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Seroprevalence and risk factors of Coxiella burnetii infection among high-risk population in center of Iran, a neglected health problem
Acta Tropica 169 (2017) 107–111
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Seroprevalence and risk factors of Coxiella burnetii infection among high-risk population in center of Iran, a neglected health problem Zary Nokhodian a , Behrooz Ataei a , Abdolreza Moradi b , Majid Yaran c , Shervin Gaffari Hoseini a , Awat Feizi d,∗ , Roya Sherkat e a
Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Isfahan Veterinary Office, Head of Public Health Supervision Section, Isfahan, Iran c Nosocomial Infection Research Center, Isfahan University of Medical Sciences, Isfahan, Iran d Department of Biostatistics and Epidemiology, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran e Acquired Immunodeficiency Research Center, Isfahan University of Medical Sciences, Isfahan, Iran b
a r t i c l e
i n f o
Article history: Received 23 October 2016 Received in revised form 2 February 2017 Accepted 2 February 2017 Available online 5 February 2017 Keywords: C. burnetii High-risk population Iran Seroprevalence
a b s t r a c t In order to evaluate the prevalence of antibodies against phase I and II antigens of Coxiella Burnetii and to identify related risk factors among high-risk groups in the center of Iran, a serological survey was performed in Isfahan County. In a cross-sectional study, 401 sera were collected from slaughterhouse workers, butchers, farmers and veterinarians in spring 2015. Samples were tested for specific immunoglobulin G (IgG) antibodies against phase I and II of C. burnetii by indirect immunofluorescence assay. A checklist was fulfilled to document demographic information. Univariate analysis and multivariable binary logistic regression model were used to analyze data. IgG antibodies against phases I and II of C. burnetii were detected in 19% and 36.9% of participants, respectively. The overall seropositivity (IgG against phase I and/or II) was 43.1%. The present study shows a high seroprevalence of C. burnetii infection among high-risk population in center of Iran. It is suggested to carry out occupational health monitoring programs for individuals who may be exposed to C. burnetii. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Q fever, a zoonotic disease caused by Coxiella burnetii, has been reported worldwide except for Antarctica and New Zealand (Anderson, 2013). The bacteria, which is a gram negative intracellular coccobacillus, can infect a wide range of animal species including domestic and wild mammals, and non-mammals such as birds, reptiles and ticks (Parker et al., 2006). Ruminants, which are the main source of human infection, excrete the bacteria into the environment where the microorganism is able to withstand tough conditions for weeks (Tozer et al., 2011). Typically, human infection is acquired by inhalation of contaminated aerosols from infected ruminants (Whitney et al., 2009). The consumption of unpasteurized dairy products, tick-bite, and person-to-person contact could be other risk factors for disease transmission (Whitney et al., 2009; Sprong et al., 2012). In this regard, the persons who have close contact with animals such as farmers, butchers, slaughterhouse
∗ Corresponding author. E-mail address: awat [email protected] (A. Feizi). http://dx.doi.org/10.1016/j.actatropica.2017.02.002 0001-706X/© 2017 Elsevier B.V. All rights reserved.
workers, livestock handlers, and veterinarians are considered to be at high risk of C. burnetii infection (Battelli, 2008; Nielsen et al., 2013). C. burnetii infection can present as acute or chronic Q fever disease, which exhibits different clinical manifestations. The three main clinical presentations of the acute disease are a flu-like syndrome, pneumonia and hepatitis; however, the majority of the infections remain asymptomatic (Norlander, 2000; Parker et al., 2006). Chronic infection may develop in about 5% of infected patients, and may become apparent months or years after an acute episode. Endocarditis is the most common complication of the chronic infection. People with cardiac valve and vascular abnormalities and immunocompromised patients have a higher risk for chronic disease. The mortality rate in chronic Q fever is estimated from 2% to 65% (Norlander, 2000; Whitney et al., 2009; Kampschreur et al., 2012). Q fever diagnosis is based on serological tests and the immunofluorescence assay (IFA) is used as the reference method for the sero-diagnosis of the disease (Vaidya et al., 2008). Although nowadays, because an IFA test is negative in the first stages of acute
108
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Q fever infection, PCR assay in combination with serologic test is recommended (Anderson, 2013). The presence of C. burnetii antibodies has been reported in livestock in numerous studies in different parts of Iran (Mostafavi et al., 2012), however few studies have been conducted on people at risk of this infection and seroprevalence of the infection is unknown in most regions of Iran (Esmaeili et al., 2014; Esmaeili et al., 2016). In addition, majority of previous studies were conducted on butchers, slaughterhouse workers and veterinary students and there is not any study on farmers, the people who have the most contact with animals. The objective of the present study was to investigate the presence of IgG antibodies against phase I and II of C. burnetii in high-risk occupational groups by IFA assay and to identify infection risk factors among these individuals in the Isfahan County, Iran.
