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Seroepidemiological and molecular investigations of infections with Crimean–Congo haemorrhagic fever virus in Kazakhstan

Open AccessPublished:October 25, 2018DOI:https://doi.org/10.1016/j.ijid.2018.10.015

      Abstract

      Objectives

      The aim of this study was to detect the seroprevalence of Crimean–Congo haemorrhagic fever virus (CCHFV) in patients with fever of unknown origin (FUO) in endemic (Kyzylorda) and non-endemic (Almaty) oblasts of Kazakhstan.

      Methods

      Paired serum samples from 802 patients with FUO were collected. Serum samples were investigated by ELISA to detect IgG and IgM antibodies against CCHFV. Sera with suspected acute infection were further investigated by RT-PCR to detect the viral RNA.

      Results

      IgG antibodies were detected in 12.7% of the sera from both oblasts. Acute infection was shown by IgM ELISA in four patients from Kyzylorda, with only one developing severe CCHF. Viral RNA was found by RT-PCR in the other three patients’ sera. Phylogenetic analysis of partial L and S segments revealed CCHFV genotype Asia 2 and a possible reassortment between the genotypes Asia 1/Asia 2. Animal husbandry, such as working with cattle and horses, was significantly associated with CCHFV seropositivity.

      Conclusions

      The antibodies and viral RNA detected in sera indicate that mild or even asymptomatic CCHFV infections are presented in Kazakhstan. This study describes the circulation of CCHFV in the so far non-endemic Almaty oblast for the first time. In conclusion, physicians treating patients with FUO in Kazakhstan should be aware of mild CCHF.

      Keywords

      Introduction

      Crimean–Congo haemorrhagic fever (CCHF) is an acute viral tick-borne zoonotic disease caused by the Crimean–Congo haemorrhagic fever virus (CCHFV). Recently, the taxonomy of the former family Bunyaviridae was revised, and CCHFV is now grouped as a member of the genus Orthonairovirus, family Nairoviridae, order Bunyavirales (). The genome of CCHFV consists of three segments of RNA, with the large (L) segment encoding the RNA-dependent RNA polymerase, the medium (M) segment encoding two surface glycoproteins, and the small (S) segment encoding the nucleocapsid protein (,
      • Schmaljohn C.S.
      • Nichol S.T.
      Bunyaviridae.
      ).
      The main vectors of CCHFV are ticks of the genus Hyalomma, thus the geographical distribution of the virus follows that of Hyalomma ticks (
      • Hoogstraal H.
      The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa.
      ;
      • Ergönül O.
      Crimean-Congo hemorrhagic fever.
      ;
      • Messina J.P.
      • Pigott D.H.
      • Golding N.
      • Duda K.A.
      • Brownstein J.S.
      • Weiss D.J.
      • et al.
      The global distribution of Crimean-Congo hemorrhagic fever.
      ). Presently CCHF is recognized in more than 30 countries of Asia, Southeast Europe, and Africa, as well as in the Middle East, with an emerging trend in the Balkans (
      • Ergönül O.
      Crimean-Congo hemorrhagic fever.
      ,
      • Shayan S.
      • Bokaean M.
      • Shahrivar Chinikar M.S.
      Crimean-Congo hemorrhagic fever.
      ,
      • Elevli M.
      • Ozkul A.
      • Civilibal M.
      • Midilli K.
      • Gargili A.
      • Duru N.S.
      A newly identified Crimean-Congo hemorrhagic fever virus strain in Turkey.
      ,
      • Whitehouse C.A.
      Crimean-Congo hemorrhagic fever.
      ). CCHF has a serious impact on public health and threatens the health system globally, as the disease manifests in severe haemorrhagic forms with a mortality rate ranging between 5% and 30% (
      • Whitehouse C.A.
      Crimean-Congo hemorrhagic fever.
      ,
      • Ertugrul B.
      • Uyar Y.
      • Yavas K.
      • Turana C.
      • Oncua S.
      • Saylaka O.
      • et al.
      An outbreak of Crimean-Congo hemorrhagic fever in western Anatolia, Turkey.
      ).
      CCHF is an endemic infection in Kazakhstan, with approximately 10 clinically apparent cases registered annually; the average lethality rate between 1948 and 2013 was 14.8% (
      • Onishchenko G.
      • Tumanova I.
      • Vyshemirskiĭ O.
      • Kuhn J.
      • Seregin S.
      • Tiunnikov G.
      • et al.
      [ELISA and RT-PCR-based research of viruses in the ticks collected in the foci of Crimean-Congo fever in Kazakhstan and Tajikistan in 2001-2002].
      ). The high density of the population in the southern parts of Kazakhstan, the favourable climate for vectors, and the migration of animals and wild birds carrying infected ticks from neighbouring areas of Uzbekistan and Tajikistan that are endemic for CCHFV, make the south of Kazakhstan favourable for CCHF (
      • Onishchenko G.
      • Tumanova I.
      • Vyshemirskiĭ O.
      • Kuhn J.
      • Seregin S.
      • Tiunnikov G.
      • et al.
      [ELISA and RT-PCR-based research of viruses in the ticks collected in the foci of Crimean-Congo fever in Kazakhstan and Tajikistan in 2001-2002].
      ,
      • Nurmakhanov T.
      • Sansyzbaev Y.
      • Atshabar B.
      • Deryabin P.
      • Kazakov S.
      • Zholshorinov A.
      • et al.
      Crimean-Congo haemorrhagic fever virus in Kazakhstan (1948-2013).
      ). The main endemic areas in the south of Kazakhstan are the oblasts of Zhambyl and Kyzylorda, from where 119 clinically apparent cases of CCHF were registered during 2000–2013, and the Asia 1 and Asia 2 strains are the subtypes circulating in the country (
      • Nurmakhanov T.
      • Sansyzbaev Y.
      • Atshabar B.
      • Deryabin P.
      • Kazakov S.
      • Zholshorinov A.
      • et al.
      Crimean-Congo haemorrhagic fever virus in Kazakhstan (1948-2013).
      ,
      • Knust B.
      • Medetov J.
      • Kyraubaev K.
      • Bumburidi Y.
      • Erickson B.
      • MacNeil A.
      • et al.
      Crimean-Congo hemorrhagic fever, Kazakhstan, 2009-2010.
      ,
      • Atkinson B.
      Thesis. The characterisation of arboviral zoonoses in Central Asia. September 2016.
      ,
      • Yashina L.
      • Petrova I.
      • Seregin S.
      • Vyshemirskii O.
      • Lvov D.
      • Aristova V.
      • et al.
      Genetic variability of Crimean-Congo haemorrhagic fever virus in Russia and Central Asia.
      ). CCHFV can also cause unspecific fever without any pathognomonic signs (
      • Christova I.
      • Younan R.
      • Taseva E.
      • Gladnishka T.
      • Trifonova I.
      • Ivanova V.
      • et al.
      Hemorrhagic fever with renal syndrome and crimean-congo hemorrhagic fever as causes of acute undifferentiated febrile illness in Bulgaria.
      ,
      • Bodur H.
      • Akinci E.
      • Ascioglu S.
      • Onguru D.
      • Uycer Y.
      Subclinical infections with Crimean chemorrhagic fever, Turkey.
      ), and this presentation has not yet been investigated in Kazakhstan.
      The aim of this study was to investigate, for the first time, patients suffering from fever of unknown origin (FUO) to identify infection with CCHFV. The investigation was undertaken in the known endemic region of Kyzylorda oblast as well as in the non-endemic Almaty oblast in Kazakhstan.

