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Crimean-Congo hemorrhagic fever: seroprevalence and risk factors among humans in Achaia, western Greece
International Journal of Infectious Diseases 17 (2013) e1160–e1165
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International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Crimean-Congo hemorrhagic fever: seroprevalence and risk factors among humans in Achaia, western Greece Maria Sargianou a,b, George Panos a, Andreas Tsatsaris c, Charalambos Gogos a, Anna Papa b,* a b c
Division of Infectious Diseases, Department of Internal Medicine, Patras University General Hospital, Patras, Greece Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, 54006, Greece Laboratory of GeoInformatics, Department of Surveying Engineering, Technological Educational Institute of Athens, Athens, Greece
A R T I C L E I N F O
S U M M A R Y
Article history: Received 16 May 2013 Received in revised form 28 June 2013 Accepted 20 July 2013
Background: The Crimean-Congo hemorrhagic fever virus (CCHFV) presents a wide distribution, with the Balkan Peninsula being among the endemic regions. To date, only one CCHF case has been reported in Greece; however, based on seroprevalence data, there is evidence that CCHFV circulates in the country. Achaia is a prefecture in western Greece that has not previously been studied for CCHFV. Objectives: The aim of this study was to estimate the seroprevalence of CCHFV in humans in Achaia Prefecture, Greece, and to assess possible factors playing a role in seropositivity. Methods: A total of 207 serum samples from people of all age groups, from both urban and rural areas, were prospectively collected and tested for IgG antibodies against CCHFV. Results: The overall seroprevalence was 3.4%, with significant differences among municipalities. An agro-pastoral occupation, contact with sheep and goats, former tick bite, increasing age, and living at an altitude of 400 m, on specific land cover types, were significantly associated with CCHFV seropositivity. Conclusions: A relatively high seroprevalence was detected in a previously unstudied region of Greece, where CCHFV infection seems to occur mainly through tick bites. Further investigations are needed to identify the circulating CCHFV strains in Greece, in order to gain a better understanding of CCHFV ecology and epidemiology in the country. ß 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Corresponding Editor: Hakan Leblebicioglu, Samsun, Turkey Keywords: Crimean-Congo hemorrhagic fever Seroprevalence Epidemiology Greece Risk factors
1. Introduction Crimean-Congo hemorrhagic fever (CCHF) is a viral disease that is asymptomatic in infected animals, but may cause serious health problems in humans. CCHF patients present with fever and nonspecific symptoms, which often progress to a serious hemorrhagic syndrome, with a case fatality rate of up to 30%.1 The CCHF virus (CCHFV) is transmitted to humans through a bite from, or the crushing of, infected ticks, and through direct contact with the blood or tissues of viremic patients or livestock. Consequently, specific occupations are at greater risk of CCHFV infection, such as farmers, stockbreeders, butchers, veterinarians, and healthcare workers.2–4 In general, residents of rural areas and persons performing outdoor activities are more prone to CCHFV infection.5–8 CCHFV is highly divergent and presents the most wide geographic range among all the medically important tick-borne viruses; cases have been described in several areas of Africa, Asia,
* Corresponding author. Tel.: +30 2310 999006; fax: +30 2310 999151. E-mail address: [email protected] (A. Papa).
and Europe. Several endemic foci exist in the Balkan Peninsula, where the disease occurs in sporadic or epidemic form.9–11 The viral strains circulating in Greece do not show the same pathogenicity: the first CCHFV strain in Greece (AP92) isolated from Rhipicephalus bursa ticks collected from goats, has not been related to any severe clinical case thus far,12,13 while the single CCHF human case reported in 2008 was caused by a different strain (Rhodopi strain, GenBank accession number EU871766). This Rhodopi strain was genetically similar to respective Balkan, Turkish, and Russian strains, differing from the AP92 strain by 25% in the S RNA segment.13 The significant presence of human CCHFV antibodies among the Greek population, as reported in previous studies, is contradictory to the low number of diagnosed CCHF cases, a disparity that may be explained by a low pathogenicity of the prevalent strain (AP92, or others as yet unidentified), suggesting that this phenomenon might be strainrelated.14,15 Achaia is a Greek prefecture (population 309 694)16 in the north of the Peloponnese Peninsula, in southern Greece (Figure 1), where the local climate is moderate to temperate, with a mean temperature of 17–18 8C near the sea, and some degrees lower in the mountainous areas. This type of climate, along with the local
1201-9712/$36.00 – see front matter ß 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijid.2013.07.015
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Figure 1. Areas of residence of CCHFV IgG-positive persons in Achaia Prefecture, western Greece (inset). The size of the circle indicates the number of people with a positive test result at each location.
geomorphology and vegetation, potentially favors the survival and spread of Hyalomma spp ticks, the most common vector of the virus,17 but also the spread of Rhipicephalus spp ticks, which are associated with the AP92 strain.12 Since this region has not been studied previously for CCHFV, the aim of the present study was to estimate the seroprevalence in humans in an attempt to detect possible endemic foci of the virus, and to assess possible risk factors for the acquisition of CCHFV infection. 2. Methods A cross-sectional study was conducted in the regional unit of Achaia. A representative sample of 258 human sera was collected prospectively between March and July 2012 (only four individuals refused to participate in the study). The sample size was estimated using Epi Info software, with the assumption of an expected seroprevalence of 4% in Greece.14 The participants were selected randomly, regardless of CCHF risk factors, among persons without signs of infectious disease, referred for routine blood testing or for blood donation to Patras University General Hospital and to four additional primary healthcare centers (in Chalandritsa, Kalavrita, Kato Achaia, and Klitoria). Fifty-one samples were rejected from the final cohort as they failed to fulfill the inclusion criterion (residents only from Achaia Prefecture). Thus, 207 samples were included in the study from residents of the following municipalities: six from Egialia, 27 from Erimanthos, 17 from Kalavrita, 130 from Patra, and 27 from West Achaia. Information regarding the virus and the purpose of the present study was provided by physicians, and written consent was obtained from all participants before sampling. A standardized questionnaire employed in previous studies was used to collect
demographics and information on potential CCHF risk factors, such as animal contact and tick bite.15 The study was approved by the Bioethics Committee of the Medical School of the University of Patras. 2.1. Inclusion criteria and management of samples Individuals aged over 14 years without any profound symptoms of infectious disease were included in the study. Only persons residing in Achaia were included in the study (n = 207). Serum samples were stored at 80 8C until their transportation with ice packs to the National Reference Laboratory for Arboviruses at Aristotle University of Thessaloniki, where testing was performed. 2.2. Serological assays Serum samples were tested for CCHFV IgG antibodies by ELISA (Vector-Best, Koltsovo, Novosibirsk, Russia), in accordance with the manufacturer’s instructions. All the ELISA-positive samples were tested for CCHFV IgM antibodies by the same commercial ELISA. In addition, the ELISA-positive serum samples were further tested by indirect immunofluorescence assay (IFA) (Euroimmun IIFT: Crimean-Congo fever virus Mosaic 2 (IgG); Medizinische Labordiagnostika AG, Lu¨beck, Germany); this technique enables the discrimination between CCHFV nucleoprotein-specific (N) and CCHFV glycoprotein Gc-specific (GPC) IgG antibodies. 2.3. Data analysis The data were analyzed using IBM SPSS v. 20 (IBM Corp., Armonk, NY, USA). The association between seropositivity and
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categorical variables was estimated by Chi-square test or Fisher’s exact test (when the expected count was <5), while the Student’s ttest was performed for continuous variables. A p-value of <0.05 was set as the threshold defining a covariate as a statistically significant risk factor. Univariate logistic regression analysis was performed to calculate the odds ratio (OR) and the 95% confidence interval (95% CI), and to identify possible risk factors for inclusion in the final multivariate model. Variables with a p-value of <0.05 in the univariate regression analysis were retained in the multivariate model, which was built with a stepwise forward selection. Statistical results mentioned in the text refer to the univariate analysis, if not specified otherwise. 2.4. Mapping In order to map the sites where seropositive persons were detected, the GIS software ArcGIS v.9.3.1 (Esri, Redlands, CA, USA) was used. The following layers were implemented: (1) the location of the sites by transfer of the information from Google Earth; (2) the digital elevation model using corresponding information from the Greek Databank of Hydrological and Meteorological Information (http://www.hydroscope.gr) that represents the relief of the Greek State at a scale of 1:50 000 (cell size 25 m 25 m); (3) the land cover of the area of focus using the CORINE Land Cover Program (http://www.eea.europa.eu/publications/COR0-landcover); and (4) the administrative division (prefectures (NUTS 3) and municipalities (NUTS 4)), with the corresponding annotation of the areas.
