|Year : 2022 | Volume
| Issue : 2 | Page : 172-177
Seroprevalence against Toscana virus in Spain: The case of the autonomous community of Madrid
Jesús García Martinez1, Sonsoles García García2, Stefan Walter3, Ruth Gil- Prieto3, Daniel López Lacomba4, Patricia Marín-García5, Ángel Gil-de-Miguel3, Alba González-Escalada6
1 Area of Microbiology, Hospital Universitario de Fuenlabrada, Cº Molino, 2. 28942-Fuenlabrada, Madrid, Spain
2 Rey Juan Carlos University, Av. Atenas s/n. 28922, Alcorcón, Madrid, Spain
3 Area of Preventive Medicine and Public Health, Rey Juan Carlos University, Av. Atenas s/n. 28922, Alcorcón, Madrid, Spain
4 Area of Haematology, Hospital Universitario de Fuenlabrada, Cº Molino, 2. 28942-Fuenlabrada, Madrid, Spain
5 Area of Medical Immunology, Rey Juan Carlos University, Av. Atenas s/n. 28922, Alcorcón, Madrid, Spain
6 Area of Medical Microbiology, Rey Juan Carlos University, Av. Atenas s/n. 28922, Alcorcón, Madrid, Spain
|Date of Submission||15-Nov-2021|
|Date of Acceptance||13-Dec-2021|
|Date of Web Publication||08-Sep-2022|
Area of Medical Microbiology, Rey Juan Carlos University, Av. Atenas s/n. 28922, Alcorcón, Madrid
Source of Support: None, Conflict of Interest: None
Background & objectives: The Toscana virus (TOSV) is a neurotropic arbovirus that is transmitted through the bite of some Phlebotomus species. In 2009, the largest outbreak of leishmaniasis described so far in Europe, occurred in the Autonomous Community of Madrid, Spain, which was related to the population increase of P. perniciosus in this region.
Methods: A seroprevalence study was conducted to determine the circulation of TOSV among the population of this geographic area. A total of 516 sera were collected in two different stages: 2007 (before the leishmaniasis outbreak) and 2018–19 (representative of the current situation). In the sera, presence of IgG antibodies against TOSV was determined by commercial ELISA.
Results: The overall seroprevalence was 34.5%. The anti-TOSV IgG level was significantly higher in the samples collected in 2007 (41.5%) than 2018–19 (27.3%).
Interpretation & conclusion: The results of this study show a very active TOSV circulation in the region that is greater than expected. The lower seroprevalence figures in 2018–19 may be related to the vector and environmental control measures that were put in place as a result of the leishmaniasis outbreak of 2009. This highlights the importance of such strategies to reduce the incidence of TOSV infection and other vector-borne diseases.
Keywords: Toscana virus; seroprevalence; Phlebotomus; Spain
|How to cite this article:|
Martinez JG, García SG, Walter S, Gil- Prieto R, Lacomba DL, Marín-García P, Gil-de-Miguel &, González-Escalada A. Seroprevalence against Toscana virus in Spain: The case of the autonomous community of Madrid. J Vector Borne Dis 2022;59:172-7
|How to cite this URL:|
Martinez JG, García SG, Walter S, Gil- Prieto R, Lacomba DL, Marín-García P, Gil-de-Miguel &, González-Escalada A. Seroprevalence against Toscana virus in Spain: The case of the autonomous community of Madrid. J Vector Borne Dis [serial online] 2022 [cited 2022 Sep 28];59:172-7. Available from: https://www.jvbd.org/text.asp?2022/59/2/172/335771
| Introduction|| |
Toscana virus (TOSV) is an arbovirus belonging to the species sandfly fever Naples phlebovirus (genus Phlebovirus, family Phenuiviridae) that is transmitted through the bite of some species of Phlebotomus; P. perniciosus being its main vector,. It is distributed throughout the Mediterranean basin and is the only neurotropic virus that belongs to this species. Human infection with TOSV is usually asymptomatic, although it can also occur as a self-limiting febrile syndrome (known as “pappataci fever”) or as a neurological condition in the form of meningitis, encephalitis, meningoencephalitis, or peripheral neuropathy generally of benign prognosis, which occurs mainly during the summer, coinciding with the period of greatest vector activity,,.