      Materials and methods

      Paired serum samples (day 0, day 10–14) were collected from adult patients suffering from FUO in nine hospitals of Almaty oblast and four hospitals of Kyzylorda oblast in Kazakhstan, from April to October 2014 and 2015, during the season of tick activity (Figure 1). Patients with FUO were defined as having a temperature >37.5 °C (ear) for more than 3 days and by exclusion of rheumatic diseases. Participants completed an interview-based questionnaire consisting of 47 questions on socio-demographic characteristics, living conditions, livestock and vector habitat factors, and clinical symptoms. The recruitment, enrolment, questionnaire, sampling methods, and laboratory investigations were reviewed and approved by the ethics committees in Kazakhstan and Germany.
      Figure 1
      Figure 1Crimean–Congo haemorrhagic fever antibodies in patients with fever of unknown origin investigated in the two oblasts of Almaty (A.) and Kyzylorda (K.) in Kazakhstan; regions where seropositive patients (▲), acute infections (☆), and seronegative patients (△) were found.
      Blood was centrifuged and serum samples were stored in two aliquots, one at −20 °C and one at −80 °C, until use. The sera were screened for the presence of IgG and IgM antibodies against CCHFV using commercial ELISA tests (Vector-Best, Novosibirsk, Russia) according to the manufacturer’s instructions. Briefly, in a first step, the second serum samples of all participants were screened for IgG by ELISA (dilution 1:100, as indicated by the kit instructions). Next, for the IgG-positive subset, the second sample was retested and the first sample was tested for IgG. First and second serum samples with an optical density (OD) difference of ≤±0.3 were considered to indicate IgG from a previous infection. All other sera were titrated in log2 steps (1:100, 1:200, up to 1:3200). A four-fold titre change was considered to indicate acute infection. Following this, the samples for which the first serum tested IgG-negative were investigated by IgM ELISA. Lastly, IgM-negative and all IgM-positive first serum samples were further investigated by molecular methods.
      The QiAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany) was used for RNA extraction from 140 μl of serum, following the kit instructions. The presence of CCHFV RNA in the sera was determined by real-time reverse transcription PCR (rtRT-PCR) performed using the method of
      • Atkinson B.
      • Chamberlain J.
      • Logue H.C.
      • Cook N.
      • Bruce C.
      • Dowall D.S.
      • et al.
      Development of a real-time RT PCR assay for the detection of Crimean-Congo hemorehagic fever virus.
      by optimizing the protocol with the QuantiTect virus kit (Qiagen, Hilden, Germany) on a Rotor-Gene Q cycler (Qiagen, Hilden, Germany). Samples positive for viral RNA in the RT-PCR were then analyzed by CCHFV-specific RT-PCR with primers targeting the S and L segments, as described in detail previously (
      • Woelfel R.
      • Paweska T.J.
      • Petersen N.
      • Globbelaar A.A.
      • Leman A.P.
      • Hewson R.
      • et al.
      Virus detection and monitoring of viral load in Crimean-Congo hemorrhagic fever virus patients.
      ,
      • Filippone C.
      • Marianneau P.
      • Murri S.
      • Mollard N.
      • Avsic-Zupanc T.
      • Chinikar S.
      • et al.
      Molecular diagnostic and genetic characterization of highly pathogenic viruses: application during Crimean-Congo haemorrhagic fever virus outbreaks in Eastern Europe and the Middle East.
      ). RT-PCR products were purified and sequenced by Sanger method with the ABI Prism Big Dye Terminator V3.1 Cycle Sequencing Kit and 3500xl Genetic Analyzer machine, using the initial primers of the RT-PCR amplification. Retrieved sequences were compared with known sequences from GenBank using BLAST 2.2.32 (
      • Zhang Z.
      • Schwartz S.
      • Wagner L.
      • Miller W.
      A greedy algorithm for aligning DNA sequences.
      ,
      • Morgulis A.
      • Coulouris G.
      • Raytselis Y.
      • Madden T.
      • Agarwala R.
      • Schäffer A.
      Database indexing for production mega BLAST searches.
      ). Phylogenetic trees were constructed in MEGA 6 (
      • Tamura K.
      • Stecher G.
      • Peterson D.
      • Filipski A.
      • Kumar S.
      MEGA6: molecular evolutionary genetics analysis version 6.0.
      ) with the maximum likelihood method based on the Kimura two-parameter model (
      • Kimura M.
      A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences.
      ).
      Questionnaire responses from CCHFV-positive patients were compared with those from CCHFV infection-negative participants. Statistical analyses were conducted using R 3.3.3 (
      • R Core Team
      R: a language and environment for statistical computing.
      ). Logistic regressions were used to evaluate the independent risk factors for seropositivity. The criterion for statistical significance was p< 0.05.

