Katerina Tsergouli

Diagnostic Assays for Crimean-Congo Hemorrhagic Fever

On-site testing would diminish time, costs, and risks involved in handling of highly infectious materials.

Seroprevalence of Crimean-Congo Hemorrhagic Fever Virus, Bulgaria

To the Editor: Crimean-Congo hemorrhagic fever (CCHF) is endemic in southern Russia, southeastern Europe, Africa, the Middle East, and southwestern Asia (1). The incidence and spread of the disease have increased in recent years. In Bulgaria, located on the Balkan Peninsula, CCHF is endemic. The disease was first described in the country in 1952 (2). Since then, a mandatory reporting system has been introduced. Most of Bulgaria is an ecologically favorable environment for CCHF virus (CCHFV) circulation in nature. In the 1970s, numerous virologic and serologic studies were performed by Vasilenko et al., who showed that the most affected age group was 21–50 years and that most of those with CCHF were male (65%) (cited in [3]). A genetic study showed that CCHFV strains in Bulgaria cluster together with strains from other Balkan countries and Russia (2). A vaccine consisting of chloroform-inactivated CCHFV was developed in 1974, and the currently used vaccine strain, isolated from a Bulgarian patient, was characterized genetically (4). In the last 10 years, <10 CCHF cases have been registered annually in Bulgaria. Although the number of cases is lower than previously, the disease has spread into new areas (southeast, northeast, south-central provinces). In 2008, a cluster of cases was observed in southwestern Bulgaria (Blagoevgrad district), a low-risk CCHF area (5). Since then, a substantial number of cases have been reported in this district. During the past 4 years (2008–2011), 30 CCHF cases have been registered in Bulgaria, 12 from Blagoevgrad district, 8 from Burgas district, 4 each from Haskovo and Sliven districts, and 1 each from Kardjali and Shumen districts. To estimate the current situation on CCHFV seroprevalence in both disease-endemic and -nonendemic areas in Bulgaria, we tested serum samples for CCHFV IgG antibodies using a commercially available ELISA kit (Vector Best, Novosibirsk, Russia). The serum samples were collected prospectively during 2011 from 1,018 healthy persons (50.2% male) from 13 districts: Sofia (n = 116), Blagoevgrad (n = 100), Pazardjik (n = 52), Stara Zagora (n = 36), Smolyan (n = 46), Yambol (n = 60), Haskovo (n = 108), Kardjali (n = 50), Sliven (n = 50), Burgas (n = 200), Shumen (n = 50), Ruse (n = 100), and Pleven (n = 50); they were then tested for CCHFV IgG antibodies with a commercially available ELISA kit (Vector Best). The median age of participants was 48 years (range 2–89 years). Persons previously vaccinated against CCHFV were excluded from the study. Twenty-eight persons (2.8%) had IgG antibodies to CCHFV. The highest seroprevalence was observed in Burgas (7.6%), followed by Kardjali (6%), Pazardjik (5.8%), and Haskovo (4.6%) districts (Figure). Low seroprevalence levels were detected in Sliven (2%), Blagoevgrad (1%), and Ruse (1%) districts. Generally, these results are consistent with the number of reported cases in different districts. Notably, Kardjali and Pazardjik districts showed high CCHFV seroprevalence but single reported cases in the last years. However, these regions were among the main endemic foci in the past. In contrast, the low seroprevalence rate found in district of Blagoevgrad conflicts with the high number of diagnosed CCHF cases, but this district has been at low risk for many years. Figure Prevalence rates for Crimean-Congo hemorrhagic fever virus in various districts of Bulgaria. F.Y.R.M., Former Yugoslav Republic of Macedonia. Multivariate analysis showed that having a former tick bite and farming were significant risk factors, while age and sex were not related to seropositivity (Table). Although no significant difference was seen among age groups, none of the samples from persons 0–19 years of age were seropositive, whereas seroprevalence levels were increasing in those 20–59 years (2.65%) and 60–89 years (3.37%). This increase would be expected because the probability of contacting the virus increases with age. The main risk factor for the 20–29 year age group was the tick bite, and farming and contact with animals were incriminated in the older age groups. Table Univariate and multivariate regression analysis of CCHFV seropositivity in human population, Bulgaria* A similar study conducted in Greece, a neighboring country, showed an overall seroprevalence of 4.2%; slaughtering and agricultural activities were significant risk factors for CCHFV seropositivity (6). Notably, the seroprevalence levels in the Greek districts Rodopi and Evros (4.95% and 4.49%, respectively) were similar to those in neighboring Bulgarian districts Kardjali and Haskovo (6% and 4.6%, respectively). We found that the risk for seropositivity was increased 5.4-fold in persons bitten by ticks. Increased tick aggressiveness in years that have favorable climatic conditions results in high rates of attacks on humans and an increased number of tick-borne diseases (7). A recent survey for CCHFV in ticks in Haskovo, Kardzhali, and Stara Zagora districts showed that 4.83%, 2.09%, and 1.46%, respectively, were infected by CCHFV, and that the most infected tick was Hyalomma marginatum (8). These results coincide with those of the current study because Kardzhali and Haskovo were among the districts with the highest seropositivity. Because of the increasing spread of CCHFV in new foci, public health awareness of this problem is essential. Studies giving information about the spread and ecology of the virus can provide the necessary data for risk assessment analysis and even for prediction of epidemics.

