Seroprevalence of Crimean–Congo Hemorrhagic Fever Virus in Erzincan Province,Turkey,Relationship with Geographic Features and Risk Factors

Abstract

To determine the seroprevalence and risk factors associated with Crimean–Congo hemorrhagic fever virus (CCHFV) in residents of Erzincan, Turkey. Although CCHFV is endemic in Erzincan, this is the first study to evaluate its seroprevalence in this region. This study included a total of 372 subjects, 174 of whom had been exposed to or bitten by ticks, 145 of whom worked with livestock, and 53 of whom resided in the city and did not have exposure to livestock. Data on CCHFV IgG and IgM antibodies were extracted from serum samples collected from all subjects using an ELISA. All samples were tested for CCHFV IgG and CCHFV IgM. Only IgM-positive samples were processed for detection of viral RNA through RT-PCR. Using seropositive cases only, we performed spatial analyses to evaluate correlations between seroprevalence and geographic location (i.e., proximity to rivers, altitude, and slope angle of land). In this study, 14.0% (52/322) of the total subjects were positive for CCHFV IgG. Seven of the individuals were positive both for CCHFV IgG and CCHFV IgM. Of these seven, only one sample tested positive for CCHFV RNA. Individuals who worked with livestock in the rural areas and had a history of tick exposure were statistically more likely to test positive for CCHFV IgG than individuals from the city and not exposed to ticks (p < 0.05). Seroprevalence was affected by geographic characteristics, including distance to rivers, altitude, and slope angle of land. We observed a high seroprevalence of CCHFV in Erzincan, which is similar to that observed in other endemic regions of Turkey. CCHFV seroprevalence rates are found to be quite high in the people who live in the sloping fields at certain heights and where there are a lot of rivers and streams.

Introduction

Crimean–Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by the CCHF virus (CCHFV), a virus of the genus Nairovirus in the family of Bunyaviridae (Bente et al. 2013, Horne and Vanlandingham 2014, Nurmakhanov et al. 2015). Humans become infected through tick bites, by direct contact with a CCHFV-positive individual, or by contact with the blood or tissues from viremic livestock (Oncü 2013, Jääskeläinen et al. 2014).

CCHF, an important hemorrhagic viral disease that occurs in humans, is associated with a 30% mortality rate and causes sporadic cases or outbreaks of severe illness across vast geographic areas, such as western China to the Middle East (Bente et al. 2013, Lumley et al. 2014). CCHFV is endemic to the Central and Northeastern Anatolia regions as well as to the Southern Black Sea region of Turkey. The disease has continued to spread across Turkey, with the emergence of several cases in other provinces, including Kastamonu, Bartın, Ankara, Çankırı, Bolu, and Balıkesir (Gozalan et al. 2007, Yilmaz et al. 2008, 2009).

In Turkey, the first cases of CCHF were reported in the Kelkit Valley, but cases are now encountered every year, resulting in numerous mortalities in many regions, including Erzincan (Gozalan et al. 2007, Akduman et al. 2009, Günaydın et al. 2010). To date, there have not been any epidemiological studies on the prevalence of CCHFV in Erzincan, Turkey. Therefore, the objective of this study was to determine the seroprevalence, identify its geographic characteristics, and clarify other risk factors associated with CCHFV in Turkey.

Materials and Methods

Location of study

The Erzincan province, which is situated in the north part of the Eastern Anatolia region of Turkey at 39° 45′ 12" North and 39° 20′ 28" East, consists of nine districts (Capital, Çayırlı, İliç, Kemah, Kemaliye, Otlukbeli, Refahiye, Tercan, and Üzümlü). The climate of Erzincan is affected by those of the Black Sea Region and Eastern Anatolia. The province has an area of 11909 km² and is covered by mountains and plateaus. The Karasu River divides the region from east to west. In addition, a dam is located in the Tercan district. The densest population is located in Kelkit Valley, which has a suitable climate and vegetation, contains several sources of water, and provides the resources necessary for agriculture and animal husbandry.

Ethics statement

This research was approved by the Ethics Committee of Erzincan University, Turkey. Physicians provided information about the purpose of this study as well as about CCHFV. Written consent was obtained from all participants before the start of the study. An in-person interview and questionnaire were used for data collection. Participants were asked to complete a questionnaire about their place of residence, age, sex, occupation, and history of exposure to ticks or tick bites (during last 5 years), as well as other activities or data related to an increased risk of CCHF.

