Eili Huhtamo

Early diagnosis of dengue in travelers: Comparison of a novel real-time RT-PCR, NS1 antigen detection and serology

BACKGROUND The increased traveling to dengue endemic regions and the numerous epidemics have led to a rise in imported dengue. The laboratory diagnosis of acute dengue requires several types of tests and often paired samples are needed for obtaining reliable results. Although several diagnostic methods are available, proper comparative data on their performance are lacking. OBJECTIVES To compare the performance of novel methods including a novel pan-DENV real-time RT-PCR and a commercially available NS1 capture-EIA in regard to IgM detection for optimizing the early diagnosis of DENV in travelers. STUDY DESIGN A panel of 99 selected early phase serum samples of dengue patients was studied by real-time RT-PCR, NS1 antigen ELISA, IgM-EIA, IgG-IFA and cell culture virus isolation. RESULTS The novel real-time RT-PCR was shown specific and sensitive for detection of DENV-1-4 RNA and suitable for diagnostic use. The diagnostic rate using combination of RNA and IgM detection was 99% and using NS1 and IgM detection 95.9%. The results of RNA and NS1 antigen detection disagreed in 15.5% of samples that had only RNA or NS1 antigen detected. CONCLUSIONS The diagnostic rates of early samples are higher when either RNA or NS1 antigen detection is combined with IgM detection. Besides the differences in the RNA and NS1 detection assays, the observed discrepancy of results could suggest individual variation or differences in timing of these markers in patient serum.

Characterization of a novel flavivirus from mosquitoes in northern europe that is related to mosquito-borne flaviviruses of the tropics.

ABSTRACT A novel flavivirus was isolated from mosquitoes in Finland, representing the first mosquito-borne flavivirus from Northern Europe. The isolate, designated Lammi virus (LAMV), was antigenically cross-reactive with other flaviviruses and exhibited typical flavivirus morphology as determined by electron microscopy. The genomic sequence of LAMV was highly divergent from the recognized flaviviruses, and yet the polyprotein properties resembled those of mosquito-borne flaviviruses. Phylogenetic analysis of the complete coding sequence showed that LAMV represented a distinct lineage related to the Aedes sp.-transmitted human pathogenic flaviviruses, similarly to the newly described Nounané virus (NOUV), a flavivirus from Africa (S. Junglen et al., J. Virol. 83:4462-4468, 2009). Despite the low sequence homology, LAMV and NOUV were phylogenetically grouped closely, likely representing separate species of a novel group of flaviviruses. Despite the biological properties preferring replication in mosquito cells, the genetic relatedness of LAMV to viruses associated with vertebrate hosts warrants a search for disease associations.

Zika virus infection in a traveller returning from the Maldives, June 2015

We report a Zika virus (ZIKV) infection in a patient with fever and rash after returning to Finland from Maldives, June 2015. The patient had dengue virus (DENV) IgG and IgM antibodies but pan-flavivirus RT-PCR and subsequent sequencing showed presence of ZIKV RNA in urine. Recent association of ZIKV with microcephaly highlights the need for laboratory differentiation of ZIKV from DENV infection and the circulation of ZIKV in areas outside its currently known distribution range.

Sindbis Virus Infection in Resident Birds, Migratory Birds, and Humans, Finland

Resident grouse may be involved in the epidemiology of SINV in humans.

Novel insect-specific flavivirus isolated from northern Europe

Mosquitoes collected in Finland were screened for flaviviral RNA leading to the discovery and isolation of a novel flavivirus designated Hanko virus (HANKV). Virus characterization, including phylogenetic analysis of the complete coding sequence, confirmed HANKV as a member of the “insect-specific” flavivirus (ISF) group. HANKV is the first member of this group isolated from northern Europe, and therefore the first northern European ISF for which the complete coding sequence has been determined. HANKV was not transcribed as DNA in mosquito cell culture, which appears atypical for an ISF. HANKV shared highest sequence homology with the partial NS5 sequence available for the recently discovered Spanish Ochlerotatus flavivirus (SOcFV). Retrospective analysis of mitochondrial sequences from the virus-positive mosquito pool suggested an Ochlerotatus mosquito species as the most likely host for HANKV. HANKV and SOcFV may therefore represent a novel group of Ochlerotatus-hosted insect-specific flaviviruses in Europe and further afield.