2. Materials and methods 2.1. Study design, area and samples In spring 2015, a cross-sectional study was conducted among high-risk populations in the Isfahan County, which is the largest County in Isfahan Province, located in the center of Iran and southeast of Tehran (Fig. 1). This county has thirteen cities, covers an area of 10,5937 km2 , and has a human population of 21,741,72. About 10% of the population lives in rural areas and the primary occupation of them are agriculture and animal husbandry. The urban people are engaged in industrial and service jobs. About 30,0000 small ruminants (sheep and goat) and 10,0000 large ruminants (cattle and calf) are registered in veterinary head office of Isfahan country. The camel farming is not an important activity in this area. There are two types of ruminant herds in Isfahan County: traditional and commercial herds. All of small ruminants and 60% of large ruminants are kept on the traditional farms. Traditional farms, which are located in the villages, do not usually have suitable buildings for keeping animals; in fact, farmer share part of his house with animals. Staffs do not have academic education and standard equipment such as milking machines is not used in these farms. Traditional farms do not have a health license from governmental veterinary services and the quality of animal feed (which are usually prepared by farmers), water sources and milk quality/hygiene are not evaluated by these services. Forty percent of large ruminants are kept in the commercial farms, which have a health license from governmental veterinary services. There are strict rules for location of the farm, farm buildings and other farming structures, minimum space per animal and buying new animals in these farms. The health of herds and dairy products is regularly evaluated by relevant agencies. In this study, slaughterhouse workers, butchers, farmers and veterinarians were considered as high-risk groups. According to the veterinary head office of the Isfahan County records, there are 808 butchers and slaughterhouse workers who are involved in slaughtering of animals and meet processing, 94 veterinarians who contact with cattle, sheep and goats, and a large number of farmers working in 2399 livestock farms. There are four slaughterhouses in the county that only cattle, sheep, and goat from both types of the herds are killed in a traditional way in those slaughterhouses. On the basis of the literature review and prevalence rate of 0.3 (Esmaeili et al., 2014), 5% allowed error and 95% confidence, a minimum of 338 samples were needed. We included 401 samples to compensate for passible loss of blood samples during the Lab procedure. The probability proportional to size was used as sampling method, therefore 99 slaughterhouse workers and butchers, 12 veterinarians and 290 farmers were enrolled in the study. For the sample selection in each occupational group, at first, an alpha-
betical list was made and then samples were randomly selected from the list. 290 farms were randomly selected from the list prepared by veterinary head office of the Isfahan County records (this list contained both types of the farms) and in each selected farm, a farmer was randomly selected. The aims of the study were explained to the participants and an informed consent was obtained from each subject. It was highlighted that the information would remain confidential and participation in the survey was voluntary. The research protocol was reviewed and approved by the Ethics Committee of Isfahan University of Medical Sciences (Project number: 293393). 2.2. Procedures and variable assessment Five ml of blood was obtained from each participant and the samples were transferred to the laboratory of Isfahan Infectious Diseases and Tropical Medicine Research Center, during 2–5 h, under refrigerated conditions. The samples were centrifuged and the separated serums were stored at −70 ◦ C until used. A checklist was filled out to assess information relevant to each person such as age, sex, education, occupation, years of occupational experience, hygienic precautions taken by the subject during contact with animals (including changing boot/clothes, washing, and disinfecting hands), and consumption of unpasteurized milk. Sera were tested for both IgG antibodies against phase I and phase II of C. burnetii using IFA assay (Vircell, S.L., Spain) according to the manufacturer’s instructions. Briefly, serum samples were diluted in 1:64 phosphate-buffered saline and were added to slide wells containing either C. burnetti antigen phase I or phase II, thus a cut-off value of 1:64 was considered for both antibodies. The slides were incubated at 37 ◦ C in a humidified chamber and after washing were dried at room temperature. Fluorescein isothiocyanate (FITC) conjugated antihuman IgG was added to each well and slides were incubated and washed again and finally air dried. A small drop of mounting medium was added to each well, slides were covered and were viewed with fluorescence microscope Eurostar III plus (Germany) at 400× magnification. Positive and negative controls were processed in parallel. Each test was performed in duplicates. Seropositivity was defined as the positive IgG antibody against phase I and/or II of C. burnetii. 2.3. Statistical analysis The data were analyzed using SPSS software (version 15, SPSS Inc., Chicago, IL, USA). Quantitative and categorical data were expressed as mean ± SD and frequency (percentage). Univariate (Chi-square test and T test for qualitative and quantitative variables, respectively) and multivariable binary logistic regression analyses were conducted for evaluating the association between potential risk factors and positivity for IgG antibodies. Whereas age and years of occupational experience were closely correlated, age was not included in the analysis. Those variables which were significant at p < 0.1 in univariate analyses were entered in multivariable analysis. P-Values less than 0.05 were considered as statistically significant. The odds ratio (OR) and 95% confidence interval (CI) were used to present results. 3. Results The mean age of the participants were 37.1 ± 12.7 years ranging from 18 to 81, and the majority of the subjects were male (96.5%). About 28.9% of the participants were less educated, 58.6% had less than 12 years of formal education and 12.5% had higher education. The mean duration of occupational experience was 20.9 ± 16.7 years. Table 1 summarizes the general characteristics of the participants.
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Fig. 1. Map of Iran, black lines delineates the 31 provinces of Iran. The shaded area shows the Isfahan County, the largest County in Isfahan Province.