      Results

      In total, 950 patients were enrolled in the study, but only 802 patients with FUO met the criteria of having paired serum samples as well as completing the questionnaire; these 802 patients were therefore included in the study (Table 1). Among all samples tested by IgG ELISA, 102 (12.7%) showed a positive IgG result in the serum pair, indicating a recent or past infection. In total, 59 samples exhibited a difference in OD of ≤±0.3 between the first and second serum sample, 17 serum pairs had a low IgG titre (1:100), 22 serum pairs had a medium titre (1.200–1:400), and four serum pairs had a high titre (1:800–1:3200) (Table 2).
      Table 1Results of CCHFV ELISA and RT-PCR in patients with FUO investigated in two oblasts in Kazakhstan (2014–2015).
      OblastNumber of samples testedNumber of seronegative samplesNumber of IgG-positive samples (%)Number of IgM-positive samplesNumber of RT-qPCR-positive samples
      1st serum2nd serum
      Almaty37833444 (11.6)000
      Kyzylorda42436258 (13.6)133
       Total802696102 (12.7)4
      Three samples had IgM and RNA.
      3
      Three samples had IgM and RNA.
      CCHFV, Crimean–Congo haemorrhagic fever virus; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription PCR; RT-qPCR; reverse transcription quantitative PCR; FUO, fever of unknown origin.
      a Three samples had IgM and RNA.
      Table 2Results for the 102 samples that tested positive for CCHFV IgG by ELISA.
      Not including the four samples with positive IgM and from patients with acute infection.
      Result ELISA (2nd/1st serum)Number of samples
      Low titre (≥1:100/≥1:100)
      No titration, as the difference in OD was ±0.3.
      59
      Low titre (1:100/1:100)
      Titrated, as the difference in OD was >0.3.
      17
      Medium titre (1:200–1:400/1:200–1:400)22
      High titre (1:800–1:3200/1:800–1:3200)4
       Total102
      CCHFV, Crimean–Congo haemorrhagic fever virus; ELISA, enzyme-linked immunosorbent assay; OD, optical density.
      a Not including the four samples with positive IgM and from patients with acute infection.
      b No titration, as the difference in OD was ±0.3.
      c Titrated, as the difference in OD was >0.3.
      Of all 802 samples included in the screening for acute infection using IgM ELISA, four samples (0.5%) were positive for IgM antibodies. PCR was performed on these four samples and the 14 samples with negative IgM but elevated IgG (>1:200) titres. Three of these samples (0.4%) from patients with FUO and that were positive for IgM were also positive for CCHFV RNA. The total seroprevalence in the oblast of Almaty was 11.6% (44/378), with seropositive samples originating from five of the nine hospitals (Tekeli, Taldykorgan, Usharal, Kabanbay, and Almaty City, ranging from 4.8% to 21%); the total seroprevalence in the oblast of Kyzylorda was 14.6% (62/424), with positive samples from all four hospitals (Kyzylorda, Syrdariya, Zhanakordan, and Shiely, ranging from 9.6% to 30%) (Figure 1). Out of the four IgM-positive patients, three only had a mild infection with CCHFV with a temperature >37.5 °C (ear) for more than 5 days (samples Syrdariya 6, Shiely 26, and Kyzylorda 121). One patient developed fever 5 days after a tick bite on her left shoulder. She had thrombocytopenia and leukocytopenia, and developed severe CCHF with haemorrhages 4 days after the onset of fever (sample Kyzylorda 43) (Table 3). This patient was hospitalized for 10 days and recovered. In the analyses of the partial S segment, all three detected CCHFV RNAs from Kazakhstan clustered in subgroup Asia 2. Analysis of the partial L segments of the samples of the patient with the severe form of CCHF, Kyzylorda 43, revealed a clustering in subgroup Asia 2 with strains from Tajikistan (AY20893, KX013444) and a strain obtained in 1971 from Kazakhstan (KX01354453). However, the partial L segment from sample Shiely 26 clustered in subgroup Asia 1 with strains from Iraq, Afghanistan, and India. Therefore, for sample Shiely 26, a reassortment between the two Asian subtypes of CCHFV is present (Figure 2).
      Table 3CCHFV identified by sequencing of the partial S and L segments in patients with FUO in Kyzylorda oblast in Kazakhstan (2014–2015).
      Patient numberAge (years)/sexDevelopment of haemorrhagic feverS segment genotypeAccession numberL segment genotypeAccession number
      Shiely 2673/MAsia 2MG974102Asia 1MG974103
