Seroprevalence of Crimean-Congo Hemorrhagic Fever in Sistan-Va
Jpn. J. Infect. Dis., 59, 326-328, 2006
Short Communication Seroprevalence of Crimean-Congo Hemorrhagic Fever in Sistan-va-Baluchestan Province of Iran Shahrokh Izadi, Kourosh Holakouie-Naieni1*, Seyed Reza Majdzadeh1, Sadegh Chinikar2, Abolhassan Nadim1, Fatemeh Rakhshani and Badakhshan Hooshmand3 Department of Epidemiology and Biostatistics, School of Public Health, Zahedan University of Medical Sciences, Zahedan; 1Department of Epidemiology and Biostatistics, School of Public Health and Institute of Health Research, Tehran University of Medical Sciences; 2Arbovirus Laboratory, Pasteur Institute of Iran; and 3Zoonosis Department, Center for Disease Control, Ministry of Health and Medical Education of I.R. Iran, Tehran, Iran (Received February 6, 2006. Accepted June 23, 2006)
SUMMARY: During the years 2000 to 2004, of 248 serologically confirmed cases of Crimean-Congo hemor- rhagic fever (CCHF) that occurred in several parts of Iran, 169 were reported from Sistan-va-Baluchestan province. To assess the seroprevalence of CCHF virus infection within the Zahedan and Zabol districts of the Sistan-va-Baluchestan province in Iran, 300 subjects were sampled from the general population. In addition to blood sampling, a questionnaire was completed for every subject. All but just 3 of our 300 sampled subjects participated in blood sampling, and just 7 out of the 297 serum samples were found to be IgG ELISA positive. The point estimate of the seroprevalence was 0.024 (95% confidence interval: 0.003 -0.044). A history of keeping livestock in houses (even for short periods) showed an association with seropositivity (P = 0.018). It seems that even occasional contact with livestock could be effective in transmission of the virus.
Crimean-Congo hemorrhagic fever (CCHF) is a viral hem- orrhagic fever of the Nairovirus group. Although primarily a zoonosis, sporadic cases and outbreaks of CCHF affecting humans do occur. Humans who become infected with CCHF acquire the virus from direct contact with blood or other infected tissues from infected livestock, or they may become infected from a tick bite (1). In 1970 Chomacov et al. pub- lished the first report about CCHF in Iran, when 45 of 100 sheep sera that were sent from Tehran abattoir to Moscow (Institute of Poliomyelitis and Viral Encephalitides) reacted positively for CCHF virus infection (2). In 1975 a large-scale serologic study was performed with the cooperation of Yale University, mostly in the northern half of Iran. In this study, where ‘agar gel diffusion precipitation’ tests were used, 13% of human sera, 38% of cattle sera, and 18% of sheep sera were seropositive for CCHF (3). In 1978, the CCHF virus was isolated for the first time from engorged Alveonasus lahorensis in the northeastern Fig. 1. Zabol and Zahedan districts in Sistan-va-Baluchestan province, Iran. region of Iran (4). After that, there was no report of the disease until 1999, when the disease reemerged, and for the next 5 years the number of cases increased annually. From 7 the border. Afghan refugees have mostly settled in the urban June 2000 to 15 October 2004, of 683 serum samples of CCHF regions. suspected cases from several regions of Iran, sent to the This study was designed to determine the seroprevalence national reference laboratory of arboviral diseases in the of CCHF infection in the Zabol and Zahedan districts. The Pasteur Institute of Iran, Tehran, 248 sera were positive for primary health care (PHC) coverage in Zahedan and Zabol is anti-CCHF virus antibodies. Of these, 169 seropositive cases 84 and 95%, repectively. The sampling began on 10 January were reported from the Zabol and Zahedan districts located 2002 and lasted until 20 March 2002. The only inclusion in the northern part of Sistan-va-Baluchestan province (Fig- criterion was being a dweller of the study area. There were ure 1) (5). This province is located in the neighborhood of no age or geographical restrictions in selecting the study Afghanistan and Pakistan to the east and is the residential subjects, and the sampling method was a modified form of area of many Afghan refugees. It is also the passage route of the classical probability-proportional-to-size (PPS) cluster legal and illegal traders and nomads between two sides of sampling (6). During an interview with every individual a questionnaire was completed and a blood sample was taken. *Corresponding author: Mailing address: P.O. Box 6446, Tehran The variables considered within the questionnaire were: age, 14155, I.R. Iran.Tel: +98-21-895-1402, Fax: +98-21-895-1397, sex, living environment (rural/urban), education years, occu- E-mail: [email protected] pation, history of tick bites, history of contact with livestock,