CCHFV infection (Table 2). A statistically significant negative association of altitude <80 m with CCHFV seropositivity (OR 0.059, 95% CI 0.007–0.498, p = 0.009) was also found, most likely due to the presence of urban centers at this level, such as the city of Patras. Seroprevalence differed significantly among municipalities (p = 0.001): four of the seven seropositive persons were detected in the Erimanthos municipality (4/27, 15%), and one each in the municipalities of Egialia (1/6, 17%), Kalavrita (1/17, 6%), and West Achaia (1/27, 4%), while no seropositive person was found in the Patra municipality (0/130, 0%). Six land cover types were observed in the study: continuous urban fabrics, discontinuous urban fabrics, vineyards, areas with complex cultivation patterns, non-irrigated arable lands, and lands principally occupied by agriculture with significant areas of natural vegetation. The significantly related land types were non-irrigated arable land (OR 24.25, 95% CI 1.807–325.389, p = 0.016) and land principally occupied by agriculture with significant areas of natural vegetation (OR 24.25, 95% CI 3.399– 173.023, p = 0.001). Concerning occupation, four of the seven seropositive persons were principally occupied with either farming or animal husbandry, or both; two of the others were housewives and one was a retiree. All seven tended sheep and four of them also tended goats (Table 1). Multivariate logistic regression analysis showed that former tick bite (OR 18.3, 95% CI 2.22–151, p = 0.007), an agro-pastoral occupation (OR 6.99, 95% CI 1.01–48.4, p = 0.049), and living at an altitude of 400 m (OR 17, 95% CI 2.48–117, p = 0.004) were significantly associated with CCHFV seropositivity.
3. Results In total, seven of the 207 (3.4%) study subjects were found to carry CCHFV IgG antibodies; four were male and three female. The sites where seropositive persons were detected are shown in Figure 1. Demographic and laboratory data of the seven seropositive persons are presented in Table 1. CCHFV IgM antibodies were not detected in any of these persons. Furthermore, all were positive by IgG IFA (Figure 2). The amount of immunofluorescence was proportional to the optical density (OD) of the ELISA (Table 1). A strong granular immunofluorescence was observed in one of the samples obtained from a 75-year male from West Achaia, suggestive of a relatively recent infection (however, no IgM antibodies were detected). Univariate logistic regression analysis showed that increased age, an agro-pastoral occupation, contact with sheep and goats, former tick bite, living at an altitude of 400 m, living on a specific land type, and living in the Erimanthos municipality were significantly associated with CCHFV seropositivity in Achaia (p < 0.05); gender, slaughtering, hunting, and frequently walking in forests did not present a statistically significant association with
4. Discussion Achaia is a prefecture in southern Greece. A 3.4% CCHFV seroprevalence was detected among residents of the region. Seropositive individuals were encountered in rural areas in four of the five municipalities, most of whom (4/7) lived in Erimanthos, a municipality in central Achaia (8877 population, population density 15.25/km2).16 The local economy of Erimanthos is based almost entirely on livestock farming, which places it first in the animal-farming ranking among the municipalities of Achaia; animal farming primarily involves sheep and goats, mainly kept under semi-free range conditions,17 facilitating their infestation with ticks. This area presents the highest livestock density, calculated at 320.716/km2, compared to densities ranging from 44.423 to 165.546/km2 in the other municipalities of Achaia.16,17 Erimanthos also presents the highest livestock/human population density ratio. Approximately 65% of the local land is grassland for livestock grazing, while the rest consists of croplands.17 Total annual rainfall was estimated at 800–1000 mm in 2012.18
Table 1 Characteristics of CCHFV-seropositive individuals No.
1 2 3 4 5 6 7
Sex
F M F M F M M
Age
53 61 80 77 80 45 75
Municipality
Egialia Erimanthos Erimanthos Erimanthos Erimanthos Kalavrita W. Achaia
Village
Kouloura Chrisopigi Helliniko Chrisavgi Kakavouleika Likouria Petas
Altitude (m)
67 671 560 438 400 767 162
Land covera
112 243 242 243 211 112 242
Agro-pastoral occupation
No Yes No Yes No Yes Yes
Contact with:
Tick bite
Sheep
Goats
Cattle
Yes Yes Yes Yes Yes Yes Yes
No Yes No Yes Yes Yes No
No No No No No No No
Yes No Yes Yes Yes Yes No
ELISA IgM
Neg Neg Neg Neg Neg Neg Neg
ELISA IgG (AU) Positive >0.2
0.85 2.412 Out of range 1.290 0.626 1.2 Out of range
IFA IgG N antigen
GPC antigen
+ ++ ++ ++
++ +++
+/ +++
+ + +++
CCHFV, Crimean-Congo hemorrhagic fever virus; IFA, immunofluorescence assay; F, female; M, male. a Land cover types: 112, discontinuous urban fabric; 211, non-irrigated arable land; 242, complex cultivation patterns; 243, land principally occupied by agriculture with significant areas of natural vegetation.