Orthomyxo-, paramyxo- and flavivirus infections in wild waterfowl in Finland

BackgroundScreening wild birds for viral pathogens has become increasingly important. We tested a screening approach based on blood and cloacal and tracheal swabs collected by hunters to study the prevalence of influenza A, paramyxo-, flavi-, and alphaviruses in Finnish wild waterfowl, which has been previously unknown. We studied 310 blood samples and 115 mixed tracheal and cloacal swabs collected from hunted waterfowl in 2006. Samples were screened by RT-PCR and serologically by hemagglutination inhibition (HI) test or enzyme-linked immunosorbent assay (ELISA) for influenza A (FLUAV), type 1 avian paramyxo-(APMV-1), Sindbis (SINV), West Nile (WNV) and tick-borne encephalitis (TBEV) virus infections.ResultsFLUAV RNA was found in 13 tracheal/cloacal swabs and seven strains were isolated. Five blood samples were antibody positive. Six APMV-1 RNA-positive samples were found from which four strains were isolated, while two blood samples were antibody positive. None of the birds were positive for flavivirus RNA but three birds had flavivirus antibodies by HI test. No antibodies to SINV were detected.ConclusionWe conclude that circulation of both influenza A virus and avian paramyxovirus-1 in Finnish wild waterfowl was documented. The FLUAV and APMV-1 prevalences in wild waterfowl were 11.3% and 5.2% respectively, by this study. The subtype H3N8 was the only detected FLUAV subtype while APMV-1 strains clustered into two distinct lineages. Notably, antibodies to a likely mosquito-borne flavivirus were detected in three samples. The screening approach based on hunted waterfowl seemed reliable for monitoring FLUAV and APMV by RT-PCR from cloacal or tracheal samples, but antibody testing in this format seemed to be of low sensitivity.

A Single Dose of Vero Cell–Derived Japanese Encephalitis (JE) Vaccine (Ixiaro) Effectively Boosts Immunity in Travelers Primed With Mouse Brain–Derived JE Vaccines

The new Japanese encephalitis vaccine (JE-VC, Ixiaro) has replaced mouse brain–derived vaccines (JE-MB) associated with serious safety concerns. A single dose of JE-VC effectively boosted immunity in JE-MB–primed travelers. Current recommendations for booster vaccination should be reevaluated.

Japanese encephalitis in a Finnish traveler on a two-week holiday in Thailand

Japanese encephalitis (JE) virus is a mosquito-borne flavivirus, and one of the leading causes of epidemic encephalitis in Southeast Asia. Reports of symptomatic JEV encephalitis in tourists have been rare. We describe a case of symptomatic JE transmitted in 2004 during a short two-week trip to common tourist attractions in Thailand.

Novel flaviviruses from mosquitoes: mosquito-specific evolutionary lineages within the phylogenetic group of mosquito-borne flaviviruses.

Novel flaviviruses that are genetically related to pathogenic mosquito-borne flaviviruses (MBFV) have been isolated from mosquitoes in various geographical locations, including Finland. We isolated and characterized another novel virus of this group from Finnish mosquitoes collected in 2007, designated as Ilomantsi virus (ILOV). Unlike the MBFV that infect both vertebrates and mosquitoes, the MBFV-related viruses appear to be specific to mosquitoes similar to the insect-specific flaviviruses (ISFs). In this overview of MBFV-related viruses we conclude that they differ from the ISFs genetically and antigenically. Phylogenetic analyses separated the MBFV-related viruses isolated in Africa, the Middle East and South America from those isolated in Europe and Asia. Serological cross-reactions of MBFV-related viruses with other flaviviruses and their potential for vector-borne transmission require further characterization. The divergent MBFV-related viruses are probably significantly under sampled to date and provide new information on the variety, properties and evolution of vector-borne flaviviruses.

Approach to non-invasive sampling in dengue diagnostics: exploring virus and NS1 antigen detection in saliva and urine of travelers with dengue.

BACKGROUND Dengue diagnostics currently relies on serum and plasma tests. Although the proof of concept for detecting dengue virus (DENV) RNA and nonstructural protein 1 (NS1) antigen from urine and saliva has been demonstrated, few studies have explored their use in diagnostics. OBJECTIVES To investigate the occurrence, excretion kinetics, and diagnostic potential of DENV-RNA and NS1 antigen in the urine and saliva of dengue patients. STUDY DESIGN We examined serial serum, urine (n=50) and saliva (n=48) samples of 14 Finnish travelers with dengue. All samples were analyzed by NS1 ELISA and DENV RT-PCR, and the first and last serum specimens were tested for DENV IgG and IgM. In addition, biochemical parameters were studied from the urine and clinical and laboratory data of the patients were collected. RESULTS DENV-NS1 protein and RNA proved detectable from saliva and urine using tests developed for serum samples. RNA/NS1 detection showed a diagnostic sensitivity of 64%/54% and 60%/56% for urine and saliva, respectively. RNA analyses performed on days 7-13 after onset of symptoms revealed the sensitivity for urine (72%) to be greater than for serum (31%) or saliva (50%). The concentration of urine samples had no impact on RNA detection. CONCLUSIONS Noninvasive sampling enables an alternative approach to dengue diagnostics. The performance of the NS1 antigen assay may be improved by optimizing it for urine and saliva samples. The prolonged excretion of DENV-RNA in urine extends the sampling time window for molecular diagnostics and surveillance.