Table 1 Serologic information and demographic characteristics of the high risk population in Isfahan County. Variable
Samples tested (percentage)
IgG I Positive (percentage)
IgG II Positive (percentage)
Total Seropositivity (percentage)
P-value*
0.568
Sex
Male female
387(96.5) 14(3.5)
75(19.4) 1(7.1)
143(37) 5(35.7)
168(43.4) 5(35.7)
Occupational group
Veterinarians Farmer Slaughterhouse workers and Butchers
12(3) 290 (72.3) 99(24.7)
0 59(20.3) 17(17.2)
2(16.7) 100(34.5) 46(46.5)
2(16.7) 121(41.7) 50(50.5)
0.050
Years of occupational experience
1–9 10–19 ≥20
115(28.7) 97(24.2) 189(47.1)
13(11.3) 16(16.5) 47(24.9)
37(32.2) 32(33) 79(41.8)
39(33.9) 40(41.2) 94(49.7)
0.024
History of working with small ruminants
no Yes
155(38.7) 246(61.3)
20(12.9) 56(22.8)
50(32.3) 98(39.8)
55(35.5) 118(48)
hygienic precautions during contact with animals
nothing Changing clothes/boots and/or washing hands and/or disinfecting hands
54(13.5) 347(86.5)
19(35.2) 57(16.4)
19(35.2) 129(37.2)
27(50) 146(42.1)
0.27
consumption of unpasteurized milk
yes no
340(84.8) 61(15.2)
71(20.9) 5(8.2)
124(36.5) 24(39.3)
148(43.5) 25(41)
0.712
*
0.014
P value for total sreopositivity. The variables with p < 0.2 were included in multivariable analysis.
Prevalence of IgG antibodies against phase I and II of C. burnetii were 19% and 36.9% respectively. The overall seropositivity (presence of antibodies against phase I and/or phase II) was 43.1%. The
mean age of seropositive and seronegative people was 38.6 ± 13.3 and 35.9 ± 12.2 years respectively. In univariate analyses, occupational groups, years of occupational experience and history of
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Table 2 Multivariable binary logistic regression analysis of risk factors possibly associated with seropositivity for C. burnetii in high-risk population in Isfahan County. Variable
Odds Ratio (95% CI)
P- value
Occupation group
Veterinarians Farmer Slaughterhouse workers and Butchers
0.22 (0.05–1.07) 0.83(0.51–1.34) 1
0.06 0.44
Years of occupational experience
1–9 10–19 ≥20
1 1.20(0.68–2.14) 1.53(0.91–2.58)
Yes No
1.46(0.91–2.32) 1
History of working with small ruminants
working with small ruminants were associated with C. burnetii seropositivity at p < 0.05 (Table 1). These variables were included in the multivariable binary logistic regression analysis. No statistically significant association was observed between included covariates and antibody positivity in binary logistic regression analysis (Table 2). 4. Discussion The present study reveals a relatively high seroprevalence of C. burnetii infection in center of Iran among high-risk population (an overall seroprevalence of 43.1%). In Iran, few studies have evaluated the presence of antibodies against C. burnetii as an occupational hazard and different infection rates have been reported. In a study in Kurdistan Province, Western Iran, IgG antibodies were assessed in hunters and their families, butchers, and health care workers. This survey reported that 27.8% of participants had IgG antibodies against phase I and/or II of the bacterium (Esmaeili et al., 2014). In another study in Sistan va Baluchestan, South-Eastern Iran, the overall seroprevalence was 22.5% among butchers and slaughterhouse workers (Esmaeili et al., 2016). In Kerman, southern of Iran, 68% of slaughterhouse workers and 34.7% of veterinary students were positive for IgG II (Khalili et al., 2014; Khalili et al., 2015). Sistan va Baluchestan and Kurdistan provinces have share borders with Afghanistan and Iraq respectively and illegal transport of animals in these regions is common. But the veterinary head office of the Isfahan County controls all of imported animals; moreover the animal husbandry is one of the most important industries in Kurdistan province, but Isfahan is an industrial region with lower rates of animal husbandry, thus we expected to have lower seropositivity in Isfahan county compared to Sistan va Baluchestan and Kurdistan provinces. Of course, the lower rates in those two studies might be due to variations in methods and sampling; in the present study, we used IFA assay which is a reference method to assess seroprevalence of C. burnetii and is more sensitive than ELISA. However, in the aforementioned studies ELISA assay was used which might underestimate the seroprevalence rates. Moreover, in our study larger sample size was used that could make a better estimation of the status of infection. In other countries, several studies have been done with varying results; in a study in Turkey, IgG antibody against phase II of C. burnetii was detected in 7.8% of people with high-risk occupations (Cetinkaya et al., 2000). The prevalence of IgG antibody against phase II was 13.5% among the Japanese veterinarians (Abe et al., 2001). In Denmark, among 195 women with occupational exposure to livestock, IgG antibodies against phase I and II of C. burnetii were detected in 43 and 89 individuals respectively (Nielsen et al., 2013). In another study, more than 22% of American veterinarians had antibodies against C. burnetii (Whitney et al., 2009). The probability of exposure of the workers with zoonotic agents depends on numerous factors such as the health status of the animals, type of the activity, frequency of close contact with animals, and type of hygiene precautions taken by workers that usually are varying
