Immunoglobulin M seropositivity for Toscana virus in a random population sample in Sicily

SUMMARY

Objectives

High Toscana virus (TOSV) antibody seropositivity rates have been documented in the last decade, especially in the Mediterranean area. It is unclear if these rates are associated with a recent or past exposure to the virus. This is of importance, as primary infection can cause neurologic complications, especially in adults. The aim of the present study was to assess the current active TOSV circulation in western Sicily.

Methods

A cross-sectional seroprevalence study was conducted on 271 individuals aged 4–92 years, sampled from the general population of a small city. Each participant completed a self-administered questionnaire and provided serum, which was analyzed for the presence of specific anti-TOSV IgM and IgG.

Results

Anti-TOSV IgM was detected in eight (3.0%) participants, of whom only three had anti-TOSV IgG. The prevalence of anti-TOSV IgM was highest in subjects aged 25–34 and 35–44 years (7.1% and 4.8%, respectively). All subjects positive for anti-TOSV IgM were resident in the suburban area.

Conclusions

The detection of IgM documented the circulation of TOSV, a Phlebovirus, in a random population sample of Sicilian adults. The highest risk of TOSV seroconversion in subjects living in the suburbs appears to suggest a high density of TOSV vectors in peri-urban areas.

1. Introduction

Toscana virus (TOSV) is a sandfly virus belonging to the genus Phlebovirus of the family Bunyaviridae. This virus is endemic in several European areas, and particularly in some Mediterranean countries, including Italy,¹ France,² Cyprus,³ Spain,⁴ Portugal,⁵ and Turkey.⁶ The basic lifecycle of TOSV is not completely defined, but several studies have reported that it can be transmitted to humans by the bite of phlebotomine sandflies, mainly Phlebotomus perniciosus,⁷ which are considered to be more active in warm and humid environments. Populations living in extra-urban areas and with frequent outdoor activities have been documented to have a higher risk of TOSV exposure.^6,8

TOSV infections are self-limiting and can be asymptomatic,⁹ or can manifest in a 3- to 5-day fever and relatively mild headache, with muscle and joint pains.¹⁰ Neurological manifestations such as meningitis and encephalitis have been reported as rare complications of TOSV infection.¹¹ In particular, TOSV-associated neurologic complications have been detected more frequently in adults than in children.¹² This could have important public health implications, since the reduced use of dichlorodiphenyltrichloroethane (DDT) has been associated with an increasing abundance of some Phlebovirus vectors in some countries of the Mediterranean area.¹³ Notably, the reemergence of TOSV in these endemic countries would affect many adults who are susceptible to TOSV infection and, consequently, at higher risk of complications.

Previous studies in Sicily have found that approximately two-thirds of subjects aged between 25 and 44 years do not show anti-TOSV IgG positivity, but circulation of the virus in the population could be deduced by the presence of anti-TOSV IgG seroprevalence among children.⁸ Thus, the aim of the present study was to assess and estimate the current active TOSV circulation in a random population sample in western Sicily, with a particular focus on adults, who may be susceptible to neurologic complications.

2. Materials and methods

2.1. Study design, participants, and questionnaire

The study was carried out from September 2009 to July 2010 in Calatafimi-Segesta, a small city located in the province of Trapani, western Sicily, Italy. Calatafimi-Segesta has an overall surface area of about 154 km², and it is situated at an altitude of 338 m above sea level. Criteria for exclusion of subjects from the study were: residence other than in Calatafimi-Segesta, age ≤4 years, severe cognitive dysfunction, death before recruitment, and inability of the general practitioner to reach each subject within the recruitment period.

As reported in detail elsewhere,⁸ a random sample of 271 eligible subjects were successfully recruited and enrolled into the study. The subjects provided a venous blood sample and were interviewed using a standardized questionnaire to obtain information on sociodemographics (e.g., age, sex, home postal address, job, etc.), anthropometrics, and lifestyle.

Before participation, all subjects were asked to give their informed consent. The study was approved by the Institutional Review Board of the AOUP “P. Giaccone”, Palermo, Italy.

2.2. Serological tests

Serum samples were stored at 4 °C for a maximum of 24 h or processed immediately; alternatively they were aliquoted and frozen at −20 °C for future testing. All laboratory tests were performed using blinded methods, in specialized laboratories that met national and international quality control standards.

All serum samples were tested for the presence of specific anti-TOSV IgM using an enzyme immunoassay (EIA) with recombinant N protein (IgG/IgM TOSV virus detection kit; Diesse, Siena, Italy), following the procedures described by the manufacturer. Samples showing a borderline value were further analyzed by immunofluorescence assay (IFA) to detect anti-TOSV virus IgM using a procedure described elsewhere.¹⁴ An immunoblot assay (Recom-Line Bunyavirus IgG/IgM, Mikrogen Diagnostik, Germany) was also performed for the confirmation of TOSV IgM.

2.3. Statistical analyses

Data were analyzed using the R statistical software package.¹⁵ Absolute and relative frequencies and 95% confidence intervals (CI) were calculated for qualitative variables. All quantitative variables were summarized as mean ± standard error. A geographic map of Calatafimi-Segesta was obtained by and constructed with Google Earth^™, running on Microsoft Windows operating systems.¹⁶ The IgM-positive cases were geo-localized via their home postal address using the mapping application Google Maps^™.17

3. Results

Of the 271 recruited subjects, 145 (53.5%) were females with an average age of 49.1 ± 1.6 years, and 126 (46.5%) were males with an average age of 49.4 ± 2 years. A total of five females (3.4%) aged 13–43 years and three males (2.4%) aged 45–66 years were seropositive for anti-TOSV IgM. Of the anti-TOSV IgM seropositive subjects, three had anti-TOSV IgG, one was a student, one was unemployed, one was a lawyer, one was a housewife, one was a home builder, two were otherwise employed, and one was retired.