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Figure 2. Indirect immunofluorescence assay (Euroimmun IIFT: Crimean-Congo fever virus Mosaic 2 IgG) on a serum sample from a CCHFV IgG-positive person: (A) no cells; (B) uninfected cells; (C) N antigen; (D) GPC antigen.
In contrast, none of the 130 sampled residents of the Patra municipality (213 984 population, population density 639.03/km2) tested CCHFV IgG-positive. The local land is classified as continuous urban fabric, and neither animal farming nor agriculture (which mainly consists of olive groves) are as developed as in Erimanthos.17 The city of Patras, the capital of the prefecture and third largest city in population in Greece, is located at an altitude of 20 m, with total annual rainfall ranging between 700 and 900 mm.19 Since the average high and low temperatures do not differ much between the municipalities of Erimanthos and Patra,18,19 it is likely that components other than climate have a greater impact on CCHFV circulation (reflecting the seropositivity), such as the type and density of vegetation, the land use, the livestock density (and livestock/human population density ratio), and the more frequent ownership of livestock. In general, it was observed that seropositive individuals resided in the foothills of semi-mountainous or mountainous areas, and 5/ 7 lived in areas at an altitude of 400 m. This can be attributed to the fact that pasture lands, sheep farms and ranches are mostly located at these altitudes. This finding is in agreement with a study conducted in Turkey during 2007–2011, where it was found that 94.4% of acute CCHF cases lived at an altitude of 300 m, and 84.9% at an altitude between 600 and 950 m.6 Although according to the multivariate model increased age was not found to be a significant independent predisposing factor
to seropositivity, it was observed that the median age differed greatly between IgG-positive and IgG-negative persons (75 and 46 years, respectively). The mean OR for risk of infection is 1.056 for each additional year of age, which amounts to a cumulative mean OR of 1.72 per decade. Agro-pastoral occupation served as a significant and independent risk factor for acquisition of viral infection in Achaia; this association was not observed in Russia, where occupation was not believed to strictly correlate with CCHFV seropositivity.20 Four of the seven seropositive individuals reported either farming or animal husbandry, or both, as their principal occupation, underlining the importance of this factor in CCHFV infection in this region. This significant relationship between employment in farming or animal husbandry and CCHFV infection has also been stated in studies from neighboring countries, such as Albania, Bulgaria, and Turkey.10,21,22 Tick bite and tending livestock, especially sheep and goats (which are often tick-infested), were found to play a key role in CCHFV human infection in Achaia. In other studies, tending cattle has also been found to be associated with CCHFV seropositivity;23 this was not seen in the present study, most probably because the density of cattle in Achaia is much lower (2.95/km2) than that of sheep (110.87/km2) and goats (49.91/km2). Thus, it seems that residents of this area are mostly infected through tick bites during tending of sheep and goats. Infection after contact with infected animal blood or tissues through slaughtering seems less probable
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Table 2 Univariate logistic regression analysis of risk factor contribution to CCHFV infection
Age, years, median (IQR) Missing data Sex Female Male Municipality Erimanthos Other Agro-pastoral occupation Yes No (other) Missing data Contact with goats Yes No Contact with sheep Yes No Contact with cows Yes No Hunting Yes No Missing data Slaughtering Yes No Missing data Visit to woods Yes No Missing data Tick bite Yes No Altitude Altitude 400 m Altitude <400 m
IgG positive (n = 7)
IgG negative (n = 200)
p-Value
75 (53–80) 0
46 (32–62) 10
0.018
1.056
1.009–1.104
0.922
0.927
0.202–4.250
3 (42.9%) 4 (57.1%)
82 (41%) 118 (59%)
4 (57.1%) 3 (42.9%)
23 (11.5%) 177 (88.5%)
4 (57.1%) 3 (42.9%) 0
32 (16.4%) 163 (83.6%) 5
4 (57.1%) 3 (42.9%)
28 (14%) 172 (86%)
7 (100%) 0 (0%)
32 (16%) 168 (84%)
0 (0%) 7 (100%)
8 (4%) 192 (96%)
1 (16.7%) 5 (83.3%) 1
27 (13.5%) 173 (86.5%) 0
2 (28.6%) 5 (71.4%) 0
37 (18.6%) 162 (81.4%) 1
2 (33.3%) 4 (66.7%) 1
67 (34.2%) 129 (65.8%) 4
5 (71.4%) 2 (28.6%)
43 (21.5%) 157 (78.5%)
5 (71.4%) 2 (28.6%)
25 (12.5%) 175 (87.5%)
OR
95% CI
0.001a 0.003
10.261
2.159–48.767
0.015
6.792
1.450–31.813
0.008
8.190
1.740–38.563
0.824
1.281
0.144–11.393
0.513
1.751
0.327–9.381
0.965
0.963
0.172–5.391
0.010
9.128
1.711–48.691
0.001
17.500
3.221–95.080
<0.001b
1.000b
CCHFV, Crimean-Congo hemorrhagic fever virus; OR, odds ratio; CI, confidence interval; IQR, interquartile range. a Pearson’s Chi-square test. b Estimated by Fisher’s exact test.
in Achaia, although it was found to be the primary risk factor in other parts of Greece.14 This reflects how socio-demographic and environmental factors interweave and contribute to the acquisition of CCHFV infection through different routes, even within the borders of a country; this reaffirms the complexity of the life cycle and ecology of the virus. All seven seropositive persons were CCHFV IgM-negative, and none recalled any illness resembling CCHF (fever accompanied by hemorrhagic manifestations). This finding may support the preexisting theory that CCHFV seropositivity in Greece is due to an unnoticed infection caused by non-pathogenic or low pathogenic viral strains. The CCHFV IgG seroprevalence in the Achaia Prefecture is similar to that observed in northeastern Greece15 where the fatal case was observed in June 2008.13 Identification of 537 ticks removed from humans who were referred for this reason to two hospitals in northeastern Greece, showed that the vast majority (81.5%) were Rhipicephalus sanguineus, while Hyalomma marginatum accounted for 5.2%.24 Molecular screening of ticks is needed in Achaia Prefecture, and in Greece in general, to identify the circulating CCHFV strains. Recently a study on 3671 adult volunteers reported from Turkey, covering most of the endemic regions, showed a seroprevalence of 10%, and it was demonstrated that 88% of CCHF
infections were subclinical.25 It has to be mentioned that AP92-like strains have been detected in Turkey26 and they might play a role in the subclinical infections and the increased seroprevalence. Studies in ticks and serosurveys in animals are needed to gain an insight into the CCHFV ecology and epidemiology in the country. Clinicians should include CCHF in the differential diagnosis of an acute febrile case, even without thrombocytopenia, during the season when Hyalomma and Rhipicephalus ticks are active, especially when the patient has an agro-pastoral occupation.