Cytokines as biomarkers of Crimean-Congo hemorrhagic fever.

Crimean‐Congo hemorrhagic fever (CCHF) is a potentially severe disease caused by CCHF virus. As in other viral hemorrhagic fevers, it is considered that the course and outcome of the disease depend on the viral load and the balance among the immune response mediators, and that a fatal outcome is the result of a “cytokine storm.” The level of 27 cytokines was measured in serum samples taken from 29 patients during the acute phase of the disease. Two cases were fatal. Among survivors, significant differences between severe and non‐severe cases were observed in the levels of IP‐10, and MCP‐1, while the levels of IL‐1b, IL‐5, IL‐6, IL‐8, IL‐9, IL‐10, IL‐15, IP‐10, MCP‐1, TNF‐α, and RANTES differed significantly between fatal and non‐fatal cases (P < 0.05). RANTES was negatively correlated with the outcome of the disease. A striking similarity with the cytokine patterns seen in Ebola virus disease was observed. A weak Th1 immune response was seen. The viral load was positively correlated with IL‐10, IP‐10, and MCP‐1 levels, and negatively correlated with the ratio IL‐12/IL‐10. Especially IP‐10 and MCP‐1 were significantly associated with the viral load, the severity and outcome of the disease, and they could act as biomarkers and, probably, as potential targets for treatment strategies design. J. Med. Virol. 88:21–27, 2016.

Vascular Endothelial Growth Factor Levels in Dobrava/Belgrade Virus Infections

The levels of vascular endothelial growth factor-A (VEGF) were estimated in 102 serum samples from 63 hospitalized Greek patients with hemorrhagic fever with renal syndrome (HFRS) caused by Dobrava/Belgrade virus. Significantly higher VEGF levels were seen in the severe when compared with non-severe cases (mean values 851.96 pg/mL and 326.75 pg/mL, respectively; p = 0.003), while a significant difference was observed among groups based on the day after the onset of illness. In both severe and non-severe cases, VEGF peaked in the second week of illness; however, elevation of VEGF in the severe cases started later and remained high until convalescence, suggesting that the role of VEGF was associated with repair of vascular damage rather than with increased permeability.