Blood sample collection

Blood samples were collected from 372 individuals from nine districts and 95 villages. Blood samples were centrifuged at 1610 × g for 10 min, and serum was separated and stored in several aliquots at −20°C until assayed. The individuals in the study were divided into three groups. The first group consisted of 174 subjects exposed to or bitten by ticks. The second group included 145 subjects who were unexposed to ticks but were involved in animal husbandry in rural areas. The third group consisted of 53 healthy individuals who resided in the city and were not exposed to livestock or ticks.

Serological assays

Serum samples were tested for CCHFV IgG and CCHFV IgM antibodies using an enzyme-linked immunosorbent assay (ELISA; Vector-Best, Novosibirsk, Russia), in accordance with the manufacturer's instructions. In addition, CCHFV IgM-positive serum samples were further tested by real-time RT-PCR for the presence of CCHFV RNA. Human serum was diluted 1:100 in a dilution buffer (90% SDB and 10% SPSD-buffer). Briefly, serum dilutions (100 μL/well) were incubated for 1 h at 37°C. All washing steps were performed with PBS-Tween. After washing the plates, we added 100 μL/well of peroxidase-labeled antibodies to human IgG and incubated it for 30 min at 37°C. After washing the plates, 100-μL/well TMB solution (tetramethylbenzidine) was added and incubated for 25 min. After incubation, the reaction was stopped with H₂SO₄. Absorbance was read at 450 nm (reference wavelength 620–655 nm) with an Epoch spectrophotometer using Gen5 data analysis software (BioTek Instruments, Inc., Winooski, VT).

Molecular analyses

Viral RNA was isolated from blood sera using a High Pure Viral Nucleic Acid Kit (Roche Diagnostics GmbH, Mannheim, Germany). Viral RNA was dissolved in 30 μL of elution buffer. The presence of CCHFV RNA was tested using a TaqMan-based one-step reverse transcriptase PCR (real time RT-PCR), as described by Yapar et al. (2005). Briefly, 5 μL of viral RNA was added to 20 μL of a mixture containing 5 pmol of each primer, 4 pmol of TaqMan probe, 0.2 mM of each dNTP (containing dUTP), and 6 mM of magnesium chloride. The cycles were performed as follows: 42°C for 40 min, 95°C for 10 min, and 40 PCR cycles (15 s at 95°C, 1 min at 60°C). The assay was run on a Perkin–Elmer 7700 Sequence Detection System (Applied BioSystems, Foster City, CA) by using the combination of reverse transcriptase (MBI Fermentas, St Leon-Rot, Germany) and Hot Start Taq DNA polymerase (Bioron GmBH, Munich, Germany). Detailed data analyses for copies/mL were performed, as detailed in a previous study (Yapar et al. 2005).

Mapping

This study used ArcGIS 10.1 software (ESRI, Redlands, CA) and the Google Earth program (Google, Mountain View, CA) to draw and analyze maps. We started our analysis by first drawing the Erzincan province, followed by its county maps, rivers, and tributaries. Next, baseline data were created using the ArcGIS Basemap OpenStreetMap service. Then, locations of the seropositive cases were identified in the Google Earth program and transferred to the ArcMAP software. In addition, we determined correlations between seropositive cases and altitude, slope angle of the location, and proximity to rivers. To determine the geographic correlations with seropositive samples, we obtained data from ASTER GDEM (the Advanced Spaceborne Thermal Emission and Reflection Radiometer Global Digital Elevation Model), the National Aeronautics and Space Administration (NASA), and the Ministry of Economy, Trade, and Industry (METI). For spatial analyses, we performed buffer analyses for rivers and tributaries (Buffer, Multiple Ring Buffer).

Statistical analyses

We used the Statistical Package for the Social Sciences (SPSS) version 21 for Windows (SPSS, Inc., Chicago, IL) for statistical analyses. Chi-square tests were used to determine significant differences between positive CCHF rates and risk factors associated with this infection. A p < 0.05 was considered statistically significant.

Results

This study included 228 females and 144 males. CCHFV IgG antibodies were observed in 18.1% of females and in 13.4% of males (p > 0.05). The mean age was 51.4 ± 17 (min: 17, max: 86, n = 372) in the total sample. We observed differences in age and prevalence of CCHFV IgG antibodies: ≤20 years old (0%), 21–40 (19.2%), 41–60 (30.8%), and >61 (50%). However, differences among age groups were not statistically significant (Table 1).

Table 1.