With regard to the seasons, it is noteworthy that the number of serum samples positive for TOSV IgM collected in winter (one in January, two in February, and one in March out of 112; 3.6%) was similar to that obtained analyzing the serum samples collected during the other months of the year (4/159; 2.5%).

The age-related distribution of anti-TOSV IgM seropositivity is shown in Table 1. Subjects aged 25–34 and 35–44 years had the highest prevalence of anti-TOSV IgM seropositivity (7.1% and 4.8%, respectively). Finally, as shown in Figure 1, all anti-TOSV IgM seropositive subjects were resident in the suburbs of the city.

Table 1.

Age-related distribution of IgM anti-TOSV seropositivity observed in inhabitants of Calatafimi-Segesta

Age group, years	n/Total	TOSV IgM positives
		%	95% CI
4–14	1/23	4.3	0.1–21.9
15–24	0/16	0	0.0–20.6
25–34	2/28	7.1	0.9–23.5
35–44	2/42	4.8	0.6–16.2
45–54	1/45	2.2	0.1–11.8
55–64	1/49	2.0	0.1–10.9
65–74	1/40	2.5	0.1–13.2
>74	0/28	0	0.0–12.3

TOSV, Toscana virus; 95% CI, 95% confidence interval.

Figure 1.

Geographic map indicating the locations of residence of subjects seropositive for anti-TOSV IgM in Calatafimi-Segesta, western Sicily.

* Each red balloon equals to one IgM anti-TOSV seropositive

4. Discussion

Studies conducted in central and southern Italy have found an increase in abundance of Phlebotomus sandflies, especially in urban areas.¹³ The high prevalences of TOSV seropositivity found in Italy,¹ as well as in other European countries,¹⁸ could be linked to this increase in Phlebovirus vectors. In Sicily, high anti-TOSV IgG seroprevalence rates have been reported in the general population,⁸ and, of particular concern, in the elderly.^8,19 Although positivity for IgG does not distinguish between current and past virus infection, an IgG seroprevalence of 4.3% in subjects aged <15 years⁸ does suggest active circulation of TOSV and its related vectors. The present study attempted to address this hypothesis more directly.

Our results support the hypothesis that TOSV transmission is active, showing that about 3.0% of the Sicilian population sample analyzed was positive for TOSV IgM, indicating a recent exposure to the virus. A similar IgM seroprevalence has been reported recently in a sample of blood donors from southeastern France,²⁰ and an even higher TOSV IgM reactivity (8.2%) has been documented in Turkish blood donors.⁶ Considering that our study showed an IgM seroprevalence of 4.8–7.1% in the 25–44 years age group, an age group typical for blood donors, our data appear to be more like those recorded in Turkey than those reported in France. Notably, this relatively high seroprevalence in adults may have clinical relevance, since adults have been demonstrated to be more susceptible to TOSV-associated neurologic complications. Di Nicuolo et al. found TOSV detected by PCR in seven of 126 patients with aseptic meningitis or encephalitis;¹¹ the median age of patients was 23 (range 16–44) years. The TOSV IgM-positive healthy individuals considered in this study appeared to be substantially older (median age 39 years, range 13–66 years). Further studies on the correlation between TOSV infection and the occurrence of neurologic disorders in the various age groups are needed.

The present study also provides insight into the ecologic distribution of the vectors involved in the maintenance of the basic lifecycle and transmission of TOSV. Several authors have reported that the virus is mainly transmitted by P. perniciosus, a sandfly that is generally considered to be primarily adapted to sylvatic environments, as well as to urban and peri-urban areas.²¹ This adaptation does not appear evident in our study, since a higher risk of TOSV seroconversion seemed to be present only in subjects living in suburban areas. This finding could be explained by the distribution of sandfly ecological niches, which are determined by the availability of larval breeding sites and vertebrate hosts that are quite common in suburban areas. Moreover, the horizontal dispersion of the sandfly is known to be limited because of its short flight range.²² This model is supported by previous findings that have shown a higher TOSV seroprevalence in Sicilian subjects who have a pet living outdoors and in owners of an extra-urban holiday residence, suggesting a relationship with the suburban environment, and possibly with domestic animals that may be reservoirs contributing to the basic maintenance cycles of the virus.^8,23

Finally, it should be noted that anti-TOSV IgM seroprevalence was similar in specimens drawn in the winter and in the milder seasons. This observation may be explained by the fact that the climate in Sicily was not very cold or harsh during the winter of 2010. Several studies have demonstrated transovarial transmission of TOSV among phlebotomines,^24,25 which might contribute to maintaining the virus year-round. It is possible that even in winter, infected larvae can find a suitable, warm habitat that enables them to develop and then to bite people living nearby.

The possibility of non-specificity of our results must be considered. It was not possible to confirm seroconversion with a second serum sample drawn from the people who we found to be TOSV IgM-positive. Nonetheless, we note that the results obtained by EIA were confirmed by IFA and immunoblotting. We acknowledge that these methods do not exclude the possibility of cross-reaction of these sera with sandfly fever Naples virus, since there is high homology between the N protein of this virus and TOSV and there is no gold standard method for serological testing of TOSV antibodies.

The possibility of cross-reactivity, the lack of longitudinal observations, the relatively small sample size, and restriction to the general population of a small city of western Sicily must be considered potentially important limitations. Nevertheless, our study does provide further knowledge on Phlebovirus infections, documenting the current active TOSV circulation in a general population and, particularly, in adults.