Neuroinvasive Phlebovirus Infection in Greece: A Case Report

Objective: Sandfly fever phleboviruses are endemic in Mediterranean countries. We report a febrile phlebovirus case in a Greek patient who presented signs of neuroinvasive infection. Methods: In summer 2010, a 20-year-old male was admitted to hospital with fever and lethargy; he was a resident of central Macedonia, northern Greece, where a large outbreak of West Nile virus (WNV) infections occurred at that time. Since there was no laboratory evidence of WNV infection, the patients serum and cerebrospinal fluid were tested for a probable phlebovirus infection. Results: High titers of IgM and IgG antibodies against Toscana virus were detected in serum and cerebrospinal fluid, while the titers against sandfly fever Naples virus were lower; no reactivity was detected against sandfly Sicilian and Cyprus viruses. Since neutralization assays were not performed and PCR resulted in being negative, it was concluded that the causative agent was a phlebovirus of the sandfly fever Naples serocomplex. Conclusion: The present case confirms results from previous seroprevalence studies showing that phleboviruses of the sandfly fever Naples serocomplex are present in Greece and provides evidence that they cause febrile neuroinvasive disease in humans, prompting for inclusion of phleboviral infections in the differential diagnosis of acute febrile cases during the time when sandflies are active.

Crimean-Congo Hemorrhagic Fever: Tick-Host-Virus Interactions

Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans by bite of infected ticks or by direct contact with blood or tissues of viremic patients or animals. It causes to humans a severe disease with fatality up to 30%. The current knowledge about the vector-host-CCHFV interactions is very limited due to the high-level containment required for CCHFV studies. Among ticks, Hyalomma spp. are considered the most competent virus vectors. CCHFV evades the tick immune response, and following its replication in the lining of the ticks midgut, it is disseminated by the hemolymph in the salivary glands and reproductive organs. The introduction of salivary gland secretions into the host cells is the major route via which CCHFV enters the host. Following an initial amplification at the site of inoculation, the virus is spread to the target organs. Apoptosis is induced via both intrinsic and extrinsic pathways. Genetic factors and immune status of the host may affect the release of cytokines which play a major role in disease progression and outcome. It is expected that the use of new technology of metabolomics, transcriptomics and proteomics will lead to improved understanding of CCHFV-host interactions and identify potential targets for blocking the CCHFV transmission.

Phlebovirus infections in Greece

Sandfly fever viruses are endemic in the Mediterranean region causing to humans asymptomatic or mild infections to severe neurological syndromes. In order to investigate the epidemiology of phlebovirus infections in Greece, samples from 499 patients with acute febrile illness (50.2% accompanied by neurological symptoms) were tested during 2010–2014 by serological and molecular methods. Phlebovirus infection was detected in 40 (8%) patients, 25 of them presenting acute neurological infection. Most cases were observed in the summer, with a peak in August. Increased number of cases was observed in 2013, and three of them were observed in Athens. Toscana virus lineage C RNA was detected in one encephalitis case, while the serological results showed that most cases were caused by phleboviruses belonging to the sandfly fever Naples virus serocomplex. This study provided the first insight into the epidemiology of phleboviral disease in Greece. J. Med. Virol. 87:1072–1076, 2015.

Molecular epidemiology of Dobrava-Belgrade virus in Greece

In order to gain an insight into the genetic relatedness of the Dobrava-Belgrade virus (DOBV) in Greece, a phylogenetic analysis was performed based on all currently available DOBV sequences obtained from hospitalized cases with hemorrhagic fever with renal syndrome (HFRS). Most cases occurred in northwestern and north central part of the country. Two sequence datasets consisted of 41 S and 12 M partial DOBV RNA segment sequences were analyzed. All DOBV strains belong to Dobrava genotype which is associated with the rodent Apodemus flavicollis. In both phylogenetic trees (S and M segments), two main clusters of Greek strains could be distinguished. Phylogenetic analysis showed a spatial rather than temporal relation of the strains, since their genetic clustering was highly associated with the geographic distribution of the cases. Besides previous characterized endemic foci, novel ones have been identified, expanding our knowledge on the epidemiology of HFRS in Greece.