Seroprevalence and Associated Risk Factors for CCHFV

	CCHF IgG			CCHF IgM
	Positive (%)	Negative (%)		Positive (%)	Negative (%)
	n:52 (14.0)	n:320 (86.0)	p	n:7 (1.9)	n:365 (98.1)	p
Sex
Male	35 (67.3)	193 (60.3)	0.361	5 (71.4)	223 (61.1)	0.711
Female	17 (32.7)	127 (39.7)		2 (28.6)	142 (38.9)
Age
≤20	0 (—)	16 (5.0)	0.172	0 (—)	16 (4.3)	0.321
21–40	10 (19.2)	78 (24.4)		2 (28.6)	86 (23.6)
41–60	16 (30.8)	127 (39.7)		4 (57.1)	139 (38.1)
61<	26 (50.0)	99 (30.9)		1 (14.3)	124 (34.0)
Livestock
Yes	46 (88.5)	231 (72.2)	0.015	6 (85.7)	271 (74.2)	0.683
No	6 (11.5)	89 (27.8)		1 (14.3)	94 (25.8)
Tick contact
Yes	30 (57.7)	144 (45.0)	0.100	5 (71.4)	169 (46.3)	0.259
No	22 (42.3)	176 (55.0)		2 (28.6)	196 (53.7)
Tick bite
Yes	21 (40.4)	96 (30.0)	0.148	5 (71.4)	112 (30.7)	0.034
No	31 (59.6)	224 (60.0)		2 (28.6)	253 (69.3)
Living area
Rural	48 (92.3)	249 (77.8)	0.015	7 (100)	290 (79.5)	0.353
Urban	4 (7.7)	71 (22.2)		0 (—)	75 (20.5)

Figures in bold were considered statistically significant.

CCHFV, Crimean–Congo hemorrhagic fever virus.

Among all subjects, 14.0% (52/372) tested positive for CCHFV IgG and 1.9% (7/372) tested positive for both CCHFV IgG and IgM. CCHFV IgG prevalence differed among individuals with a history of CCHFV exposure or tick bites (16.7% [29/174]), individuals who worked with livestock but had not been exposed to CCHFV (12.4% [18/145]), and individuals who lived within the city and had not been exposed to CCHFV (9.4% [5/53]). There was a statistically significant difference between the prevalence of CCHFV among individuals working within the livestock industry or having exposure to CCHFV and those unexposed to CCHFV or residing within the city (p < 0.05) (Table 2).

Table 2.

The Relationship Between Livestock and Tick Exposure

	CCHF IgG			CCHF IgM
	Positive	Negative	p	Positive	Negative	p
Tick exposure (n = 174)
Livestock
Yes (n = 132)	29	103	0.002	5	127	0.338
No (n = 42)	1	41		—	42
Livestock (n = 277)
Tick exposure
Yes (n = 132)	29	103	0.024	5	127	0.106
No (n = 145)	17	128		1	144

Figures in bold were considered statistically significant.

CCHFV IgG-positive subjects were categorized as follows: 92.3% (48/52) lived in rural areas, 88.5% (46/52) worked with livestock, 61.5% (32/52) had a history of CCHFV exposure, and 98% (51/52) had recent tick bites. Living in rural areas and working with livestock were both significantly related to CCHFV prevalence according to a comparison with recent tick bites (p < 0.05) (Table 1).

Seven samples were found positive for CCHF IgG and IgM CCHF. Of the subjects who tested positive for CCHF IgG and CCHFV IgM, five had a history of a recent tick bite (p < 0.05) (Table 1). RT-PCR was performed to assess the presence of CCHFV RNA in CCHFV IgM-positive serum samples. Only one RT-PCR sample was positive for CCHFV RNA. The individual who tested positive for CCHFV RNA had been bitten by a tick 15 days before this study and had suffered a febrile illness. Although a small number of the total subject pool, those who tested positive for both CCHFV IgG and IgM consisted mainly of subjects exposed to tick bites. However, the associations between tick bites and testing positive for CCHFV IgG and IgM were not statistically significant.

Our analysis of the prevalence of CCHFV IgG-positive subjects and the regions in which they lived revealed the following rates of prevalence by region: 6.7% lived in Çayırlı, 12.1% in the central district, 13.8% in Üzümlü, 17.2% in Refahiye, 17.4% in Kemah, and 37.0% in Tercan. Individuals living in Tercan had significantly higher prevalence of CCHFV IgG than did those subjects living in other regions of Erzincan (p < 0.01) (Table 1).