      Kyzylorda 12117/MAsia 2MG974101NA
      NA=no amplification in several different L-gene RT-PCRs.
      Kyzylorda 4322/F+Asia 2MG974100Asia 2MG974104
      CCHFV, Crimean–Congo haemorrhagic fever virus; FUO, fever of unknown origin; M, male; F, female.
      a NA = no amplification in several different L-gene RT-PCRs.
      Figure 2
      Figure 2Phylogenetic analysis of the partial small (S, 180 nt) and large (L, 220 nt) gene fragments obtained from Crimean–Congo haemorrhagic fever virus strains analyzed in this study (★). Reference strains belong to different genogroups, as retrieved from GenBank. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Numbers next to branches indicate the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates).
      The questionnaires showed that CCHFV-seropositive patients had only unspecific and mild symptoms such as headache (93.4%), fever (98.1%), weakness (76.4%), exanthema (31.1%), and diarrhoea (12.3%). Table 4 summarizes the results of the statistical analyses. Among the seropositive patients, the rate of seropositivity was higher in males (60.3%) than in females (39.6%). The mean ± standard deviation age of the seropositive patients was 35 ± 16 years. The median age of the seropositive patients was 30 years. Univariate logistic regression analysis (Table 4) did not identify a statistically significant difference in the prevalence of seropositivity between male and female patients. Seropositivity was also unrelated to age. Urban areas tended to have lower seropositivity than rural areas; however this result was only statistically significant at the 10% level. Regarding the risk factors, animal husbandry and occupation as a farm worker with animals increased the prevalence of seropositivity (p< 0.05). However, neither a history of tick bites nor the presence of birds affected seropositivity. Finally, a sub-analysis of the animal husbandry category identified that a higher frequency of contact with all animals for which data were collected (sheep, goats, cattle, horses, cats, and poultry) led to an increased prevalence of seropositivity, with the exception of pigs; however the effects were only statistically significant for cattle and horses (data not shown in detail). Comparable results were also found using univariate ordinary least squares (OLS) regression with robust standard errors.
      Table 4Characteristics of CCHFV seropositivity in patients with FUO in Kazakhstan (2014–2015).
      p-Values based on heteroscedasticity-robust standard errors.
      VariablesSeronegative IgG (n = 696)Seropositive (n = 106)Univariate logistic regression explaining seropositivity
      Logistic (Change in OR)OLS (Change in probability)
      Sex−0.071 (p> 0.05)−0.008 (p> 0.05)
       Male42764
       Female26142
       NA80
      Age (years)0.005 (p> 0.05)0.001 (p> 0.05)
       Mean3535
       SD1615
      History of tick bites−0.253 (p> 0.05)−0.028 (p> 0.05)
       Yes15920
       No53186
       NA60
      Animal husbandry0.508 (p< 0.05)
      Significant, p<0.05.
      0.059 (p< 0.05)
      Significant, p<0.05.
       Yes29048
       No39958
       NA70
      Birds or nests at home0.061 (p> 0.05)0.008 (p> 0.05)
       Yes9719
       No39997
       NA/does not know4090
      House location−0.364 (p> 0.05)−0.042 (p> 0.05)
       Rural area32860
       Urban area36246
       NA60
      Occupation
       Student/pupil97170.446 (p> 0.05)0.048 (p> 0.05)
       Farmer/farm worker
        Plants1120.495 (p> 0.05)0.186 (p< 0.05)
        Animals1041.283 (p< 0.05)
      Significant, p<0.05.
      0.186 (p< 0.05)
      Significant, p<0.05.
        Forestry810.063 (p> 0.05)0.006 (p> 0.05)
        Housekeeper2760.665 (p> 0.05)0.079 (p> 0.05)
        Unskilled labourer4080.579 (p> 0.05)0.066 (p> 0.05)
        Driver2050.813 (p> 0.05)0.100 (p> 0.05)
        Administration/academic professional5580.264 (p> 0.05)0.027 (p> 0.5)
        Business man/woman1720.005 (p> 0.05)0.0009 (p> 0.05)
        Nurse/physician/pharmacist191−1.089 (p> 0.05)−0.085 (p> 0.05)
        Other profession124140.013 (p> 0.05)0.001 (p> 0.05)
        Unemployed137240.443 (p> 0.05)0.048 (p> 0.05)
      CCHFV, Crimean–Congo haemorrhagic fever virus; FUO, fever of unknown origin; OR, odds ratio; OLS, univariate ordinary least squares; NA, not available; SD, standard deviation.
      a p-Values based on heteroscedasticity-robust standard errors.
      * Significant, p< 0.05.

