Sero-Epidemiological Survey of Crimean-Congo Hemorrhagic Fever among the Human Population of the Punjab Province in Pakistan

Dear Editor,

Crimean-Congo hemorrhagic fever (CCHF), caused by the CCHF virus (CCHFV), is a tick-borne zoonotic infection characterized by myalgia, high-grade fever ([ 38 C), headache, nausea, bleeding from the body cavities, and in 10%–50% of cases, results in death (Swanepoel et al. 1989). As CCHFV belongs to the Nairoviridae family, the virus can be transmitted to humans through the bite of infected ticks or by contact with the tissues or blood of infected animals (Bente et al. 2013).

Since the first case of CCHF in Pakistan in 1970, the number of clinical cases has increased annually (Yousaf et al. 2018). Like other resource-limited countries worldwide, most diagnoses of this disease in Pakistan are limited to clinical signs and symptoms (Haider et al. 2016; Hasan et al. 2013). Consequently, despite the endemicity of the disease in various regions of Pakistan, the majority of cases remain either undiagnosed and/or misdiagnosed, due to lack of diagnostic facilities. In order to highlight the presence of subclinical forms of the disease or underestimation of actual disease status and associated risk factors, we conducted a sero-epidemiological study involving patients originating from areas with and without a history of the disease.

We reported the presence of anti-CCHFV IgG from 1052 collected blood serum samples in selected districts of the Punjab Province of Pakistan (see Supplementary Materials and Methods for data collection and immunological assay details) over a period of 8 months (from October 2016 to May 2017) and found an association between seroprevalence and different categorical variables. We found a lower CCHFV seropositivity (2.09%, 95% CI 1.23–2.96), which is not surprising, as previous studies have found that CCHFV seropositivity varies even in endemic places. For instance, using sera of individuals that had a subclinical form of infection or were clinically healthy, the percentage of patients which were seropositive was found to be 0.5% in India (Mourya et al. 2019), 3.7% in Bulgaria (Christova et al. 2013), 4.9% in Greece (Sidira et al. 2012), 9.3% in Kosovo (Fajs et al. 2014) and 10%– 19.6% in Turkey (Ertugrul et al. 2012; Koksal et al. 2014). Such variations in the study outcomes may be attributed to the sampling and subsequent analysis strategies employed previously. For instance, some of these studies included areas with a frequent occurrence of the disease reported in the past, while other studies involved a comparison between areas with a frequent occurrence to areas with no prevalence. For example, studies from Turkey were exclusive to patients originating from disease-endemic areas, whereas, similar to our study, both endemic and nonendemic areas were explored in the study from Kosovo.

Herein, we report a higher seroprevalence in regions having a history of exposure (n = 20/741, 2.70%, 95% CI 1.58–3.97), that was four times higher (OR = 4.29, 95% CI 0.99–18.45, P = 0.03) than that found in regions without any history of CCHF (n = 2/311, 0.64%, 95% CI 0.25–1.54) (Table 1). Among the districts with a history of CCHF, the Chakwal district had the highest prevalence (7.45%, 95% CI 2.14–12.75), followed by the Mianwali (3.48%, 95% CI 0.39–7.37), Rawalpindi (3.09%, 95% CI 0.35–6.54), Lahore (2.63%, 95% CI 0.09–5.18), and Dera Ghazi Khan districts (1.98%, 95% CI 0.74–4.70), whereas, the lowest prevalence was found to be in the Attock district (1.01%, 95% CI 0.94–2.92) and no seropositivity was found in the Rajanpur district (Fig. 1). Similarly, in areas with no previous exposure, the seroprevalence was present in the Jhelum district (1.90%, 95% CI 0.71–4.52), while in the Bahawalpur and Rahim Yar Khan districts, no CCHFV seroprevalence was identified, as shown in Fig. 1. We found 7 of 10 districts with seropositive individuals, 2 districts without, and 1 district with a history of CCHF, indicating that sub-clinical forms of infection are also present in Pakistan and should, therefore, be included when conducting a mass-scale surveillance study in the future. However, seropositivity to CCHFV was more prevalent in areas with a history of CCHF.

Figure 1.  Geographic distribution of CCHFV seropositive and seronegative districts of Punjab Province.

In order to assess a relationship between risk factors and seropositivity to CCHFV in humans, different categorical variables, including age, gender, geographic characteristics (endemic/non-endemic), and occupational history, were analyzed (Table 1). Our results showed that among seropositive individuals, females were more common (n = 6, 2.29%, 95% CI 0.56–4.77) than males (n = 16, 2.02%, 95% CI 1.06–3.07) but we found a lack of gender predisposition against CCHFV seroprevalence ((P > 0.05). This is contradictory to previous observations from India (Mourya et al. 2019) which may be due to the differences in the environment of the study participants in each country. For instance, both males and females included in our study had equal exposure to agriculture and livestock, however, females were involved mainly in livestock rearing and management. Similar observations have been reported by another study, where the percentage of seropositive patients was found to be higher in females who had direct contact with animals and, therefore, were exposed to tick-bites (Lwande et al. 2012). On the other hand, in the referenced Indian study, males were more exposed to livestock and agricultural activities than females.

The seroprevalence was 6 and 2.7 times higher in individuals living in rural areas (OR = 6.04, 95% CI 1.40–26.01, P = 0.013) and livestock farmers (OR = 2.72, 95% CI 1.10–6.57, P = 0.029), respectively. The percentage of seropositivity was higher in individuals involved in agricultural and livestock-related activities (3.20%, 95% CI 1.77–4.46) than others (1.20%, 95% CI 0.23–1.41). These results are not surprising, as farmers are more exposed to ticks and subsequent tick-bites than other individuals and therefore have more chances of contracting the disease.

In our present study the seroprevalence was found to be higher in older patients aged 41–55 years (2.94%, 95% CI 1.47–4.59) than younger ones aged 18–40 years (1.39%, 95% CI 0.44–2.38). This may be due to the fact that older individuals have more exposure time to livestock animals and because of their agriculture-related profession. Similar findings were reported by an earlier study in Iran (Mostafavi et al. 2017), where individuals with a longer duration of employment and exposure to livestock animals had significantly higher levels of seropositivity than other individuals. Additionally, a previous study from India reported that the risk of CCHF seropositivity was 3 times higher in older individuals (40–60 years) than in younger ones (< 40 years) (Mourya et al. 2019).

Our study has several limitations. As most of the individuals were illiterate and had a lack of exposure to scientific studies, they were hesitant to share the details of their work history or were unaware of any history of tick bites. Although they claimed to have a history of fever, due to the lack of access to a nearby appropriate diagnostic facility, they were not sure of any typical causative agent and had symptomatic treatments. As it evaluated samples originating from areas with and without a history of CCHF during a period when there was no epidemic, the study provides an assessment of seropositivity and its association with various categorical variables for individuals with a history of fever. Future studies with a larger cohort of individuals are required to ascertain the number of both clinical and sub-clinical forms of infection along with an exploration of ticks in a nearby setting.

Authors would like to thank all involved personnel in primary & secondary health-care centers in Punjab that contributed to sampling.