Rickettsia aeschlimannii in Hyalomma marginatum Ticks, Germany

To the Editor: Rickettsia spp. of the spotted fever group cause worldwide emerging human infections known as tick-borne rickettsioses (1). Data on the occurrence and prevalence of Rickettsia in Germany are still limited (2). Six Rickettsia species have been reported to date (2). R. helvetica, R. felis, R. massiliae, and R. monacensis were detected with a relatively low prevalence in Ixodes ricinus ticks collected in southern Germany (2); R. raoultii was identified with high prevalence in the rapidly expanding area where D. reticulatus ticks are found (2). R. raoultii was recently recognized as an agent of tick-borne lymphadenopathy/Dermacentor-borne necrosis and erythema lymphadenopathy (3). Low prevalence of another tick-borne lymphadenopathy agent, R. slovaca, in Dermacentor marginatus ticks collected in southern Germany was recently reported (4)

2nd International External Quality Control Assessment for the Molecular Diagnosis of Dengue Infections

Dengue viruses (DENV) are the most widespread arthropod-borne viruses which have shown an unexpected geographic expansion, as well as an increase in the number and severity of outbreaks in the last decades. In this context, the accurate diagnosis and reliable surveillance of dengue infections are essential. The laboratory diagnosis of dengue relies on the use of several methods detecting markers of DENV infection present in patient serum. Molecular diagnosis methods are usually rapid, sensitive, and simple when correctly standardized. Moreover, PCR-based diagnosis techniques are able to readily detect DENV during the acute phase of the disease and may assume an important role in dengue diagnosis and surveillance. Different reverse transcriptase PCR (RT-PCR) methods have been developed and are currently available and should be standardized in each laboratory to maintain high quality performance. In this work an External quality assessment (EQA) activity has been carried out to evaluate the accuracy and quality of laboratory data for the molecular diagnosis and surveillance of dengue, which involved worldwide dengue reference laboratories. In conclusion, RT-PCR techniques for dengue diagnosis applied by the participating laboratories demonstrated the need of further improvement in most laboratories

First International External Quality Assessment Study on Molecular and Serological Methods for Yellow Fever Diagnosis

Objective: We 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 Design: For 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-YFVspecific 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. Results: Thirty-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. Conclusion: This 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

Differentiation of Medically Important Euro-Asian Tick Species Ixodes ricinus, Ixodes persulcatus, Ixodes hexagonus, and Dermacentor reticulatus by Polymerase Chain Reaction

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

EQA results of the 28 participating laboratories in the serological panel.

<p>The score reflects the quality of the diagnostic results: +, positive results; −, negative result; nd, not done; eq, equivocal. NT, seroneutralization test; HAI, haemagglutination inhibition test; Wrong results are marked in grey.</p>a<p>EUROIMMUN;</p>b<p>In-house assay;</p>c<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036291#pone.0036291-Takacs1" target="_blank">[25]</a>;</p>d<p>Bernhard Nocht Institut für Tropenmedizin, Hamburg;</p>e<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036291#pone.0036291-Talarmin1" target="_blank">[32]</a>;</p>f<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036291#pone.0036291-Ansari1" target="_blank">[33]</a>;</p>g<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036291#pone.0036291-Forshey1" target="_blank">[34]</a>.</p

Logit analysis of laboratory tests with a correct result (y axis) for YFV-17D related to viral RNA concentration in positive samples (x axis).

<p>Data points represent individual samples in the panel. Thick line is the regression line calculated on the basis of a logit model (dose–response curve), and thin lines are 95% confidence intervals.</p