Acknowledgements We thank the participants who responded to the call of the study. The assistance of Dr M. Karakantza and the clinicians of the Patras University General Hospital Blood Bank, and of Dr G. Mantzouranis and the clinicians of the primary healthcare facilities, is highly appreciated. Funding: The present work was supported by the FP7 project ‘‘ANTIcipating the Global Onset of Novel Epidemics’’ (ANTIGONE), Grant Agreement No. 278976. Ethical approval: The current study was approved by the Bioethics Committee of the Medical School of the University of Patras. Conflict of interest: No conflict of interest to declare.
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References 1. Papa A. Crimean-Congo hemorrhagic fever and hantavirus infections. In: Maltezou HC, Gikas A, editors. Tropical and emerging infectious diseases. India: Research Signpost; 2010. p. 49–73. 2. Mardani M, Rahnavardi M, Rajaeinejad M, Naini KH, Chinikar S, Pourmalek F, et al. Crimean-Congo hemorrhagic fever among health care workers in Iran: a seroprevalence study in two endemic regions. Am J Trop Med Hyg 2007;76:443–5. 3. Izadi S, Naieni KH, Madjdzadeh SR, Nadim A. Crimean-Congo hemorrhagic fever in Sistan and Baluchestan Province of Iran, a case–control study on epidemiological characteristics. Int J Infect Dis 2004;8:299–306. 4. Ergonul O, Zeller H, Kilic S, Kutlu S, Kutlu M, Cavusoglu S, et al. Zoonotic infections among veterinarians in Turkey: Crimean-Congo hemorrhagic fever and beyond. Int J Infect Dis 2006;10:465–9. 5. Yilmaz GR, Buzgan T, Irmak H, Safran A, Uzun R, Cevik MA, et al. The epidemiology of Crimean-Congo hemorrhagic fever in Turkey, 2002–2007. Int J Infect Dis 2009;13:380–6. 6. Sisman A. Epidemiologic features and risk factors of Crimean-Congo hemorrhagic fever in Samsun Province, Turkey. J Epidemiol 2013;23:95–102. Epub ahead of print. 7. Vatansever Z, Uzun R, Estrada-Pena A, Ergonul O. Crimean-Congo hemorrhagic fever in Turkey. In: Ergonul O, Whitehouse CA, editors. Crimean-Congo hemorrhagic fever: a global perspective. Netherlands: Springer; 2007. p. 59–74. 8. Grard G, Drexler JF, Fair J, Muyembe JJ, Wolfe ND, Drosten C, et al. Re-emergence of Crimean-Congo hemorrhagic fever virus in Central Africa. PLoS Negl Trop Dis 2011;5:e1350. 9. Papa A, Christova I, Papadimitriou E, Antoniadis A. Crimean-Congo hemorrhagic fever in Bulgaria. Emerg Infect Dis 2004;10:1465–7. 10. Papa A, Velo E, Papadimitriou E, Cahani G, Kota M, Bino S. Ecology of the Crimean-Congo hemorrhagic fever endemic area in Albania. Vector Borne Zoonotic Dis 2009;9:713–6. 11. Christova I, Di Caro A, Papa A, Castilletti C, Andonova L, Kalvatchev N, et al. Crimean-Congo hemorrhagic fever, southwestern Bulgaria. Emerg Infect Dis 2009;15:983–5. 12. Papadopoulos O, Koptopoulos G. Crimean-Congo hemorrhagic fever (CCHF) in Greece: isolation of the virus from Rhipicephalus bursa ticks and a preliminary serological survey. Zentbl Bakteriol Hyg 1980;1:189–93. 13. Papa A, Dalla V, Papadimitriou E, Kartalis GN, Antoniadis A. Emergence of CrimeanCongo haemorrhagic fever in Greece. Clin Microbiol Infect 2010;16:843–7.
e1165
14. Sidira P, Maltezou HC, Haidich AB, Papa A. Seroepidemiological study of Crimean-Congo haemorrhagic fever in Greece, 2009–2010. Clin Microbiol Infect 2012;18:E16–9. 15. Papa A, Tzala E, Maltezou HC. Crimean-Congo hemorrhagic fever virus, northeastern Greece. Emerg Infect Dis 2011;17:141–3. 16. Hellenic Statistical Authority. Announcement Census 2011—resident population. Piraeus, Greece; 2012. Available at: http://www.statistics.gr/portal/page/ portal/ESYE/BUCKET/A1602/PressReleases/ A1602_SAM01_DT_DC_00_2011_02_F_EN.pdf (Last accessed 20 March 2013). 17. Hoogstraal H. The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. J Med Entomol 1979;15:307–417. 18. KTADA in collaboration with the National Observatory of Athens. Annual climatological summary, Chalandritsa, 2012. Available at: http://penteli.meteo.gr/stations/chalandritsa/NOAAPRYR.TXT (Last accessed 20 March 2013). 19. National Observatory of Athens. Annual climatological summary, Patras, 2012. Available at: http://penteli.meteo.gr/stations/patra/NOAAPRYR.TXT (Last accessed 20 March 2013). 20. Butenko AM, Karganova GG. Crimean-Congo hemorrhagic fever in Russia and other countries of the former Soviet Union. In: Ergonul O, Whitehouse CA, editors. Crimean-Congo hemorrhagic fever: a global perspective. Netherlands: Springer; 2007. p. 99–114. 21. Gunes T, Engin A, Poyraz O, Elaldi N, Kaya S, Dokmetas I, et al. Crimean-Congo hemorrhagic fever virus in high-risk population, Turkey. Emerg Infect Dis 2009;15:461–4. 22. Christova I, Gladnishka T, Taseva E, Kalvatchev N, Tsergouli K, Papa A. Seroprevalence of Crimean-Congo hemorrhagic fever virus, Bulgaria. Emerg Infect Dis 2013;19:177–9. 23. Ertugrul B, Kirdar S, Ersoy OS, Ture M, Erol N, Ozturk B, et al. The seroprevalence of Crimean-Congo haemorrhagic fever among inhabitants living in the endemic regions of Western Anatolia. Scand J Infect Dis 2012;44:276–81. 24. Papa A, Chaligiannis I, Xanthopoulou K, Papaioakim M, Papanastasiou S, Sotiraki S. Ticks parasitizing humans in Greece. Vector Borne Zoonotic Dis 2011;11: 539–42. ¨ ngu¨ru¨ P, Uyar Y. Subclinical infections with 25. Bodur H, Akinci E, Ascioglu S, O Crimean-Congo hemorrhagic fever virus, Turkey. Emerg Infect Dis 2012;18: 640–2. 26. Midilli K, Gargili A, Ergonul O, Elevli M, Ergin S, Turan N, et al. The first clinical case due to AP92 like strain of Crimean-Congo hemorrhagic fever virus and a field survey. BMC Infect Dis 2009;9:90.