Finally, we examined the correlation of seropositive cases with geographic characteristics, including rivers, altitude, and slope angle. We noted the following relationships between the prevalence of seropositive cases and distance to rivers: 44.2% cases lived less than 1000 meters away from the rivers, 19.2% were between 1001 and 2000 meters away, 13.5% were 2001–3000 meters away, 9.6% were 3001–4000 meters away, 1.9% were 4001–5000 meters away, and 11.6% of them were more than 5500 meters away from the rivers. Seroprevalence was also affected by other geographic characteristics, including the slope angle of the land as well as the specific altitude. Specifically, 50% of seropositive subjects resided on land with a slope angle of 0–5°, 3.8% were 5.1–10°, 11.6% were 10.1–20°, 19.2% were 15.1–20°, 7.7% were 20.1–25°, and remaining 7.7% were living on the slope of angle of 25.1–30°. The area of focus in this study had an altitude ranging from 817 to 3518 meters above sea level. Individuals positive for CCHFV IgG tended to live at an altitude between 1152 and 1985 meters, with the majority (61.5%) living at an altitude between 1100 and 1500 meters (Fig. 1). Altitudes, slope angles, and distance to river of living areas of the individuals are also given in Table 4.

FIG. 1.

Distribution of CCHF IgG-positive persons in the region.

Discussion

CCHFV is one of the most common tick-borne viral infections worldwide. CCHF is a fatal disease prevalent in Asia, Africa, and Europe (Ergonul 2012). In the last decade, an increase in the incidence of this disease has been observed, specifically in southeast Europe (Vorou 2009, Mertens et al. 2013), and CCHF is currently considered to be at an epidemic level throughout Europe (e.g., Greece, Kosovo, Albania, Bulgaria, and Turkey) (Vescio et al. 2012).

A regional study in Greece conducted by Papa et al. (2013) observed a 14.4% prevalence of CCHF. Another study from Greece, conducted by Sidira et al. (2012), examined 1611 cases from across the country and tested their serum CCHFV antibodies using an ELISA. Sidira et al. (2012) observed a prevalence of 4% and noted the differences in prevalence among cities. The Thesprotia region had the highest prevalence of CCHFV antibody-positive individuals, 27.5%. Similarly, Christova et al. (2013) examined 1018 healthy individuals in various provinces across Bulgaria and reported that 28% of the population was positive for CCHFV antibodies. The regions with the highest prevalence were Burgaz (7.6%) and Kardjali (6%). In studies conducted in Kosovo, the rates of individuals with CCHFV antibodies ranged from 4% to 24% in healthy individuals (Humolli et al. 2010, Fajs et al. 2014).

There is also a high prevalence of CCHF in southeast Europe, especially in Turkey. In 2002, Turkey was identified as a country in which CCHF was endemic (Yagci-Caglayik et al. 2014). For example, more than 8000 confirmed cases have been reported in the last decade in Turkey (Ergonul and Battal 2014). Of these cases, 95% were reported from the central and eastern Anatolia regions (Yilmaz et al. 2008). In studies involving endemic regions in Turkey, Ertugrul et al. (2012) reported that 19.6% of the population was CCHFV IgG-positive, Köksal et al. (2014) reported a rate of 13.6%, and Bodur et al. (2012) reported a rate of 10.0%. In a study including seven regions of Turkey, 2.3% of the population tested positive for CCHFV IgG (Yagci-Caglayik et al. 2014). In this study, focused on Erzincan, located in northeast Turkey, we observed that 13.9% of the population was positive for CCHFV IgG, which is similar to rates reported in other endemic areas. Our study is one of the first to report the prevalence of CCHF in healthy individuals living in Erzincan, a region in which this illness is endemic.

Individuals who work with livestock (e.g., farmers, butchers, and shepherds) and those living in rural areas are at highest risk for CCHF (Sarginou et al. 2013, Gergova and Kamarinchev 2014, Sharifi-Mood et al. 2014). In this study, 88% (46/52) of the individuals who tested positive for CCHFV IgG worked with livestock and 92% (48/52) lived in rural areas (Table 3). In addition, tick exposure was significantly more common in individuals who work with livestock. On the basis of our results, we hypothesize that the high rate of CCHF and seropositivity in rural areas is likely due to the coexistence of many risk groups.

Table 3.