      Discussion

      CCHFV is endemic in many regions such as Africa, parts of Eastern and Southern Europe, and Central Asia (
      • Ergönül O.
      Crimean-Congo hemorrhagic fever.
      ,
      • Shayan S.
      • Bokaean M.
      • Shahrivar Chinikar M.S.
      Crimean-Congo hemorrhagic fever.
      ,
      • Elevli M.
      • Ozkul A.
      • Civilibal M.
      • Midilli K.
      • Gargili A.
      • Duru N.S.
      A newly identified Crimean-Congo hemorrhagic fever virus strain in Turkey.
      ,
      • Whitehouse C.A.
      Crimean-Congo hemorrhagic fever.
      ). Clinically apparent moderate and severe haemorrhagic forms of CCHF have been described for approximately 70 years in the south of Kazakhstan, and official recording of clinically apparent cases of CCHF was implemented in 1965. Despite awareness of the moderate and severe haemorrhagic forms of CCHF, no accurate data exist on the burden of disease in the south of Kazakhstan or in so far non-endemic regions of the country (
      • Onishchenko G.
      • Tumanova I.
      • Vyshemirskiĭ O.
      • Kuhn J.
      • Seregin S.
      • Tiunnikov G.
      • et al.
      [ELISA and RT-PCR-based research of viruses in the ticks collected in the foci of Crimean-Congo fever in Kazakhstan and Tajikistan in 2001-2002].
      ). The clinical pattern of CCHFV infection can differ from unspecific symptoms such as FUO to fatal haemorrhagic fever (
      • Ergönül O.
      Crimean-Congo hemorrhagic fever.
      ,
      • Elevli M.
      • Ozkul A.
      • Civilibal M.
      • Midilli K.
      • Gargili A.
      • Duru N.S.
      A newly identified Crimean-Congo hemorrhagic fever virus strain in Turkey.
      ,
      • Ertugrul B.
      • Uyar Y.
      • Yavas K.
      • Turana C.
      • Oncua S.
      • Saylaka O.
      • et al.
      An outbreak of Crimean-Congo hemorrhagic fever in western Anatolia, Turkey.
      ,
      • Christova I.
      • Younan R.
      • Taseva E.
      • Gladnishka T.
      • Trifonova I.
      • Ivanova V.
      • et al.
      Hemorrhagic fever with renal syndrome and crimean-congo hemorrhagic fever as causes of acute undifferentiated febrile illness in Bulgaria.
      ,
      • Bodur H.
      • Akinci E.
      • Ascioglu S.
      • Onguru D.
      • Uycer Y.
      Subclinical infections with Crimean chemorrhagic fever, Turkey.
      ). FUO is a significant public health issue as it is rarely properly diagnosed and the treatment of patients is therefore insufficient. There is a need to distinguish CCHF from other infections in the course of infection and to determine the burden of disease for preventive strategies (
      • Christova I.
      • Younan R.
      • Taseva E.
      • Gladnishka T.
      • Trifonova I.
      • Ivanova V.
      • et al.
      Hemorrhagic fever with renal syndrome and crimean-congo hemorrhagic fever as causes of acute undifferentiated febrile illness in Bulgaria.
      ,
      • Bodur H.
      • Akinci E.
      • Ascioglu S.
      • Onguru D.
      • Uycer Y.
      Subclinical infections with Crimean chemorrhagic fever, Turkey.
      ,
      • Karimov S.K.
      • Kiryushchenko T.V.
      • Usebaeva G.K.
      • Rogovaya S.G.
      [Contribution to the study of Crimean hemorrhagic fever in southern regions of Kazakhstan].
      ).
      This study provides serological and molecular evidence of mild forms of CCHF for the first time, with a prevalence of 12.7% (n = 102/802; Table 1) in patients with FUO investigated in two oblasts in Kazakhstan. The oblast Kyzylorda in which 13.6% seropositivity (IgG, together with IgM 14.1%) was found for samples originating from four regional hospitals, is a well-known endemic region for clinically apparent CCHF infections (
      • Nurmakhanov T.
      • Sansyzbaev Y.
      • Atshabar B.
      • Deryabin P.
      • Kazakov S.
      • Zholshorinov A.
      • et al.
      Crimean-Congo haemorrhagic fever virus in Kazakhstan (1948-2013).
      ,
      • Knust B.
      • Medetov J.
      • Kyraubaev K.
      • Bumburidi Y.
      • Erickson B.
      • MacNeil A.
      • et al.
      Crimean-Congo hemorrhagic fever, Kazakhstan, 2009-2010.
      ,
      • Atkinson B.
      Thesis. The characterisation of arboviral zoonoses in Central Asia. September 2016.
      ,
      • Yashina L.
      • Petrova I.
      • Seregin S.
      • Vyshemirskii O.
      • Lvov D.
      • Aristova V.
      • et al.
      Genetic variability of Crimean-Congo haemorrhagic fever virus in Russia and Central Asia.
      ). However, the study results also showed that mild CCHFV infections with moderate and non-specific symptoms were present in patients with FUO in Kyzylorda, as well as in those presenting to five hospitals in the non-endemic Almaty oblast, including the city of Almaty. These data cannot be explained by immigration, as only individuals residing in the same oblast for the last 10 years were included and three-fourths (n = 31/40) of the patients were also born in Almaty oblast (data not shown in detail). Previous seroprevalence studies with complement fixation were performed in the 1970s in humans and animals in southern Kazakhstan and indicated that CCHFV infection might be present in southern Kazakhstan, in Kyzylorda and eventually in Almaty oblast, causing moderate symptoms in humans, i.e. a mild clinical presentation of acute febrile illness with no clinical hallmark, or asymptomatic infection (
      • Gaeta G.B.
      • Fusco F.M.
      • Nardiello S.
      Fever of unknown origin: a systematic review of the literature for 1995-2004.
      ). The results of the present study strengthen the assumptions made in this earlier study.
      The rate of seropositivity of 4.8% up to 30% found in FUO patients presenting to the 13 hospitals included in this study corresponds with previously reported rates for CCHFV endemic regions in Eastern and Southern Europe, Africa, and other parts of Asia (
      • Ergönül O.
      Crimean-Congo hemorrhagic fever.
      ,
      • Shayan S.
      • Bokaean M.
      • Shahrivar Chinikar M.S.
      Crimean-Congo hemorrhagic fever.