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International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Crimean-Congo hemorrhagic fever: seroprevalence and risk factors among humans in Achaia, western Greece Maria Sargianou a,b, George Panos a, Andreas Tsatsaris c, Charalambos Gogos a, Anna Papa b,* a b c
Division of Infectious Diseases, Department of Internal Medicine, Patras University General Hospital, Patras, Greece Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, 54006, Greece Laboratory of GeoInformatics, Department of Surveying Engineering, Technological Educational Institute of Athens, Athens, Greece
A R T I C L E I N F O
S U M M A R Y
Article history: Received 16 May 2013 Received in revised form 28 June 2013 Accepted 20 July 2013
Background: The Crimean-Congo hemorrhagic fever virus (CCHFV) presents a wide distribution, with the Balkan Peninsula being among the endemic regions. To date, only one CCHF case has been reported in Greece; however, based on seroprevalence data, there is evidence that CCHFV circulates in the country. Achaia is a prefecture in western Greece that has not previously been studied for CCHFV. Objectives: The aim of this study was to estimate the seroprevalence of CCHFV in humans in Achaia Prefecture, Greece, and to assess possible factors playing a role in seropositivity. Methods: A total of 207 serum samples from people of all age groups, from both urban and rural areas, were prospectively collected and tested for IgG antibodies against CCHFV. Results: The overall seroprevalence was 3.4%, with significant differences among municipalities. An agro-pastoral occupation, contact with sheep and goats, former tick bite, increasing age, and living at an altitude of 400 m, on specific land cover types, were significantly associated with CCHFV seropositivity. Conclusions: A relatively high seroprevalence was detected in a previously unstudied region of Greece, where CCHFV infection seems to occur mainly through tick bites. Further investigations are needed to identify the circulating CCHFV strains in Greece, in order to gain a better understanding of CCHFV ecology and epidemiology in the country. ß 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Corresponding Editor: Hakan Leblebicioglu, Samsun, Turkey Keywords: Crimean-Congo hemorrhagic fever Seroprevalence Epidemiology Greece Risk factors
1. Introduction Crimean-Congo hemorrhagic fever (CCHF) is a viral disease that is asymptomatic in infected animals, but may cause serious health problems in humans. CCHF patients present with fever and nonspecific symptoms, which often progress to a serious hemorrhagic syndrome, with a case fatality rate of up to 30%.1 The CCHF virus (CCHFV) is transmitted to humans through a bite from, or the crushing of, infected ticks, and through direct contact with the blood or tissues of viremic patients or livestock. Consequently, specific occupations are at greater risk of CCHFV infection, such as farmers, stockbreeders, butchers, veterinarians, and healthcare workers.2–4 In general, residents of rural areas and persons performing outdoor activities are more prone to CCHFV infection.5–8 CCHFV is highly divergent and presents the most wide geographic range among all the medically important tick-borne viruses; cases have been described in several areas of Africa, Asia,
* Corresponding author. Tel.: +30 2310 999006; fax: +30 2310 999151. E-mail address: [email protected] (A. Papa).
and Europe. Several endemic foci exist in the Balkan Peninsula, where the disease occurs in sporadic or epidemic form.9–11 The viral strains circulating in Greece do not show the same pathogenicity: the first CCHFV strain in Greece (AP92) isolated from Rhipicephalus bursa ticks collected from goats, has not been related to any severe clinical case thus far,12,13 while the single CCHF human case reported in 2008 was caused by a different strain (Rhodopi strain, GenBank accession number EU871766). This Rhodopi strain was genetically similar to respective Balkan, Turkish, and Russian strains, differing from the AP92 strain by 25% in the S RNA segment.13 The significant presence of human CCHFV antibodies among the Greek population, as reported in previous studies, is contradictory to the low number of diagnosed CCHF cases, a disparity that may be explained by a low pathogenicity of the prevalent strain (AP92, or others as yet unidentified), suggesting that this phenomenon might be strainrelated.14,15 Achaia is a Greek prefecture (population 309 694)16 in the north of the Peloponnese Peninsula, in southern Greece (Figure 1), where the local climate is moderate to temperate, with a mean temperature of 17–18 8C near the sea, and some degrees lower in the mountainous areas. This type of climate, along with the local
1201-9712/$36.00 – see front matter ß 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijid.2013.07.015
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Figure 1. Areas of residence of CCHFV IgG-positive persons in Achaia Prefecture, western Greece (inset). The size of the circle indicates the number of people with a positive test result at each location.
geomorphology and vegetation, potentially favors the survival and spread of Hyalomma spp ticks, the most common vector of the virus,17 but also the spread of Rhipicephalus spp ticks, which are associated with the AP92 strain.12 Since this region has not been studied previously for CCHFV, the aim of the present study was to estimate the seroprevalence in humans in an attempt to detect possible endemic foci of the virus, and to assess possible risk factors for the acquisition of CCHFV infection. 2. Methods A cross-sectional study was conducted in the regional unit of Achaia. A representative sample of 258 human sera was collected prospectively between March and July 2012 (only four individuals refused to participate in the study). The sample size was estimated using Epi Info software, with the assumption of an expected seroprevalence of 4% in Greece.14 The participants were selected randomly, regardless of CCHF risk factors, among persons without signs of infectious disease, referred for routine blood testing or for blood donation to Patras University General Hospital and to four additional primary healthcare centers (in Chalandritsa, Kalavrita, Kato Achaia, and Klitoria). Fifty-one samples were rejected from the final cohort as they failed to fulfill the inclusion criterion (residents only from Achaia Prefecture). Thus, 207 samples were included in the study from residents of the following municipalities: six from Egialia, 27 from Erimanthos, 17 from Kalavrita, 130 from Patra, and 27 from West Achaia. Information regarding the virus and the purpose of the present study was provided by physicians, and written consent was obtained from all participants before sampling. A standardized questionnaire employed in previous studies was used to collect
demographics and information on potential CCHF risk factors, such as animal contact and tick bite.15 The study was approved by the Bioethics Committee of the Medical School of the University of Patras. 2.1. Inclusion criteria and management of samples Individuals aged over 14 years without any profound symptoms of infectious disease were included in the study. Only persons residing in Achaia were included in the study (n = 207). Serum samples were stored at 80 8C until their transportation with ice packs to the National Reference Laboratory for Arboviruses at Aristotle University of Thessaloniki, where testing was performed. 2.2. Serological assays Serum samples were tested for CCHFV IgG antibodies by ELISA (Vector-Best, Koltsovo, Novosibirsk, Russia), in accordance with the manufacturer’s instructions. All the ELISA-positive samples were tested for CCHFV IgM antibodies by the same commercial ELISA. In addition, the ELISA-positive serum samples were further tested by indirect immunofluorescence assay (IFA) (Euroimmun IIFT: Crimean-Congo fever virus Mosaic 2 (IgG); Medizinische Labordiagnostika AG, Lu¨beck, Germany); this technique enables the discrimination between CCHFV nucleoprotein-specific (N) and CCHFV glycoprotein Gc-specific (GPC) IgG antibodies. 2.3. Data analysis The data were analyzed using IBM SPSS v. 20 (IBM Corp., Armonk, NY, USA). The association between seropositivity and
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categorical variables was estimated by Chi-square test or Fisher’s exact test (when the expected count was <5), while the Student’s ttest was performed for continuous variables. A p-value of <0.05 was set as the threshold defining a covariate as a statistically significant risk factor. Univariate logistic regression analysis was performed to calculate the odds ratio (OR) and the 95% confidence interval (95% CI), and to identify possible risk factors for inclusion in the final multivariate model. Variables with a p-value of <0.05 in the univariate regression analysis were retained in the multivariate model, which was built with a stepwise forward selection. Statistical results mentioned in the text refer to the univariate analysis, if not specified otherwise. 2.4. Mapping In order to map the sites where seropositive persons were detected, the GIS software ArcGIS v.9.3.1 (Esri, Redlands, CA, USA) was used. The following layers were implemented: (1) the location of the sites by transfer of the information from Google Earth; (2) the digital elevation model using corresponding information from the Greek Databank of Hydrological and Meteorological Information (http://www.hydroscope.gr) that represents the relief of the Greek State at a scale of 1:50 000 (cell size 25 m 25 m); (3) the land cover of the area of focus using the CORINE Land Cover Program (http://www.eea.europa.eu/publications/COR0-landcover); and (4) the administrative division (prefectures (NUTS 3) and municipalities (NUTS 4)), with the corresponding annotation of the areas.