0.53 0.11 0.12
from an area to another (Battelli, 2008). The heterogeneity between the prevalence in different parts of the world could be due to differences in these factors. Several risk factors were assessed in this study. Based on our results, working as a slaughterer or butcher, having longer duration of occupational experience and history of working with small ruminants were risk factors in the univariable analysis, but they were left out when the multivariable analysis was used. Our results showed that more than 50% of slaughterhouse workers and butchers are positive for IgG antibody. In the study carried out on high-risk population in western Iran, the highest rate of seropositivity was observed in butchers (Esmaeili et al., 2014). This rate was also high among butchers and slaughterhouse workers in Sistan va Baluchestan and Kerman provinces in south-eastern Iran (Khalili et al., 2014; Esmaeili et al., 2016). Among high-risk occupations in West Turkey, the highest prevalence was reported in slaughterhouse workers, followed by butchers and farmers (Berkatas et al., 2011). Another study in Turkey showed that farmers and slaughterhouse workers had highest seroprevalence among high risk professions (Cetinkaya et al., 2000). It seems that among high-risk populations, the slaughterhouses workers and butchers are more at risk. This might be due to contact of slaughterers and butchers with a high load of animals, in contrast to farmers who often contact only with their own animals. An increase in seropositivity was observed with increase in occupational experience duration, but the difference was not significant. In the study carried out in Western Iran, the prevalence of antibodies against the bacterium was associated with length of employment (Esmaeili et al., 2014). In contrast, in Japan, no significant correlation was observed between IgG seroprevalence and years of occupational experience in veterinarians (Abe et al., 2001). Workers with more duration of occupational experience usually are older and therefore are more likely to become seropositive during their lifetime. Based on our results higher seropositivity rates for C. burnetii was observed in staff with history of working with small ruminants. Most of Q fever cases in Australia, France and Netherland are linked to exposure to small ruminants. In contrast, in the United States and Denmark large ruminants are more likely to infect humans (Maurin and Raoult, 1999; Mcquiston et al., 2006; Whitney et al., 2009; Van der Hoek et al., 2010; Nielsen et al., 2013). It seems that small ruminants play an important role for disease transmission in our area. The risk of this zoonotic disease for people, who had contact with livestock, could be more precisely estimated, if general population had been included as the control group in our study. 5. Conclusion The present study shows a high seroprevalence of C. burnetii infection among high-risk population in center of Iran. It seems that occupational health monitoring programs, especially easy access to local services providing the diagnostic tests for Q fever, are needed
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for individuals who may be exposed to C. burnetii. Moreover, since acute Q fever may be manifested with influenza-like symptoms, physicians should consider the risk of infection with C. burnetii when treating high-risk patients. Conflict of interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. Acknowledgment This project has been funded by a grant from Isfahan University of Medical Sciences (Project number: 293393). References Abe, T., Yamaki, K., Hayakawa, T., Fukuda, H., Ito, Y., Kume, H., Komiya, T., Ishihara, K., Hirai, K., 2001. A seroepidemiological study of the risks of Q fever infection in Japanese veterinarians. Eur. J. Epidemiol. 17 (11), 1029–1032. Anderson, A., Diagnosis and Management of Q Fever–United States, 2013: Recommendations from CDC and the Q Fever Working Group: US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention; 2013. Battelli, G., 2008. Zoonoses as occupational diseases. Vet. Ital. 44 (4), 601–609. Berkatas, M., Ceylan, E., Yaman, G., C¸I˙ FTCI˙ , I˙ .H., 2011. Seroprevalence of coxiella burnetii antibodies in high risk groups in eastern Turkey. Turkiye Klinikleri J. Med. Sci. 31 (1), 45–50. Cetinkaya, B., Kalender, H., Ertas, H., Muz, A., Arslan, N., Ongor, H., Gurfay, M., 2000. Seroprevalence of coxiellosis in cattle, sheep and people in the east of Turkey. Aust. Vet. J. 56, 181–183. Esmaeili, S., Pourhossein, B., Gouya, M.M., Amiri, F.B., Mostafavi, E., 2014. Seroepidemiological survey of Q fever and brucellosis in Kurdistan Province, Western Iran. Vector-Borne Zoonotic Dis. 14 (1), 41–45. Esmaeili, S., Naddaf, S.R., Pourhossein, B., Hashemi Shahraki, A., Bagheri Amiri, F., Gouya, M.M., Mostafavi, E., 2016. Seroprevalence of brucellosis, leptospirosis, and Q fever among butchers and slaughterhouse workers in South-Eastern Iran. PLoS One 11 (1), e0144953.
