Development and Validation of a Protein Array for Detection of Antibodies against the Tick-Borne Pathogen Borrelia miyamotoi.

Current serological tests for the emerging tick-borne pathogen Borrelia miyamotoi lack diagnostic accuracy. To improve serodiagnosis, we investigated a protein array simultaneously screening for IgM and IgG reactivity against multiple recombinant B. miyamotoi antigens. The array included six B. miyamotoi antigens: glycerophosphodiester phosphodiesterase (GlpQ), multiple variable major proteins (Vmps), and flagellin. Sera included samples from cases of PCR-proven Borrelia miyamotoi disease (BMD), multiple potentially cross-reactive control groups (including patients with culture-proven Lyme borreliosis, confirmed Epstein-Barr virus, cytomegalovirus, or other spirochetal infections), and several healthy control groups from regions where Ixodes is endemic and regions where it is nonendemic. Based on receiver operating characteristic (ROC) analyses, the cutoff for reactivity per antigen was set at 5 µg/mL for IgM and IgG. The individual antigens demonstrated high sensitivity but relatively low specificity for both IgM and IgG. The best-performing single antigen (GlpQ) showed a sensitivity of 88.0% (95% confidence interval [CI], 78.9 to 93.5) and a specificity of 94.2% (95% CI, 92.7 to 95.6) for IgM/IgG. Applying the previous published diagnostic algorithm-defining seroreactivity as reactivity against GlpQ and any Vmp-revealed a significantly higher specificity of 98.5% (95% CI, 97.6 to 99.2) but a significantly lower sensitivity of 79.5% (95% CI, 69.3 to 87.0) for IgM/IgG compared to GlpQ alone. Therefore, we propose to define seroreactivity as reactivity against GlpQ and any Vmp or flagellin which resulted in a comparable sensitivity of 84.3% (95% CI, 74.7 to 90.8) and a significantly higher specificity of 97.9% (95% CI, 96.9 to 98.7) for IgM/IgG compared to GlpQ alone. In conclusion, we have developed and validated a novel serological tool to diagnose BMD that could be implemented in clinical practice and epidemiological studies. IMPORTANCE This paper describes the protein array as a novel serological test for the diagnosis of Borrelia miyamotoi disease (BMD), by reporting the methodology, the development of a diagnostic algorithm, and its extensive validation. With rising numbers of ticks and tick bites, tick-borne diseases, such as BMD, urgently deserve further societal and medical attention. B. miyamotoi is prevalent in Ixodes ticks across the northern hemisphere. Humans are exposed to, and infected by, B. miyamotoi and develop BMD in Asia, in North America, and to a lesser extent in Europe. However, the burden of infection and disease remains largely unknown, due to the noncharacteristic clinical presentation, together with the lack of awareness and availability of diagnostic tools. With this paper, we offer a novel diagnostic tool which will assist in assessing the burden of disease and could be implemented in clinical care.

Autochthonous Case of Rickettsia slovaca Infection in Russia.

We describe an autochthonous case of Rickettsia slovaca infection in a man 35 years of age from Russia who had tickborne lymphadenopathy. We used ELISA and quantitative PCR testing to further identify DNA and confirm diagnosis. Physicians in Russia should consider similar diseases in differential diagnoses after tick bites.

Severe babesiosis caused by Babesia divergens in a host with intact spleen, Russia, 2018.

We report a case of severe babesiosis caused by the bovine pathogen Babesia divergens with the development of multisystem failure in a splenic host. Immunosuppression other than splenectomy can also predispose people to B. divergens. There was heavy multiple invasion of up to 14 parasites inside the erythrocyte, which had not been previously observed even in asplenic hosts. The piroplasm 18S rRNA sequence from our patient was identical B. divergens EU lineage with identity 99.5-100%.

Two Cases of Dengue Fever Imported from Egypt to Russia, 2017.

In 2017, two cases of dengue fever were imported from Hurghada, Egypt, where dengue fever was not considered endemic, to Moscow. These cases show how emergence of dengue fever in popular resort regions on the coast of the Red Sea can spread infection to countries where it is not endemic.

