised 9 samples spiked with different DENV serotypes (DENV-1 to DENV-4), including 10-fold dilution series of DENV-1 and DENV-3. Two specificity controls consisted of a sample with a pool of 4 other flaviviruses and a sample with chikungunya virus. A negative control sample was also included.ResultsThirty-seven laboratories (from Europe, Middle East Asia, Asia, the Americas/Caribbean, and Africa) participated in this EQA study, and reports including 46 sets of results were returned. Performance among laboratories varied according to methodologies used. Only 5 (10.9%) data sets met all criteria with optimal performance, and 4 (8.7%) with acceptable performance, while 37 (80.4%) reported results showed the need for improvement regarding accomplishment of dengue molecular diagnosis. Failures were mainly due to lack of sensitivity and the presence of false positives.ConclusionsThe EQA provides information on each laboratory's efficacy of RT-PCR techniques for dengue diagnosis and indicates for most laboratories an urgent need to improve sensitivity and specificity.

ObjectiveWe describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis.Study DesignFor molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included.ResultsThirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols.ConclusionThis EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for improving serological and molecular diagnosis techniques and provide a follow-up of the diagnostic profiles.

External quality assurance for serological detection of chikungunya virus infection was performed to assess the diagnostic quality of expert laboratories. Of 30 participants, only six correctly analysed all reference samples with their respective tests. Thirteen laboratories gave at least 85% correct results, and 11 laboratories 75% or less. IgM antibodies were detected less frequently than IgG antibodies (p <0.001). The study provides information on the quality of different serological tests and indicates that most of the participants need to improve the sensitivity of their assays, in particular to detect IgM antibodies more reliably and be able to detect acute infections adequately.

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Understanding epidemiology of the tick-borne pathogens requires the accurate identification of the vector ticks. Morphological analysis of ticks is difficult and often leads to misidentification. Molecular techniques offer an alternative approach of tick identification. To date, no practical and reliable molecular assays for discrimination of Euro-Asian ticks are available. Our aim was to develop such an assay for discrimination between four Euro-Asian tick species of high medical importance such as Ixodes ricinus, Ixodes persulcatus, Ixodes hexagonus, and Dermacentor reticulatus. As a basis, we have chosen conventional species-specific polymerase chain reaction (PCR), a technique providing a good combination of simplicity and reliability. The DNA information available on ticks was searched for orthologous loci containing stretches of sequence dissimilarity sufficient for designing species-specific primers. ITS2 locus (second internal transcribed region of the rRNA gene cluster) was found to be the most favorable for primer design. Finally, for each of the three Ixodes species a PCR was developed amplifying only for the targeted species. One PCR amplified the entire ITS2 locus of the four species and allowed discrimination of D. reticulatus from the Ixodes species on the basis of the size difference of the respective PCR products. This PCR system was successfully tested for discrimination of the ticks at different maturation stages (larva, nymph, and adult) in engorged and unfed conditions, and therefore it may be useful for large-scale epidemiological studies. Differentiation between the closely related I. ricinus and I. persulcatus, the two species most often occurring in the tick-borne diseases in Eurasia, is of special importance.