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Kampschreur, L.M., Oosterheert, J.J., Hoepelman, A.I., Lestrade, P.J., Renders, N.H., Elsman, P., Wever, P.C., 2012. Prevalence of chronic Q fever in patients with a history of cardiac valve surgery in an area where Coxiella burnetii is epidemic. Clin. Vaccine Immunol.: CVI 19 (8), 1165–1169. Khalili, M., Mosavi, M., Diali, H.G., Mirza, H.N., 2014. Serologic survey for Coxiella burnetii phase II antibodies among slaughterhouse workers in Kerman, southeast of Iran. Asia. Pac. J. Trop. Biomed. 4 (Suppl. 1), S209–12. Khalili, M., Qorbani, A., Sharifi, H., Golchin, M., 2015. Prevalence and risk factor of Q fever among veterinary students in Iran. Trop. Biomed. 32 (4), 704–709. Maurin, M., Raoult, D., 1999. Q fever. Clin. Microbiol. Rev. 12 (4), 518–553. Mcquiston, J.H., Holman, R.C., McCall, C., Childs, J.E., Swerdlow, D.L., Thompson, H.A., 2006. National surveillance and the epidemiology of human Q fever in the United States, 1978–2004. Am. J. Trop. Med. Hyg. 75 (1), 36–40. Mostafavi, E., Rastad, H., Khalili, M., 2012. Q fever: an emerging public health concern in Iran. Asian J. Epidemiol. 5 (3), 66. Nielsen S.Y., Molbak K., Nybo Andersen, A.M. Brink Henriksen, T. Kantso, B. Krogfelt, K.A. Hjollund, N.H., 2013. Prevalence of Coxiella burnetii in women exposed to livestock animals, Denmark, 1996–2002. Euro surveillance: bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 18(28). Norlander, L., 2000. Q fever epidemiology and pathogenesis. Microbes Infect. 2 (4), 417–424. Parker, N.R., Barralet, J.H., Bell, A.M., 2006. Q fever. Lancet (London, England) 367 (9511), 679–688. Sprong, H., Tijsse-Klasen, E., Langelaar, M., De Bruin, A., Fonville, M., Gassner, F., Takken, W., Van Wieren, S., Nijhof, A., Jongejan, F., Maassen, C.B., Scholte, E.J., Hovius, J.W., Emil Hovius, K., Spitalska, E., Van Duynhoven, Y.T., 2012. Prevalence of Coxiella burnetii in ticks after a large outbreak of Q fever. Zoonoses Public Health 59 (1), 69–75. Tozer, S.J., Lambert, S.B., Sloots, T.P., Nissen, M.D., 2011. Q fever seroprevalence in metropolitan samples is similar to rural/remote samples in Queensland, Australia. Eur. J. Clin. Microbiol. Infect. Dis. 30 (10), 1287–1293. Vaidya, V., Malik, S., Kaur, S., Kumar, S., Barbuddhe, S., 2008. Comparison of PCR, immunofluorescence assay, and pathogen isolation for diagnosis of Q fever in humans with spontaneous abortions. J. Clin. Microbiol. 46 (6), 2038–2044. Van der Hoek, W., Dijkstra, F., Schimmer, B., Schneeberger, P., Vellema, P., Wijkmans, C., Ter Schegget, R., Hackert, V., Van Duynhoven, Y., 2010. Q fever in the Netherlands: an update on the epidemiology and control measures. Euro Surveill. 15 (12), 19520. Whitney, E.A., Massung, R.F., Candee, A.J., Ailes, E.C., Myers, L.M., Patterson, N.E., Berkelman, R.L., 2009. Seroepidemiologic and occupational risk survey for Coxiella burnetii antibodies among US veterinarians. Clin. Infect. Dis. 48 (5), 550–557.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Seroprevalence and risk factors of Coxiella burnetii infection among high-risk population in center of Iran, a neglected health problem Zary Nokhodian a , Behrooz Ataei a , Abdolreza Moradi b , Majid Yaran c , Shervin Gaffari Hoseini a , Awat Feizi d,∗ , Roya Sherkat e a
Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Isfahan Veterinary Office, Head of Public Health Supervision Section, Isfahan, Iran c Nosocomial Infection Research Center, Isfahan University of Medical Sciences, Isfahan, Iran d Department of Biostatistics and Epidemiology, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran e Acquired Immunodeficiency Research Center, Isfahan University of Medical Sciences, Isfahan, Iran b
a r t i c l e
i n f o
Article history: Received 23 October 2016 Received in revised form 2 February 2017 Accepted 2 February 2017 Available online 5 February 2017 Keywords: C. burnetii High-risk population Iran Seroprevalence
a b s t r a c t In order to evaluate the prevalence of antibodies against phase I and II antigens of Coxiella Burnetii and to identify related risk factors among high-risk groups in the center of Iran, a serological survey was performed in Isfahan County. In a cross-sectional study, 401 sera were collected from slaughterhouse workers, butchers, farmers and veterinarians in spring 2015. Samples were tested for specific immunoglobulin G (IgG) antibodies against phase I and II of C. burnetii by indirect immunofluorescence assay. A checklist was fulfilled to document demographic information. Univariate analysis and multivariable binary logistic regression model were used to analyze data. IgG antibodies against phases I and II of C. burnetii were detected in 19% and 36.9% of participants, respectively. The overall seropositivity (IgG against phase I and/or II) was 43.1%. The present study shows a high seroprevalence of C. burnetii infection among high-risk population in center of Iran. It is suggested to carry out occupational health monitoring programs for individuals who may be exposed to C. burnetii. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Q fever, a zoonotic disease caused by Coxiella burnetii, has been reported worldwide except for Antarctica and New Zealand (Anderson, 2013). The bacteria, which is a gram negative intracellular coccobacillus, can infect a wide range of animal species including domestic and wild mammals, and non-mammals such as birds, reptiles and ticks (Parker et al., 2006). Ruminants, which are the main source of human infection, excrete the bacteria into the environment where the microorganism is able to withstand tough conditions for weeks (Tozer et al., 2011). Typically, human infection is acquired by inhalation of contaminated aerosols from infected ruminants (Whitney et al., 2009). The consumption of unpasteurized dairy products, tick-bite, and person-to-person contact could be other risk factors for disease transmission (Whitney et al., 2009; Sprong et al., 2012). In this regard, the persons who have close contact with animals such as farmers, butchers, slaughterhouse
∗ Corresponding author. E-mail address: awat [email protected] (A. Feizi). http://dx.doi.org/10.1016/j.actatropica.2017.02.002 0001-706X/© 2017 Elsevier B.V. All rights reserved.