      ,
      • Elevli M.
      • Ozkul A.
      • Civilibal M.
      • Midilli K.
      • Gargili A.
      • Duru N.S.
      A newly identified Crimean-Congo hemorrhagic fever virus strain in Turkey.
      ,
      • Ertugrul B.
      • Uyar Y.
      • Yavas K.
      • Turana C.
      • Oncua S.
      • Saylaka O.
      • et al.
      An outbreak of Crimean-Congo hemorrhagic fever in western Anatolia, Turkey.
      ,
      • Yashina L.
      • Petrova I.
      • Seregin S.
      • Vyshemirskii O.
      • Lvov D.
      • Aristova V.
      • et al.
      Genetic variability of Crimean-Congo haemorrhagic fever virus in Russia and Central Asia.
      ,
      • Christova I.
      • Younan R.
      • Taseva E.
      • Gladnishka T.
      • Trifonova I.
      • Ivanova V.
      • et al.
      Hemorrhagic fever with renal syndrome and crimean-congo hemorrhagic fever as causes of acute undifferentiated febrile illness in Bulgaria.
      ,
      • Grekova I.
      • Kamarinchev B.
      Seroprevalence of Crimean-Congo hemorrhagic fever in the Southeastern Bulgaria.
      ,
      • Izadi S.
      • Holakou-Naieni K.
      • Majdzadeh S.R.
      • Chinikar S.
      • Nadim A.
      • Rakhshani F.
      • et al.
      Seroprevalence of Crimean-Congo hemorrhagic fever in Sistanva-Baluchestan Province of Iran.
      ). Typical signs of mild CCHFV infection are moderate fever, headache, weakness, backache, exanthema, and diarrhoea (
      • Yashina L.
      • Petrova I.
      • Seregin S.
      • Vyshemirskii O.
      • Lvov D.
      • Aristova V.
      • et al.
      Genetic variability of Crimean-Congo haemorrhagic fever virus in Russia and Central Asia.
      ,
      • Christova I.
      • Younan R.
      • Taseva E.
      • Gladnishka T.
      • Trifonova I.
      • Ivanova V.
      • et al.
      Hemorrhagic fever with renal syndrome and crimean-congo hemorrhagic fever as causes of acute undifferentiated febrile illness in Bulgaria.
      ,
      • Grekova I.
      • Kamarinchev B.
      Seroprevalence of Crimean-Congo hemorrhagic fever in the Southeastern Bulgaria.
      ,
      • Izadi S.
      • Holakou-Naieni K.
      • Majdzadeh S.R.
      • Chinikar S.
      • Nadim A.
      • Rakhshani F.
      • et al.
      Seroprevalence of Crimean-Congo hemorrhagic fever in Sistanva-Baluchestan Province of Iran.
      ). Most of these symptoms were also detectable in seropositive patients in the present study. According to the clinical classification in Kazakhstan, CCHF with moderate symptoms is considered the non-haemorrhagic form of CCHF. However, CCHF cases with moderate symptoms are not diagnosed, and only critical cases with the severe haemorrhagic form have been registered during the last years (
      • Leshinskaya E.V.
      [Clinical manifestation of Congo-Crimean hemorrhagic fever].
      ,
      • N.N
      Guidlines of CCHF treatment and diagnostics N9, implemented by the expert сouncil of the health care ministry and social development of the Republic of Kazkahstan.
      ,
      • Yegemberdiyeva R.
      Clinical and epidemiological manifestation of some natural foci transmissive infections in Kazakhstan], Thesis.
      ). The results of this study showed that a certain proportion of the population (Table 2; 59 samples) had CCHFV-specific IgG antibodies in both investigated sera without any titre change. It is supposed that this could be evidence of a previous CCHFV infection in the past that was either asymptomatic or mild, as also shown in previous studies in other countries (
      • Ergönül O.
      Crimean-Congo hemorrhagic fever.
      ,
      • Shayan S.
      • Bokaean M.
      • Shahrivar Chinikar M.S.
      Crimean-Congo hemorrhagic fever.
      ,
      • Elevli M.
      • Ozkul A.
      • Civilibal M.
      • Midilli K.
      • Gargili A.
      • Duru N.S.
      A newly identified Crimean-Congo hemorrhagic fever virus strain in Turkey.
      ,
      • Ertugrul B.
      • Uyar Y.
      • Yavas K.
      • Turana C.
      • Oncua S.
      • Saylaka O.
      • et al.
      An outbreak of Crimean-Congo hemorrhagic fever in western Anatolia, Turkey.
      ,
      • Yashina L.
      • Petrova I.
      • Seregin S.
      • Vyshemirskii O.
      • Lvov D.
      • Aristova V.
      • et al.
      Genetic variability of Crimean-Congo haemorrhagic fever virus in Russia and Central Asia.
      ,
      • Christova I.
      • Younan R.
      • Taseva E.
      • Gladnishka T.
      • Trifonova I.
      • Ivanova V.
      • et al.
      Hemorrhagic fever with renal syndrome and crimean-congo hemorrhagic fever as causes of acute undifferentiated febrile illness in Bulgaria.
      ,
      • Grekova I.
      • Kamarinchev B.
      Seroprevalence of Crimean-Congo hemorrhagic fever in the Southeastern Bulgaria.
      ,
      • Izadi S.
      • Holakou-Naieni K.
      • Majdzadeh S.R.
      • Chinikar S.
      • Nadim A.
      • Rakhshani F.
      • et al.
      Seroprevalence of Crimean-Congo hemorrhagic fever in Sistanva-Baluchestan Province of Iran.
      ).
      Four acute infections were detected in patients from Kyzylorda with only one developing a severe haemorrhagic form of CCHF. In the patient with acute CCHF, the disease also started with mild atypical symptoms. IgM is usually detectable at the time of infection, between day 7 and day 10. The study findings are consistent with those of previous studies in other endemic countries that have reported the detection of CCHFV-specific IgM or viral RNA and clinical manifestations of CCHF as mild disease with moderate fever but without any haemorrhagic syndrome or asymptomatic disease, in a certain proportion of patients (
      • Yashina L.
      • Petrova I.
      • Seregin S.
      • Vyshemirskii O.
      • Lvov D.
      • Aristova V.
      • et al.
      Genetic variability of Crimean-Congo haemorrhagic fever virus in Russia and Central Asia.
      ,
      • Christova I.
      • Younan R.
      • Taseva E.
      • Gladnishka T.
      • Trifonova I.
      • Ivanova V.
      • et al.