Comparative analysis of the circadian rhythm genes period and timeless in Culex pipiens Linnaeus, 1758 (Diptera, Culicidae).

Nucleotide sequences of the circadian rhythm genes, period and timeless, were studied for the first time in mosquitoes Culex pipiens Linnaeus, 1758. In this work we evaluated variations of the studied genome fragments for the two forms of Culex pipiens (forma "pipiens" - mosquitoes common for aboveground habitats, forma "molestus" - underground mosquitoes). We compared Culex pipiens from Russia with transatlantic Culex pipiens and subtropical Culex quinquefasciatus Say, 1823. Our results show that intraspecies variability is higher for the gene period than for the gene timeless. The revealed substitutions in nucleotide sequences and especially in amino acid sequences grouped the individuals of the two forms into distinct clusters with high significance. The detected fixed amino acid substitutions may appear essential for functioning of the circadian rhythm proteins in Culex pipiens, and may be correlated with adaptations of the taxa within the group Culex pipiens. Our results suggest that natural selection favors fixed mutations and the decrease in diversity of the genes period and timeless in mosquitoes of the Culex pipiens f. "molestus" compared with the Culex pipiens f. "pipiens", is probably correlated with adaptive features of Culex pipiens f. "molestus". The studied genome regions may be considered as promising molecular-genetic markers for identification, population and phylogenetic analysis of similar species and forms of the Culex pipiens complex.

Complete Genome Sequences of Four European Subtype Strains of Tick-Borne Encephalitis Virus from Eastern Siberia, Russia.

Three tick-borne encephalitis virus (TBEV) strains were isolated from Ixodes persulcatus ticks, and one was isolated from a shrew in the territory of eastern Siberia (Russia). The level of sequence identity compared to Neudoerfl (the European prototype strain) is 97.2 to 97.3%.

Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia.

Borrelia miyamotoi is distantly related to B. burgdorferi and transmitted by the same hard-body tick species. We report 46 cases of B. miyamotoi infection in humans and compare the frequency and clinical manifestations of this infection with those caused by B. garinii and B. burgdorferi infection. All 46 patients lived in Russia and had influenza-like illness with fever as high as 39.5°C; relapsing febrile illness occurred in 5 (11%) and erythema migrans in 4 (9%). In Russia, the rate of B. miyamotoi infection in Ixodes persulcatus ticks was 1%-16%, similar to rates in I. ricinus ticks in western Europe and I. scapularis ticks in the United States. B. miyamotoi infection may cause relapsing fever and Lyme disease-like symptoms throughout the Holarctic region of the world because of the widespread prevalence of this pathogen in its ixodid tick vectors.

Epidemiology of West Nile infection in Volgograd, Russia, in relation to climate change and mosquito (Diptera: Culicidae) bionomics.

In 1999, there was the large outbreak of West Nile fever (WNF) in Southern Russia (>500 cases in the Volgograd Province). In 2000-2004, the WNF incidence rate decreased steadily to zero, but a new outbreak occurred in 2007 (64 cases). The analysis of historical climate data for Volgograd from 1900 to present showed that the years 1999 and 2007 were the hottest ones due to a very mild "winter" (Dec.-Mar.) and a hot "summer" (June-Sep.). There are up to 15 potential WNF vectors in Volgograd, but only Culex pipiens and Culex modestus are abundant in late summer, both in urban and rural settings. Only these species are naturally attracted to and feed on both humans and birds. The RNA of pathogenic WN virus genovariant was found by reverse transcriptase polymerase chain reaction only in Culex mosquitoes at the infection rate of about 0.04%. So these species may be considered as potential WNF "bridge vectors" between birds and humans as well as main vectors in sylvatic avain cycle. Their abundance in an epidemic season was higher in the years with a mild winter and a hot summer, so this phenomenon may serve as a connecting link between a climate and WNF epidemiology. These findings give some hints on the predisposing factors for WNF epidemic as well as the possibility to predict WNF outbreaks in the temperate climate zones.