workers, livestock handlers, and veterinarians are considered to be at high risk of C. burnetii infection (Battelli, 2008; Nielsen et al., 2013). C. burnetii infection can present as acute or chronic Q fever disease, which exhibits different clinical manifestations. The three main clinical presentations of the acute disease are a flu-like syndrome, pneumonia and hepatitis; however, the majority of the infections remain asymptomatic (Norlander, 2000; Parker et al., 2006). Chronic infection may develop in about 5% of infected patients, and may become apparent months or years after an acute episode. Endocarditis is the most common complication of the chronic infection. People with cardiac valve and vascular abnormalities and immunocompromised patients have a higher risk for chronic disease. The mortality rate in chronic Q fever is estimated from 2% to 65% (Norlander, 2000; Whitney et al., 2009; Kampschreur et al., 2012). Q fever diagnosis is based on serological tests and the immunofluorescence assay (IFA) is used as the reference method for the sero-diagnosis of the disease (Vaidya et al., 2008). Although nowadays, because an IFA test is negative in the first stages of acute
108
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Q fever infection, PCR assay in combination with serologic test is recommended (Anderson, 2013). The presence of C. burnetii antibodies has been reported in livestock in numerous studies in different parts of Iran (Mostafavi et al., 2012), however few studies have been conducted on people at risk of this infection and seroprevalence of the infection is unknown in most regions of Iran (Esmaeili et al., 2014; Esmaeili et al., 2016). In addition, majority of previous studies were conducted on butchers, slaughterhouse workers and veterinary students and there is not any study on farmers, the people who have the most contact with animals. The objective of the present study was to investigate the presence of IgG antibodies against phase I and II of C. burnetii in high-risk occupational groups by IFA assay and to identify infection risk factors among these individuals in the Isfahan County, Iran.
2. Materials and methods 2.1. Study design, area and samples In spring 2015, a cross-sectional study was conducted among high-risk populations in the Isfahan County, which is the largest County in Isfahan Province, located in the center of Iran and southeast of Tehran (Fig. 1). This county has thirteen cities, covers an area of 10,5937 km2 , and has a human population of 21,741,72. About 10% of the population lives in rural areas and the primary occupation of them are agriculture and animal husbandry. The urban people are engaged in industrial and service jobs. About 30,0000 small ruminants (sheep and goat) and 10,0000 large ruminants (cattle and calf) are registered in veterinary head office of Isfahan country. The camel farming is not an important activity in this area. There are two types of ruminant herds in Isfahan County: traditional and commercial herds. All of small ruminants and 60% of large ruminants are kept on the traditional farms. Traditional farms, which are located in the villages, do not usually have suitable buildings for keeping animals; in fact, farmer share part of his house with animals. Staffs do not have academic education and standard equipment such as milking machines is not used in these farms. Traditional farms do not have a health license from governmental veterinary services and the quality of animal feed (which are usually prepared by farmers), water sources and milk quality/hygiene are not evaluated by these services. Forty percent of large ruminants are kept in the commercial farms, which have a health license from governmental veterinary services. There are strict rules for location of the farm, farm buildings and other farming structures, minimum space per animal and buying new animals in these farms. The health of herds and dairy products is regularly evaluated by relevant agencies. In this study, slaughterhouse workers, butchers, farmers and veterinarians were considered as high-risk groups. According to the veterinary head office of the Isfahan County records, there are 808 butchers and slaughterhouse workers who are involved in slaughtering of animals and meet processing, 94 veterinarians who contact with cattle, sheep and goats, and a large number of farmers working in 2399 livestock farms. There are four slaughterhouses in the county that only cattle, sheep, and goat from both types of the herds are killed in a traditional way in those slaughterhouses. On the basis of the literature review and prevalence rate of 0.3 (Esmaeili et al., 2014), 5% allowed error and 95% confidence, a minimum of 338 samples were needed. We included 401 samples to compensate for passible loss of blood samples during the Lab procedure. The probability proportional to size was used as sampling method, therefore 99 slaughterhouse workers and butchers, 12 veterinarians and 290 farmers were enrolled in the study. For the sample selection in each occupational group, at first, an alpha-