      Hemorrhagic fever with renal syndrome and crimean-congo hemorrhagic fever as causes of acute undifferentiated febrile illness in Bulgaria.
      ,
      • Grekova I.
      • Kamarinchev B.
      Seroprevalence of Crimean-Congo hemorrhagic fever in the Southeastern Bulgaria.
      ,
      • Izadi S.
      • Holakou-Naieni K.
      • Majdzadeh S.R.
      • Chinikar S.
      • Nadim A.
      • Rakhshani F.
      • et al.
      Seroprevalence of Crimean-Congo hemorrhagic fever in Sistanva-Baluchestan Province of Iran.
      ,
      • Fajs L.
      • Humoli I.
      • Saksida A.
      • Knap N.
      • Jelovsek M.
      • Korva M.
      • et al.
      Prevalence of Crimean-Congo hemorrhagic fever virus in healthy population, livestock and ticks in Kosovo.
      ,
      • Sargianou M.
      • Panos G.
      • Tsatsaris A.
      • Gogos C.
      • Papa A.
      Crimean-Congo hemorrhagic fever: seroprevalence and risk factors among humans in Achaia, western Greece.
      ). As nosocomial infections have already been described in Kazakhstan (
      • Pshenichnaya N.Y.
      • Leblebicioglu H.
      • Bozkurt I.
      • Sannikova I.V.
      • Abuova G.N.
      • Zhuravlev A.S.
      • et al.
      Crimean-Congo hemorrhagic fever in pregnancy: a systematic review and case series from Russia, Kazakhstan and Turkey.
      ), hospital personnel should also be aware that mild forms or even asymptomatic forms of CCHF with viremia exist, and early diagnostic tools must be implemented in hospitals and laboratories.
      Interestingly, factors that have been described previously as significantly associated with CCHF, such as older age, male sex, history of previous tick bite, and living in a rural residence (
      • Schmaljohn C.S.
      • Nichol S.T.
      Bunyaviridae.
      ), were found not to be significantly associated with antibodies against CCHF in the present study. The main vectors of CCHFV, Hyalomma and Dermacentor ticks, favour dry climates and vegetation where rodents are abundant (
      • Hoogstraal H.
      The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa.
      ,
      • Leblebicioglu H.
      Crimean-Congo haemorrhagic fever in Eurasia.
      ). Previous investigations have shown that both tick species are widely distributed in Kazakhstan and found in several oblasts, including Kyzylorda and Almaty oblasts (
      • Onishchenko G.
      • Tumanova I.
      • Vyshemirskiĭ O.
      • Kuhn J.
      • Seregin S.
      • Tiunnikov G.
      • et al.
      [ELISA and RT-PCR-based research of viruses in the ticks collected in the foci of Crimean-Congo fever in Kazakhstan and Tajikistan in 2001-2002].
      ,
      • Knust B.
      • Medetov J.
      • Kyraubaev K.
      • Bumburidi Y.
      • Erickson B.
      • MacNeil A.
      • et al.
      Crimean-Congo hemorrhagic fever, Kazakhstan, 2009-2010.
      ). Furthermore, a previous study showed that reported tick bites in South Kazakhstan oblast were significantly associated with the number of CCHF cases per week (
      • Knust B.
      • Medetov J.
      • Kyraubaev K.
      • Bumburidi Y.
      • Erickson B.
      • MacNeil A.
      • et al.
      Crimean-Congo hemorrhagic fever, Kazakhstan, 2009-2010.
      ). CCHFV was identified by antigen ELISA in 3% (n = 3/100 pools from 1000 ticks) of Dermacentor niveus in two districts of Kyzylorda in 2001 (
      • Yashina L.
      • Petrova I.
      • Seregin S.
      • Vyshemirskii O.
      • Lvov D.
      • Aristova V.
      • et al.
      Genetic variability of Crimean-Congo haemorrhagic fever virus in Russia and Central Asia.
      ). So, the vectors of CCHF are present in some areas of Kazakhstan. Further vector studies are needed in Kazakhstan to describe the natural foci of CCHFV in ticks (
      • Yagci-Caglayik D.
      • Korukluoglu G.
      • Uyar Y.
      Seroprevalence and risk factors of Crimean-Congo hemorrhagic fever in selected seven provinces in Turkey.
      ).
      Several domestic and wild animals, e.g., cattle, goats, and sheep, transmit CCHFV. Small mammals and birds support the transmission cycle (
      • Hoogstraal H.
      The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa.
      ;
      • Ergönül O.
      Crimean-Congo hemorrhagic fever.
      ;
      • Whitehouse C.A.
      Crimean-Congo hemorrhagic fever.
      ). This study found that the rate of seropositivity was significantly higher in people with a history of animal husbandry, such as those working with sheep, goats, cattle, horses, cats, and poultry, as well as in people with an occupation of farm worker working with animals. Previous studies have also reported that people could become infected by occasional contact with livestock. There is an overlap of seroprevalence in animals and the incidence in humans (
      • Grekova I.
      • Kamarinchev B.
      Seroprevalence of Crimean-Congo hemorrhagic fever in the Southeastern Bulgaria.
      ,
      • Izadi S.
      • Holakou-Naieni K.
      • Majdzadeh S.R.
      • Chinikar S.
      • Nadim A.
      • Rakhshani F.
      • et al.
      Seroprevalence of Crimean-Congo hemorrhagic fever in Sistanva-Baluchestan Province of Iran.
      ). Tick densities on a single farm animal can be high in CCHF endemic regions in southern Kazakhstan during the summer, with over 1500 adults and 2–3000 nymphs (
      • Onishchenko G.
      • Tumanova I.
      • Vyshemirskiĭ O.
      • Kuhn J.
      • Seregin S.
      • Tiunnikov G.
      • et al.
      [ELISA and RT-PCR-based research of viruses in the ticks collected in the foci of Crimean-Congo fever in Kazakhstan and Tajikistan in 2001-2002].
      ,
      • Nurmakhanov T.
      • Sansyzbaev Y.
      • Atshabar B.
      • Deryabin P.
      • Kazakov S.
      • Zholshorinov A.
      • et al.
      Crimean-Congo haemorrhagic fever virus in Kazakhstan (1948-2013).
      ). Therefore, information on CCHF should be provided to people working with sheep, goats, cattle, horses, cats, and poultry, as well as doctors in Kazakhstan.