326 history of slaughtering of livestock, and having a special place sion 9. for taking care of livestock at home. All the exposure histories All but just 3 of our 300 sample subjects gave their consent were considered for the past 12 months. All adults responded for blood sampling, and just 7 out of the 297 serum samples for themselves, but children who were not able to answer were positive for IgG using an ELISA test, that is, 0.024 of were assisted by one of the parents (most often the mother). the subjects were seropositive for anti-CCHF virus IgG (95% Blood samples were transferred to the national reference CI: 0.003 - 0.044). None of the seropositive subjects, their laboratory of arboviral diseases in the Pasteur Institute of first-degree relatives and households, or near friends had a Iran (under cold-chain regulations and by airplane) for IgG history of CCHF or a similar clinical picture, which led us to capture enzyme-linked immunosorbent assay (ELISA) tests. conclude that our seropositive subjects may have experi- ELISAs were performed as follows: the ELISA plates were enced a subclinical infection. Anti-CCHF virus IgG remains coated with immunized mouse ascites fluid for CCHF virus detectable for at least 5 years after infection, while IgM is proteins at an optimal dilution of 1:4,000 and incubated over- detectable mainly within the first 4 months of onset of CCHF night at 4°C. The wells were then washed with phosphate- virus infection and is used for detection and/or confirmation buffered saline containing 0.05% Tween 20 (PBST). Next, of ongoing or recent infection (7). 100 μl of appropriately diluted CCHF antigen was added to According to Hoogstraal, in epidemic situations the expected each well, and the plates were incubated for 1 h at 37°C. seroprevalence is about 10%, which is much higher than the After washing the wells with PBST, we added the sera, seroprevalence in our findings (2). In a survey performed with diluted in PBST and 3% skim milk (PBSTM), to each well nomadic tribes in Senegal the seropositivity rate was 13.1% and the plates were incubated for 1 h at 37°C. After the plates in a context of endemic transmission. The laboratory method were washed with PBST, 100 μl of peroxidase-conjugated used in that study was ELISA for CCHF virus-specific IgG goat affinity to human IgG was added to each well. After and IgM antibodies (8). However, in another survey in a South incubation (1 h at 37°C) and washing with PBST, 100 μl of African rural community, of 475 asymptomatic people on 36 a tetramethylbenzidine substrate was added to each well and farms, 6 people who had no history of disease compatible the reaction was read after 15 min at room temperature by an with CCHF were found to have antibody to CCHF (point ELISA reader at 450 nm. prevalence = 1.26%). The laboratory method in that study Since our sampling method was a kind of cluster sampling was indirect immunofluorescence antibody assay for IgG (PPS), we had to consider the design effect of the calculation and IgM (9). Considering the above results, it seems that of the confidence interval (CI) on the estimated seroprevalence. the estimated seroprevalence of CCHF virus infection in our The usual way to measure the precision of an estimate (in the study is much lower than what is expected in an epidemic or case of our study, the seroprevalence) is by its standard error endemic situation. The underlying cause of this discrepancy (usually denoted by Se). The usual formula for calculation is not clear to us and remains be elucidated. of standard error when the data (in the case of the present The most important characteristics of seropositive subjects study, the binomial data) are assumed to come from a “simple were compared with seronegative subjects (Table 1). All random sample” is Se = [p(1–p)/n]1/2. However, in cluster seropositive subjects had a positive history of keeping live- sampling this formula changes to Se = [p(1–p)D/n]1/2. In the stock in the house within the past 12 months, while only 55.6% second formula, D stands for the design effect that measures of seronegative subjects had a positive history of this, and the increase in the Se of the estimate due to the sampling this variable seems to be the most important risk factor (P = procedure used. The design effect and CI were calculated by 0.018). At the time of interviewing, 3 of 7 seropositive sub- previously published methods (6). The data were analyzed jects still kept livestock in their houses. It is worth mention- using Statistical Package for Social Scientists (SPSS) ver- ing that all these subjects were dwellers of urban areas or
Table 1. Comparison of seropositive and seronegative subjects Seropositive Seronegative Fisher exact test Variables No. (%) No. (%) P-value History of keeping livestock in houses 0.018 (within the past 12 mo) Never 0 ( 0) 128 ( 44) Sometimes 7 (100) 160 ( 56) Nationality 0.172 Iranian 4 ( 57) 219 ( 79) Afghan 3 ( 43) 58 ( 21) Sex 0.452 Male 4 ( 57) 138 ( 48) Female 3 ( 43) 152 ( 52) Age (yr) 0.1131) ≤ 15 4 ( 57) 122 ( 42) 16-401 ( 14) 133 ( 46) > 40 2 ( 29) 35 ( 12) Living environment 0.0301) Urban 3 ( 43) 153 ( 55) Urban with rural culture 4 ( 57) 37 ( 13) Good condition rural areas 0 ( 0) 32 ( 11) Poor condition rural areas 0 ( 0) 59 ( 21) 1): “Exact test” has performed according to the same method as the Fisher exact test (12).