betical list was made and then samples were randomly selected from the list. 290 farms were randomly selected from the list prepared by veterinary head office of the Isfahan County records (this list contained both types of the farms) and in each selected farm, a farmer was randomly selected. The aims of the study were explained to the participants and an informed consent was obtained from each subject. It was highlighted that the information would remain confidential and participation in the survey was voluntary. The research protocol was reviewed and approved by the Ethics Committee of Isfahan University of Medical Sciences (Project number: 293393). 2.2. Procedures and variable assessment Five ml of blood was obtained from each participant and the samples were transferred to the laboratory of Isfahan Infectious Diseases and Tropical Medicine Research Center, during 2–5 h, under refrigerated conditions. The samples were centrifuged and the separated serums were stored at −70 ◦ C until used. A checklist was filled out to assess information relevant to each person such as age, sex, education, occupation, years of occupational experience, hygienic precautions taken by the subject during contact with animals (including changing boot/clothes, washing, and disinfecting hands), and consumption of unpasteurized milk. Sera were tested for both IgG antibodies against phase I and phase II of C. burnetii using IFA assay (Vircell, S.L., Spain) according to the manufacturer’s instructions. Briefly, serum samples were diluted in 1:64 phosphate-buffered saline and were added to slide wells containing either C. burnetti antigen phase I or phase II, thus a cut-off value of 1:64 was considered for both antibodies. The slides were incubated at 37 ◦ C in a humidified chamber and after washing were dried at room temperature. Fluorescein isothiocyanate (FITC) conjugated antihuman IgG was added to each well and slides were incubated and washed again and finally air dried. A small drop of mounting medium was added to each well, slides were covered and were viewed with fluorescence microscope Eurostar III plus (Germany) at 400× magnification. Positive and negative controls were processed in parallel. Each test was performed in duplicates. Seropositivity was defined as the positive IgG antibody against phase I and/or II of C. burnetii. 2.3. Statistical analysis The data were analyzed using SPSS software (version 15, SPSS Inc., Chicago, IL, USA). Quantitative and categorical data were expressed as mean ± SD and frequency (percentage). Univariate (Chi-square test and T test for qualitative and quantitative variables, respectively) and multivariable binary logistic regression analyses were conducted for evaluating the association between potential risk factors and positivity for IgG antibodies. Whereas age and years of occupational experience were closely correlated, age was not included in the analysis. Those variables which were significant at p < 0.1 in univariate analyses were entered in multivariable analysis. P-Values less than 0.05 were considered as statistically significant. The odds ratio (OR) and 95% confidence interval (CI) were used to present results. 3. Results The mean age of the participants were 37.1 ± 12.7 years ranging from 18 to 81, and the majority of the subjects were male (96.5%). About 28.9% of the participants were less educated, 58.6% had less than 12 years of formal education and 12.5% had higher education. The mean duration of occupational experience was 20.9 ± 16.7 years. Table 1 summarizes the general characteristics of the participants.
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Fig. 1. Map of Iran, black lines delineates the 31 provinces of Iran. The shaded area shows the Isfahan County, the largest County in Isfahan Province.
Table 1 Serologic information and demographic characteristics of the high risk population in Isfahan County. Variable
Samples tested (percentage)
IgG I Positive (percentage)
IgG II Positive (percentage)
Total Seropositivity (percentage)
P-value*
0.568
Sex
Male female
387(96.5) 14(3.5)
75(19.4) 1(7.1)
143(37) 5(35.7)
168(43.4) 5(35.7)
Occupational group
Veterinarians Farmer Slaughterhouse workers and Butchers
12(3) 290 (72.3) 99(24.7)
0 59(20.3) 17(17.2)
2(16.7) 100(34.5) 46(46.5)
2(16.7) 121(41.7) 50(50.5)
0.050
Years of occupational experience
1–9 10–19 ≥20
115(28.7) 97(24.2) 189(47.1)
13(11.3) 16(16.5) 47(24.9)
37(32.2) 32(33) 79(41.8)
39(33.9) 40(41.2) 94(49.7)
0.024
History of working with small ruminants
no Yes
155(38.7) 246(61.3)
20(12.9) 56(22.8)
50(32.3) 98(39.8)
55(35.5) 118(48)
hygienic precautions during contact with animals
nothing Changing clothes/boots and/or washing hands and/or disinfecting hands
54(13.5) 347(86.5)
19(35.2) 57(16.4)
19(35.2) 129(37.2)
27(50) 146(42.1)
0.27
consumption of unpasteurized milk
yes no
340(84.8) 61(15.2)
71(20.9) 5(8.2)
124(36.5) 24(39.3)
148(43.5) 25(41)
0.712
*
0.014
P value for total sreopositivity. The variables with p < 0.2 were included in multivariable analysis.