      Phylogenetic analyses of CCHFV strains indicate at least seven different clades of CCHFV. Analyses of the S segment usually mirror the geographical distribution of the strains. So far only limited data on genetic sequences of Kazakh CCHFV strains have been published, as RT-PCR is still uncommon in Kazakhstan (
      • Onishchenko G.
      • Tumanova I.
      • Vyshemirskiĭ O.
      • Kuhn J.
      • Seregin S.
      • Tiunnikov G.
      • et al.
      [ELISA and RT-PCR-based research of viruses in the ticks collected in the foci of Crimean-Congo fever in Kazakhstan and Tajikistan in 2001-2002].
      ). The phylogenetic analyses of sequences of all three partial S segments obtained showed that they clustered within the Asia 2 subgroup, whereas in the L segment sample, Shiely 26 grouped within the Asia 1 group. Previous studies have described the presence of these two subgroups in Kazakhstan (
      • Christova I.
      • Younan R.
      • Taseva E.
      • Gladnishka T.
      • Trifonova I.
      • Ivanova V.
      • et al.
      Hemorrhagic fever with renal syndrome and crimean-congo hemorrhagic fever as causes of acute undifferentiated febrile illness in Bulgaria.
      ,
      • Atkinson B.
      • Chamberlain J.
      • Logue H.C.
      • Cook N.
      • Bruce C.
      • Dowall D.S.
      • et al.
      Development of a real-time RT PCR assay for the detection of Crimean-Congo hemorehagic fever virus.
      ,
      • Tumanova I.
      • Seregin S.
      • Vyshemirskiĭ O.
      • Gutorov V.
      • Petrova I.
      • Tiunnikov G.
      • et al.
      [Genetic monitoring of the Crimean-Congo hemorrhagic fever virus in Kazakhstan and Tajikistan in 2001-2003].
      ,
      • Iashina L.
      • Petrov V.
      • Petrova I.
      • Gutorov V.
      • Kazakov S.
      • Ospanov K.
      • et al.
      Genetic [Identification of the Crimean-Congo hemorrhagic fever virus during epidemic outbreak in Kazakhstan in 2000].
      ).
      The sample Shiely 26 in this study showed a reassorted genotype Asia 2 in the S segment and Asia 1 in the L segment. Interestingly, despite the general observation that the S segments of CCHFV strains were grouped according to the geographic distribution, Kazakhstan is not close to any of the other countries with the circulating Asia 1 subgroup, which include Iraq, Afghanistan, Oman, and India. However, segmented RNA viruses often undergo genetic reassortment (
      • Chinikar S.
      • Bouzari S.
      • Shokrgozar M.A.
      • Mostafavi E.
      • Jalali T.
      • Khakifirouz S.
      • et al.
      Genetic diversity of Crimean-Congo hemorrhagic fever virus strains from Iran.
      ,
      • Greenbaum B.D.
      • Li O.T.
      • Poon L.L.
      • Levine A.J.
      • Rabadan R.
      Viral reassortment as an information exchange between viral segments.
      ). Based on sequences of all three genomic fragments, it has been shown that CCHFV genomes are highly susceptible to reassortment and that the reassortment is statistically supported (
      • Zhou Z.
      • Deng F.
      • Han N.
      • Wang H.
      • Sun S.
      • Zhang Y.
      • et al.
      Reassortment and migration analysis of Crimean-Congo haemorrhagic fever virus.
      ). However, reassortment does not completely follow geographical factors, and it is suggested to be facilitated by the dispersal of different genotypes through migratory birds. Tick distribution by birds, animal trading, and local and long distance transport routes such as the new silk road and railways, could play a significant role in the transmission of the virus, and this may explain the presence of a reassorted Asia 1/Asia 2 type in Kyzylorda oblast (
      • Whitehouse C.A.
      Crimean-Congo hemorrhagic fever.
      ,
      • Tumanova I.
      • Seregin S.
      • Vyshemirskiĭ O.
      • Gutorov V.
      • Petrova I.
      • Tiunnikov G.
      • et al.
      [Genetic monitoring of the Crimean-Congo hemorrhagic fever virus in Kazakhstan and Tajikistan in 2001-2003].
      ,
      • Chinikar S.
      • Shah-Hosseini N.
      • Bouzari S.
      • Shokrgozar M.A.
      • Mostafavi E.
      • Jalali T.
      • et al.
      Assessment of reassortment in the S-segment genome of Crimean-Congo hemorrhagic fever virus in Iran.
      ,
      • Leblebicioglu H.
      • Eroglu C.
      • Erciyas-Yavuz K.
      • Hokelek M.
      • Acici M.
      • Yilmaz H.
      Role of migratory birds in spreading Crimean-Congo hemorrhagic fever, Turkey.
      ).
      In summary, this article reports for the first time that CCHF may appear as a mild or asymptomatic endemic infection in two oblasts of Kazakhstan. Moreover, recent CCHF infections were detected in patients with FUO in the so far non-endemic oblast of Almaty. Animal husbandry appears to be a significant factor for infection with CCHFV. The results of this study show that the distribution of CCHFV in Kazakhstan could be wider than previously thought and that physicians treating patients with FUO should also be aware of mild or asymptomatic CCHF.

      Acknowledgements

      We thank the staff of the Scientific Practical Centre for Sanitary Epidemiological Expertise and Monitoring, Kazakh National Medical University and the hospitals in the pilot regions for their technical assistance. The authors also express gratitude to the Exceed Programme of German Federal Ministry for Economic Cooperation and Development (BMZ) and the German Academic Exchange Services (DAAD) through the CIH LMU – Centre for International Health, Ludwig-Maximilians-Universität, Munich, Germany. This study was funded by the German Biosecurity Programme of the German Federal Foreign Office.

      Ethical approval

      The recruitment and enrolment of patients, questionnaire, sampling methods, and laboratory investigations were reviewed and approved by the ethics committees in Kazakhstan and Germany following international standards.

      Disclaimer

      The opinions expressed by the authors contributing to this study do not necessarily reflect the opinions of the institutes involved.

      Conflict of interest

      No conflict of interest to declare.

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