CCHFV infection (Table 2). A statistically significant negative association of altitude <80 m with CCHFV seropositivity (OR 0.059, 95% CI 0.007–0.498, p = 0.009) was also found, most likely due to the presence of urban centers at this level, such as the city of Patras. Seroprevalence differed significantly among municipalities (p = 0.001): four of the seven seropositive persons were detected in the Erimanthos municipality (4/27, 15%), and one each in the municipalities of Egialia (1/6, 17%), Kalavrita (1/17, 6%), and West Achaia (1/27, 4%), while no seropositive person was found in the Patra municipality (0/130, 0%). Six land cover types were observed in the study: continuous urban fabrics, discontinuous urban fabrics, vineyards, areas with complex cultivation patterns, non-irrigated arable lands, and lands principally occupied by agriculture with significant areas of natural vegetation. The significantly related land types were non-irrigated arable land (OR 24.25, 95% CI 1.807–325.389, p = 0.016) and land principally occupied by agriculture with significant areas of natural vegetation (OR 24.25, 95% CI 3.399– 173.023, p = 0.001). Concerning occupation, four of the seven seropositive persons were principally occupied with either farming or animal husbandry, or both; two of the others were housewives and one was a retiree. All seven tended sheep and four of them also tended goats (Table 1). Multivariate logistic regression analysis showed that former tick bite (OR 18.3, 95% CI 2.22–151, p = 0.007), an agro-pastoral occupation (OR 6.99, 95% CI 1.01–48.4, p = 0.049), and living at an altitude of 400 m (OR 17, 95% CI 2.48–117, p = 0.004) were significantly associated with CCHFV seropositivity.
3. Results In total, seven of the 207 (3.4%) study subjects were found to carry CCHFV IgG antibodies; four were male and three female. The sites where seropositive persons were detected are shown in Figure 1. Demographic and laboratory data of the seven seropositive persons are presented in Table 1. CCHFV IgM antibodies were not detected in any of these persons. Furthermore, all were positive by IgG IFA (Figure 2). The amount of immunofluorescence was proportional to the optical density (OD) of the ELISA (Table 1). A strong granular immunofluorescence was observed in one of the samples obtained from a 75-year male from West Achaia, suggestive of a relatively recent infection (however, no IgM antibodies were detected). Univariate logistic regression analysis showed that increased age, an agro-pastoral occupation, contact with sheep and goats, former tick bite, living at an altitude of 400 m, living on a specific land type, and living in the Erimanthos municipality were significantly associated with CCHFV seropositivity in Achaia (p < 0.05); gender, slaughtering, hunting, and frequently walking in forests did not present a statistically significant association with
4. Discussion Achaia is a prefecture in southern Greece. A 3.4% CCHFV seroprevalence was detected among residents of the region. Seropositive individuals were encountered in rural areas in four of the five municipalities, most of whom (4/7) lived in Erimanthos, a municipality in central Achaia (8877 population, population density 15.25/km2).16 The local economy of Erimanthos is based almost entirely on livestock farming, which places it first in the animal-farming ranking among the municipalities of Achaia; animal farming primarily involves sheep and goats, mainly kept under semi-free range conditions,17 facilitating their infestation with ticks. This area presents the highest livestock density, calculated at 320.716/km2, compared to densities ranging from 44.423 to 165.546/km2 in the other municipalities of Achaia.16,17 Erimanthos also presents the highest livestock/human population density ratio. Approximately 65% of the local land is grassland for livestock grazing, while the rest consists of croplands.17 Total annual rainfall was estimated at 800–1000 mm in 2012.18
Table 1 Characteristics of CCHFV-seropositive individuals No.
1 2 3 4 5 6 7
Sex
F M F M F M M
Age
53 61 80 77 80 45 75
Municipality
Egialia Erimanthos Erimanthos Erimanthos Erimanthos Kalavrita W. Achaia
Village
Kouloura Chrisopigi Helliniko Chrisavgi Kakavouleika Likouria Petas
Altitude (m)
67 671 560 438 400 767 162
Land covera
112 243 242 243 211 112 242
Agro-pastoral occupation
No Yes No Yes No Yes Yes
Contact with:
Tick bite
Sheep
Goats
Cattle
Yes Yes Yes Yes Yes Yes Yes
No Yes No Yes Yes Yes No
No No No No No No No
Yes No Yes Yes Yes Yes No
ELISA IgM
Neg Neg Neg Neg Neg Neg Neg
ELISA IgG (AU) Positive >0.2
0.85 2.412 Out of range 1.290 0.626 1.2 Out of range
IFA IgG N antigen
GPC antigen
+ ++ ++ ++
++ +++
+/ +++
+ + +++
CCHFV, Crimean-Congo hemorrhagic fever virus; IFA, immunofluorescence assay; F, female; M, male. a Land cover types: 112, discontinuous urban fabric; 211, non-irrigated arable land; 242, complex cultivation patterns; 243, land principally occupied by agriculture with significant areas of natural vegetation.