Prevalence of IgG antibodies against phase I and II of C. burnetii were 19% and 36.9% respectively. The overall seropositivity (presence of antibodies against phase I and/or phase II) was 43.1%. The
mean age of seropositive and seronegative people was 38.6 ± 13.3 and 35.9 ± 12.2 years respectively. In univariate analyses, occupational groups, years of occupational experience and history of
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Table 2 Multivariable binary logistic regression analysis of risk factors possibly associated with seropositivity for C. burnetii in high-risk population in Isfahan County. Variable
Odds Ratio (95% CI)
P- value
Occupation group
Veterinarians Farmer Slaughterhouse workers and Butchers
0.22 (0.05–1.07) 0.83(0.51–1.34) 1
0.06 0.44
Years of occupational experience
1–9 10–19 ≥20
1 1.20(0.68–2.14) 1.53(0.91–2.58)
Yes No
1.46(0.91–2.32) 1
History of working with small ruminants
working with small ruminants were associated with C. burnetii seropositivity at p < 0.05 (Table 1). These variables were included in the multivariable binary logistic regression analysis. No statistically significant association was observed between included covariates and antibody positivity in binary logistic regression analysis (Table 2). 4. Discussion The present study reveals a relatively high seroprevalence of C. burnetii infection in center of Iran among high-risk population (an overall seroprevalence of 43.1%). In Iran, few studies have evaluated the presence of antibodies against C. burnetii as an occupational hazard and different infection rates have been reported. In a study in Kurdistan Province, Western Iran, IgG antibodies were assessed in hunters and their families, butchers, and health care workers. This survey reported that 27.8% of participants had IgG antibodies against phase I and/or II of the bacterium (Esmaeili et al., 2014). In another study in Sistan va Baluchestan, South-Eastern Iran, the overall seroprevalence was 22.5% among butchers and slaughterhouse workers (Esmaeili et al., 2016). In Kerman, southern of Iran, 68% of slaughterhouse workers and 34.7% of veterinary students were positive for IgG II (Khalili et al., 2014; Khalili et al., 2015). Sistan va Baluchestan and Kurdistan provinces have share borders with Afghanistan and Iraq respectively and illegal transport of animals in these regions is common. But the veterinary head office of the Isfahan County controls all of imported animals; moreover the animal husbandry is one of the most important industries in Kurdistan province, but Isfahan is an industrial region with lower rates of animal husbandry, thus we expected to have lower seropositivity in Isfahan county compared to Sistan va Baluchestan and Kurdistan provinces. Of course, the lower rates in those two studies might be due to variations in methods and sampling; in the present study, we used IFA assay which is a reference method to assess seroprevalence of C. burnetii and is more sensitive than ELISA. However, in the aforementioned studies ELISA assay was used which might underestimate the seroprevalence rates. Moreover, in our study larger sample size was used that could make a better estimation of the status of infection. In other countries, several studies have been done with varying results; in a study in Turkey, IgG antibody against phase II of C. burnetii was detected in 7.8% of people with high-risk occupations (Cetinkaya et al., 2000). The prevalence of IgG antibody against phase II was 13.5% among the Japanese veterinarians (Abe et al., 2001). In Denmark, among 195 women with occupational exposure to livestock, IgG antibodies against phase I and II of C. burnetii were detected in 43 and 89 individuals respectively (Nielsen et al., 2013). In another study, more than 22% of American veterinarians had antibodies against C. burnetii (Whitney et al., 2009). The probability of exposure of the workers with zoonotic agents depends on numerous factors such as the health status of the animals, type of the activity, frequency of close contact with animals, and type of hygiene precautions taken by workers that usually are varying
0.53 0.11 0.12
from an area to another (Battelli, 2008). The heterogeneity between the prevalence in different parts of the world could be due to differences in these factors. Several risk factors were assessed in this study. Based on our results, working as a slaughterer or butcher, having longer duration of occupational experience and history of working with small ruminants were risk factors in the univariable analysis, but they were left out when the multivariable analysis was used. Our results showed that more than 50% of slaughterhouse workers and butchers are positive for IgG antibody. In the study carried out on high-risk population in western Iran, the highest rate of seropositivity was observed in butchers (Esmaeili et al., 2014). This rate was also high among butchers and slaughterhouse workers in Sistan va Baluchestan and Kerman provinces in south-eastern Iran (Khalili et al., 2014; Esmaeili et al., 2016). Among high-risk occupations in West Turkey, the highest prevalence was reported in slaughterhouse workers, followed by butchers and farmers (Berkatas et al., 2011). Another study in Turkey showed that farmers and slaughterhouse workers had highest seroprevalence among high risk professions (Cetinkaya et al., 2000). It seems that among high-risk populations, the slaughterhouses workers and butchers are more at risk. This might be due to contact of slaughterers and butchers with a high load of animals, in contrast to farmers who often contact only with their own animals. An increase in seropositivity was observed with increase in occupational experience duration, but the difference was not significant. In the study carried out in Western Iran, the prevalence of antibodies against the bacterium was associated with length of employment (Esmaeili et al., 2014). In contrast, in Japan, no significant correlation was observed between IgG seroprevalence and years of occupational experience in veterinarians (Abe et al., 2001). Workers with more duration of occupational experience usually are older and therefore are more likely to become seropositive during their lifetime. Based on our results higher seropositivity rates for C. burnetii was observed in staff with history of working with small ruminants. Most of Q fever cases in Australia, France and Netherland are linked to exposure to small ruminants. In contrast, in the United States and Denmark large ruminants are more likely to infect humans (Maurin and Raoult, 1999; Mcquiston et al., 2006; Whitney et al., 2009; Van der Hoek et al., 2010; Nielsen et al., 2013). It seems that small ruminants play an important role for disease transmission in our area. The risk of this zoonotic disease for people, who had contact with livestock, could be more precisely estimated, if general population had been included as the control group in our study. 5. Conclusion The present study shows a high seroprevalence of C. burnetii infection among high-risk population in center of Iran. It seems that occupational health monitoring programs, especially easy access to local services providing the diagnostic tests for Q fever, are needed
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