327 urban areas with rural culture (P = 0.030). This finding is in hemorrhagic fever-Congo (CHF-C) virus antibodies in contrast to our understanding of CCHF as a disease of rural man, and in domestic and small mammals, in Iran. Am. communities. A simple explanation for this discrepancy is J. Trop. Med. Hyg., 24, 353-357. the presence of special markets for animal trading on the 4. Sureau, P., Klein, J.M., Casals, J., Digoutte, J.P., Salaun, outskirts of large cities of the study area that increases the J.J., Piazak, N. and Calvo, M.A. (1980): Isolation of chance of contact of residents with infected animals. Thogoto, Wad Medani, Wanowrie and Crimean-Congo Three of the seropositive subjects were Afghan refugees, haemorrhagic fever viruses from ticks of domestic ani- 2 males (age 13 and 25) and 1 female (age 12). Whether they mals in Iran. Ann. Virol. (Inst. Pasteur), 131 E, 185-200. have become infected in Iran or in Afghanistan is not known. 5. Chinikar, S., Mazaheri, V., Mirahmadi, R., Nabeth, P., From August 1999 to September 2001, 24 symptomatic Saron, M.F., Salehi, P., Hosseini, N., Bouloy, M., serologically confirmed CCHF cases from the Zahedan and Mirazimi, A., Lundkvist, A., Nilsson, M. and Mehrabi- Zabol districts were reported to the Communicable Disease Tavana, A. (2005): A serological survey in suspected Center for Disease Control of Iran (10). The youngest among human patients of Crimean-Congo haemorrhagic fever them was 12 and the next youngest was 17, while among in Iran by determination of IgM-specific ELISA method the asymptomatic subjects of the present study there were 2 during 2000-2004. Arch. Iranian Med., 8, 52-55. cases less than 10 years old (3 and 6 years old). Even though 6. Bennett, S., Woods, T., Liyanage, W.M. and Smith, D.L. more observations are required for a consistent conclusion, it (1991): A simplified general method for cluster-sample seems that the proportion of asymptomatic infection among surveys of health in developing countries. World Health children has been higher than among adults (P = 0.045, Fisher Stat. Q., 44, 98-106. exact test). 7. Swanepoel, R. (1998): Crimean-Congo Haemorrhagic Our findings raise the suspicion that the CCHF virus could Fever. p. 311-317. In S.R. Palmer, Lord Soulsby, and be transmitted even in the case of only occasional contact with D.I.H. Simpson (ed.), Zoonosis, Biology, Clinical Prac- livestock. There are clues from other studies that are com- tice, and Public Health Control. Oxford University Press, patible with these findings, and if our suspicion is correct, Oxford. instructions for prevention of transmission of CCHF virus 8. Chapman, L.E., Wilson, M.L., Hall, D.B., LeGuenno, must be revised (10,11). B., Dykstra, E.A., Ba, K. and Fisher-Hoch, S.P. (1991): Risk factors for Crimean-Congo hemorrhagic fever in rural northern Senegal. J. Infect. Dis., 164, 686-692. ACKNOWLEDGMENTS 9. Fisher Hoch, S.P., McCormick, J.B., Swanepoel, R., Our thanks are due to all staff members of Arbovirus Van-Middlekoop, A., Harvey, S. and Kustner, H.G. Laboratory, Pasteur Institute of Iran for performing all ELISA (1992): Risk of human infections with Crimean-Congo tests; to the staff members of Sistan-va-Baluchestan Prov- hemorrhagic fever virus in a south African rural com- ince Health Center and all the staff members of Zahedan and munity. Am. J. Trop. Med. Hyg., 47, 337-345. Zabol District Health Centers for providing fieldwork and 10. Izadi, S., Holakouie-Naieni, K., Madjdzadeh, S.R. and facilities for this study. Nadim, A. (2004): Crimean-Congo hemorrhagic fever in Sistan and Baluchestan province of Iran, a case- control study on epidemiological characteristics. Int. J. REFERENCES Infect. Dis., 8, 299-306. 1. World Health Organization (2000): Crimean-Congo 11.Ozkurt, Z., Kiki, I., Erol, S., Erdem, F., Yilmaz, N., Parlak, haemorrhagic fever. Fact sheet No. 208. Revised March M., Gundogdu, M. and Tasyaran, M.A. (2006): Crimean- 2000. WHO/OMS. Online at
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