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Figure 2. Indirect immunofluorescence assay (Euroimmun IIFT: Crimean-Congo fever virus Mosaic 2 IgG) on a serum sample from a CCHFV IgG-positive person: (A) no cells; (B) uninfected cells; (C) N antigen; (D) GPC antigen.
In contrast, none of the 130 sampled residents of the Patra municipality (213 984 population, population density 639.03/km2) tested CCHFV IgG-positive. The local land is classified as continuous urban fabric, and neither animal farming nor agriculture (which mainly consists of olive groves) are as developed as in Erimanthos.17 The city of Patras, the capital of the prefecture and third largest city in population in Greece, is located at an altitude of 20 m, with total annual rainfall ranging between 700 and 900 mm.19 Since the average high and low temperatures do not differ much between the municipalities of Erimanthos and Patra,18,19 it is likely that components other than climate have a greater impact on CCHFV circulation (reflecting the seropositivity), such as the type and density of vegetation, the land use, the livestock density (and livestock/human population density ratio), and the more frequent ownership of livestock. In general, it was observed that seropositive individuals resided in the foothills of semi-mountainous or mountainous areas, and 5/ 7 lived in areas at an altitude of 400 m. This can be attributed to the fact that pasture lands, sheep farms and ranches are mostly located at these altitudes. This finding is in agreement with a study conducted in Turkey during 2007–2011, where it was found that 94.4% of acute CCHF cases lived at an altitude of 300 m, and 84.9% at an altitude between 600 and 950 m.6 Although according to the multivariate model increased age was not found to be a significant independent predisposing factor
to seropositivity, it was observed that the median age differed greatly between IgG-positive and IgG-negative persons (75 and 46 years, respectively). The mean OR for risk of infection is 1.056 for each additional year of age, which amounts to a cumulative mean OR of 1.72 per decade. Agro-pastoral occupation served as a significant and independent risk factor for acquisition of viral infection in Achaia; this association was not observed in Russia, where occupation was not believed to strictly correlate with CCHFV seropositivity.20 Four of the seven seropositive individuals reported either farming or animal husbandry, or both, as their principal occupation, underlining the importance of this factor in CCHFV infection in this region. This significant relationship between employment in farming or animal husbandry and CCHFV infection has also been stated in studies from neighboring countries, such as Albania, Bulgaria, and Turkey.10,21,22 Tick bite and tending livestock, especially sheep and goats (which are often tick-infested), were found to play a key role in CCHFV human infection in Achaia. In other studies, tending cattle has also been found to be associated with CCHFV seropositivity;23 this was not seen in the present study, most probably because the density of cattle in Achaia is much lower (2.95/km2) than that of sheep (110.87/km2) and goats (49.91/km2). Thus, it seems that residents of this area are mostly infected through tick bites during tending of sheep and goats. Infection after contact with infected animal blood or tissues through slaughtering seems less probable
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Table 2 Univariate logistic regression analysis of risk factor contribution to CCHFV infection
Age, years, median (IQR) Missing data Sex Female Male Municipality Erimanthos Other Agro-pastoral occupation Yes No (other) Missing data Contact with goats Yes No Contact with sheep Yes No Contact with cows Yes No Hunting Yes No Missing data Slaughtering Yes No Missing data Visit to woods Yes No Missing data Tick bite Yes No Altitude Altitude 400 m Altitude <400 m
IgG positive (n = 7)
IgG negative (n = 200)
p-Value
75 (53–80) 0
46 (32–62) 10
0.018
1.056
1.009–1.104
0.922
0.927
0.202–4.250
3 (42.9%) 4 (57.1%)
82 (41%) 118 (59%)
4 (57.1%) 3 (42.9%)
23 (11.5%) 177 (88.5%)
4 (57.1%) 3 (42.9%) 0
32 (16.4%) 163 (83.6%) 5
4 (57.1%) 3 (42.9%)
28 (14%) 172 (86%)
7 (100%) 0 (0%)
32 (16%) 168 (84%)
0 (0%) 7 (100%)
8 (4%) 192 (96%)
1 (16.7%) 5 (83.3%) 1
27 (13.5%) 173 (86.5%) 0
2 (28.6%) 5 (71.4%) 0
37 (18.6%) 162 (81.4%) 1
2 (33.3%) 4 (66.7%) 1
67 (34.2%) 129 (65.8%) 4
5 (71.4%) 2 (28.6%)
43 (21.5%) 157 (78.5%)
5 (71.4%) 2 (28.6%)
25 (12.5%) 175 (87.5%)
OR
95% CI
0.001a 0.003
10.261
2.159–48.767
0.015
6.792
1.450–31.813
0.008
8.190
1.740–38.563
0.824
1.281
0.144–11.393
0.513
1.751
0.327–9.381
0.965
0.963
0.172–5.391
0.010
9.128
1.711–48.691
0.001
17.500
3.221–95.080
<0.001b
1.000b
CCHFV, Crimean-Congo hemorrhagic fever virus; OR, odds ratio; CI, confidence interval; IQR, interquartile range. a Pearson’s Chi-square test. b Estimated by Fisher’s exact test.
in Achaia, although it was found to be the primary risk factor in other parts of Greece.14 This reflects how socio-demographic and environmental factors interweave and contribute to the acquisition of CCHFV infection through different routes, even within the borders of a country; this reaffirms the complexity of the life cycle and ecology of the virus. All seven seropositive persons were CCHFV IgM-negative, and none recalled any illness resembling CCHF (fever accompanied by hemorrhagic manifestations). This finding may support the preexisting theory that CCHFV seropositivity in Greece is due to an unnoticed infection caused by non-pathogenic or low pathogenic viral strains. The CCHFV IgG seroprevalence in the Achaia Prefecture is similar to that observed in northeastern Greece15 where the fatal case was observed in June 2008.13 Identification of 537 ticks removed from humans who were referred for this reason to two hospitals in northeastern Greece, showed that the vast majority (81.5%) were Rhipicephalus sanguineus, while Hyalomma marginatum accounted for 5.2%.24 Molecular screening of ticks is needed in Achaia Prefecture, and in Greece in general, to identify the circulating CCHFV strains. Recently a study on 3671 adult volunteers reported from Turkey, covering most of the endemic regions, showed a seroprevalence of 10%, and it was demonstrated that 88% of CCHF
infections were subclinical.25 It has to be mentioned that AP92-like strains have been detected in Turkey26 and they might play a role in the subclinical infections and the increased seroprevalence. Studies in ticks and serosurveys in animals are needed to gain an insight into the CCHFV ecology and epidemiology in the country. Clinicians should include CCHF in the differential diagnosis of an acute febrile case, even without thrombocytopenia, during the season when Hyalomma and Rhipicephalus ticks are active, especially when the patient has an agro-pastoral occupation.