Distribution of CCHF IgG Seroprevalence According to the Districts

	Positive (n:52)	Negative (n:320)	Total (n:372)	%	p
Çayırlı	2	28	30	6	<0.001
İliç	0	4	4	—
Kemah	4	19	23	17.3
Kemaliye	0	4	4	—
Merkez	26	188	214	12.1
Refahiye	5	24	29	17.2
Otlukbeli	0	5	5	—
Tercan	10	17	27	37.0
Üzümlü	5	31	36	13.8

The area of focus in this study had a dense tick population. Therefore, CCHF acquired by viruses with a life cycle in ticks is an expected risk associated with living in this area (Gargili et al. 2013).

The possibility of infection increases with age; thus, it is logical that several studies have reported that advanced age is a risk factor for CCHF (Sidira et al. 2012, Yagci-Caglayik et al. 2014). In our study, individuals ranging in age from 21 to 40 or 41 to 60 years old had a 19.2% and a 30.8% rate of testing positive for CCHFV IgG, respectively. Among individuals older than 61 years, 50.0% tested positive for CCHFV IgG. Although the prevalence increased with age, the difference was not statistically significant.

In nine districts in the Erzincan province evaluated for the prevalence of CCHFV IgG, we observed that 37.0% of individuals in Tercan had a statistically significant chance of testing positive for CCHFV IgG (p < 0.001). The most important characteristics distinguishing Tercan from the other districts include its focus on agricultural and livestock activities and its inclusion of more wetlands, as well as the only reservoir being located in this region. To date, the data regarding whether there is a correlation between lakes/dams and the prevalence of CCHF are inconsistent. In this study, 84.6% of individuals who tested positive for CCHF IgG lived 3500 meters from rivers and their branches (Fig. 1). Similarly, a study conducted by Tigoi et al. found that the CCHF prevalence was higher in regions with more rivers and lakes (Tigoi et al. 2015). In this study, we were able to demonstrate a correlation between the distance to rivers and the prevalence of CCHF. Nevertheless, we were unable to identify any correlation between the dam located in Tercan and the seroprevalence of CCHFV.

Studies demonstrating a correlation between CCHF and other geographic characteristics, such as the slope angle of the land, are limited. The area of focus of this study had a slope angle ranging from 0° to 72.4°. The slope angle of land within residential areas was examined, 50% of seropositive cases consisted of individuals living on land with a slope angle of 0–5°, and the data about slope angles are given in Table 4. On the basis of these results, CCHF was observed more frequently in the areas of land with a low slope angle.

Table 4.

The Relationship of CCHF IgG-Positive Persons with Distance to Rivers, Slope Degrees, and Altitude

	Seropositive cases
	n:52	%
Distance (meter)
<1000	23	44.2
1001–2000	10	19.2
2001–3000	7	13.5
3001–4000	5	9.6
4001–5000	1	1.9
>5500	6	11.6
Slope degrees (°)
0–5	26	50.0
5.1–10	2	3.8
10.1–15	6	11.6
15.1–20	10	19.2
20.1–25	4	7.7
25.1–30	4	7.7
Altitude (meter)
1100–1300	16	30,8
1300–1500	16	30.8
1500–1700	11	21.1
1700–1900	7	13.5
>1900	2	3.8

Previous studies have reported a correlation between CCHFV and altitude. However, each of these studies reported a different altitude (Sisman 2013, Gergova and Kamarinchev 2014). In our study, the area of focus was at an altitude between 817 and 3518 meters above sea level, and the majority of individuals who tested positive for CCHFV IgG lived at an altitude between 1152 and 1985 meters. Of these cases, 61.5% lived at an altitude between 1100 and 1500 meters. In our study, we found a correlation between CCHFV seropositivity and altitude.

Conclusion

In this study, we observed a high seroprevalence of CCHFV IgG in Erzincan, a region in which this disease is endemic. Working with livestock, living in rural areas, and having a history of tick exposure were identified as CCHFV risk factors. CCHFV seroprevalence was highest among individuals residing in regions with an abundance of rivers and wetlands as well as those located at a certain altitude and slope angle. On the basis of the results of this study, we suggest that educational and training programs targeted at high-risk groups be developed and implemented as preventative measures.

Footnotes

Acknowledgment

This study was supported by the Directorate of Scientific Research and Project Unit of Erzincan University under the following project number: SAG-A-300614-0090.

This study was presented at the 6th Congress of European Microbiologists (FEMS 2015) (Poster No. 1313, 7–11 June 2015, Maastricht, Netherlands).

Author Disclosure Statement

No competing financial interests exist.

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