Acknowledgements We thank the participants who responded to the call of the study. The assistance of Dr M. Karakantza and the clinicians of the Patras University General Hospital Blood Bank, and of Dr G. Mantzouranis and the clinicians of the primary healthcare facilities, is highly appreciated. Funding: The present work was supported by the FP7 project ‘‘ANTIcipating the Global Onset of Novel Epidemics’’ (ANTIGONE), Grant Agreement No. 278976. Ethical approval: The current study was approved by the Bioethics Committee of the Medical School of the University of Patras. Conflict of interest: No conflict of interest to declare.
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References 1. Papa A. Crimean-Congo hemorrhagic fever and hantavirus infections. In: Maltezou HC, Gikas A, editors. Tropical and emerging infectious diseases. India: Research Signpost; 2010. p. 49–73. 2. Mardani M, Rahnavardi M, Rajaeinejad M, Naini KH, Chinikar S, Pourmalek F, et al. Crimean-Congo hemorrhagic fever among health care workers in Iran: a seroprevalence study in two endemic regions. Am J Trop Med Hyg 2007;76:443–5. 3. Izadi S, Naieni KH, Madjdzadeh SR, Nadim A. Crimean-Congo hemorrhagic fever in Sistan and Baluchestan Province of Iran, a case–control study on epidemiological characteristics. Int J Infect Dis 2004;8:299–306. 4. Ergonul O, Zeller H, Kilic S, Kutlu S, Kutlu M, Cavusoglu S, et al. Zoonotic infections among veterinarians in Turkey: Crimean-Congo hemorrhagic fever and beyond. Int J Infect Dis 2006;10:465–9. 5. Yilmaz GR, Buzgan T, Irmak H, Safran A, Uzun R, Cevik MA, et al. The epidemiology of Crimean-Congo hemorrhagic fever in Turkey, 2002–2007. Int J Infect Dis 2009;13:380–6. 6. Sisman A. Epidemiologic features and risk factors of Crimean-Congo hemorrhagic fever in Samsun Province, Turkey. J Epidemiol 2013;23:95–102. Epub ahead of print. 7. Vatansever Z, Uzun R, Estrada-Pena A, Ergonul O. Crimean-Congo hemorrhagic fever in Turkey. In: Ergonul O, Whitehouse CA, editors. Crimean-Congo hemorrhagic fever: a global perspective. Netherlands: Springer; 2007. p. 59–74. 8. Grard G, Drexler JF, Fair J, Muyembe JJ, Wolfe ND, Drosten C, et al. Re-emergence of Crimean-Congo hemorrhagic fever virus in Central Africa. PLoS Negl Trop Dis 2011;5:e1350. 9. Papa A, Christova I, Papadimitriou E, Antoniadis A. Crimean-Congo hemorrhagic fever in Bulgaria. Emerg Infect Dis 2004;10:1465–7. 10. Papa A, Velo E, Papadimitriou E, Cahani G, Kota M, Bino S. Ecology of the Crimean-Congo hemorrhagic fever endemic area in Albania. Vector Borne Zoonotic Dis 2009;9:713–6. 11. Christova I, Di Caro A, Papa A, Castilletti C, Andonova L, Kalvatchev N, et al. Crimean-Congo hemorrhagic fever, southwestern Bulgaria. Emerg Infect Dis 2009;15:983–5. 12. Papadopoulos O, Koptopoulos G. Crimean-Congo hemorrhagic fever (CCHF) in Greece: isolation of the virus from Rhipicephalus bursa ticks and a preliminary serological survey. Zentbl Bakteriol Hyg 1980;1:189–93. 13. Papa A, Dalla V, Papadimitriou E, Kartalis GN, Antoniadis A. Emergence of CrimeanCongo haemorrhagic fever in Greece. Clin Microbiol Infect 2010;16:843–7.
e1165
14. Sidira P, Maltezou HC, Haidich AB, Papa A. Seroepidemiological study of Crimean-Congo haemorrhagic fever in Greece, 2009–2010. Clin Microbiol Infect 2012;18:E16–9. 15. Papa A, Tzala E, Maltezou HC. Crimean-Congo hemorrhagic fever virus, northeastern Greece. Emerg Infect Dis 2011;17:141–3. 16. Hellenic Statistical Authority. Announcement Census 2011—resident population. Piraeus, Greece; 2012. Available at: http://www.statistics.gr/portal/page/ portal/ESYE/BUCKET/A1602/PressReleases/ A1602_SAM01_DT_DC_00_2011_02_F_EN.pdf (Last accessed 20 March 2013). 17. Hoogstraal H. The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. J Med Entomol 1979;15:307–417. 18. KTADA in collaboration with the National Observatory of Athens. Annual climatological summary, Chalandritsa, 2012. Available at: http://penteli.meteo.gr/stations/chalandritsa/NOAAPRYR.TXT (Last accessed 20 March 2013). 19. National Observatory of Athens. Annual climatological summary, Patras, 2012. Available at: http://penteli.meteo.gr/stations/patra/NOAAPRYR.TXT (Last accessed 20 March 2013). 20. Butenko AM, Karganova GG. Crimean-Congo hemorrhagic fever in Russia and other countries of the former Soviet Union. In: Ergonul O, Whitehouse CA, editors. Crimean-Congo hemorrhagic fever: a global perspective. Netherlands: Springer; 2007. p. 99–114. 21. Gunes T, Engin A, Poyraz O, Elaldi N, Kaya S, Dokmetas I, et al. Crimean-Congo hemorrhagic fever virus in high-risk population, Turkey. Emerg Infect Dis 2009;15:461–4. 22. Christova I, Gladnishka T, Taseva E, Kalvatchev N, Tsergouli K, Papa A. Seroprevalence of Crimean-Congo hemorrhagic fever virus, Bulgaria. Emerg Infect Dis 2013;19:177–9. 23. Ertugrul B, Kirdar S, Ersoy OS, Ture M, Erol N, Ozturk B, et al. The seroprevalence of Crimean-Congo haemorrhagic fever among inhabitants living in the endemic regions of Western Anatolia. Scand J Infect Dis 2012;44:276–81. 24. Papa A, Chaligiannis I, Xanthopoulou K, Papaioakim M, Papanastasiou S, Sotiraki S. Ticks parasitizing humans in Greece. Vector Borne Zoonotic Dis 2011;11: 539–42. ¨ ngu¨ru¨ P, Uyar Y. Subclinical infections with 25. Bodur H, Akinci E, Ascioglu S, O Crimean-Congo hemorrhagic fever virus, Turkey. Emerg Infect Dis 2012;18: 640–2. 26. Midilli K, Gargili A, Ergonul O, Elevli M, Ergin S, Turan N, et al. The first clinical case due to AP92 like strain of Crimean-Congo hemorrhagic fever virus and a field survey. BMC Infect Dis 2009;9:90.