Since its first isolation in Spain in 1998, TOSV has been blamed for up to 5% of aseptic meningitis cases diagnosed in the country. After enteroviruses, TOSV is one of the most frequently implicated infectious agents in this disease. Since 1998, research on seroprevalence against TOSV has been carried out in different regions of Spain and has shown seropositivity of between 5 and 26% in the general population. Specifically, in the Autonomous Community of Madrid, overall seroprevalence rates are approximately 5.7% according to the last published study, which dates back two decades. In that study it was observed that in certain areas of Autonomous Community, such as the southwestern region, these rates were somewhat higher (between 8 and 12% seropositive subjects) than in other regions and that these rates were increasing, which is related to an active and growing circulation of TOSV in that environment [Figure 1].
|Figure 1: Map of Fuenlabrada, Madrid, Spain where the study was carried out. Green areas where vector has settled down are marked with red circles.|
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Besides Phlebovirus, phlebotomine sand flies are vectors of other infectious agents, such as Leishmania spp. In 2009, the largest outbreak of leishmaniasis described in Europe occurred in Spain, specifically in the southwest of the Autonomous Community of Madrid, in the municipality of Fuenlabrada where most of the victims resided. In this outbreak rabbits and hares unexpectedly played a fundamental role both as reservoirs of the parasite (L. infantum) and as a main source of food for the vector. This event was related to the construction, albeit in previous years, of large peri-urban green areas that constituted an ideal habitat for the overgrowth of both the lagomorph and the P. perniciosus populations which represents 75% of sand flies isolations in the area with a density of 193.6 specimens/m2,, After this outbreak a study was conducted between 2012 and 2013 on the presence of Phlebovirus in the Phlebotomus population in this geographic area, and it revealed the existence of specimens infected with TOSV, which could lead to an increased risk of human infections by this virus.
Based on these findings, taking into account the last study on seroprevalence against TOSV conducted in the Autonomous Community of Madrid dating back to 2000 (almost a decade before the onset of the leishmaniasis out-break), and noting that in the last few years aseptic meningitis of unknown aetiology has become more common in this Autonomous Community, we carried out a new study of seroprevalence in the population of the southwest region of the Autonomous Community of Madrid, Spain to obtain more accurate and updated information on the epidemiology of TOSV in this region.
| Material & Methods|| |
To determine more accurately the possible impact of increased vector density on TOSV circulation in the southwest region of the Autonomous Community of Madrid, Spain, serum samples were collected from individuals residing in the area of Fuenlabrada (municipality where the highest number of cases of leishmaniasis occurred during the 2009 outbreak). The samples were collected in two different time periods: 2007 (before the outbreak of leishmaniasis, when there were already large peri-urban parks where the vectors had settled) and 2018–2019 (representative of the current situation).
Considering the population of Fuenlabrada and the seroprevalence data of this region obtained in the only previous study (8–12%), assuming an accuracy of 3%, a confidence level of 95%, and 15% of missing values, a final sample size of 518 sera was required.
Serum samples from 2007 were obtained from surplus diagnostic material stored in the serum library of Hospital Universitario de Fuenlabrada (HUF). The only data available on the subjects from whom these sera were taken, was gender, age, and the month of collection. The serum samples from the second period of the study were obtained from healthy volunteers who signed an informed consent form while donating at the HUF blood bank. Only adult population was included in our analyses because of the lack of pediatric age serum samples from the 2007 period. Once collected, sera were stored in the Microbiology Laboratory of HUF at -20°C until being sent to the Epidemiology Laboratory of the Faculty of Health Sciences, Rey Juan Carlos University, where the serological analysis was carried out.
To find out sociodemographic factors that could be associated with an increased risk of exposure to the vector (and therefore of TOSV infection), a survey was designed to collect information about the donors from the second study stage, collecting variables such as sex, age, race, home and workplace characteristics (building floor, proximity to green areas, living with pets), personal and/ or family history of leishmaniasis, residence in the municipality of Fuenlabrada before 2009 (year of onset of the leishmaniasis outbreak), and outdoor activities (daily duration and spaces where they are done).
The serological study was performed using the commercial ELISA kit Enzywell Toscana virus IgG/ IgM (Diesse®, Italy) according to the manufacturer’s instructions. This test, with a sensitivity of 95% and a specificity of 96.5%, has been used in most previous national and international studies on seroprevalence against TOSV, so its use allows us to reliably compare the results obtained.
The independent and dependent variables of interest were described using measures of centrality and dispersion (means or medians if the distribution was asymmetric), accompanied by their corresponding 95% confidence intervals for the quantitative variables and the frequency distribution (prevalence and proportions with 95% confidence intervals) for the qualitative variables. The Pearson χ2 test was run to compare frequencies and prevalence (if the expected frequency was less than 5, Fisher’s exact test was used). After verifying the normality of the age variable through the Kolmogorov-Smirnov test, the mean ages were compared by Student’s T-test.
In all hypothesis tests, to estimate differences, associations, and relationships, these were considered significant when the p value was less than 0.05. Statistical analysis was performed in R version 3.5.
All data about study participants were anonymized and managed in accordance to the Ley Orgánica 3/2018 of December 5 regarding Protection of Personal Data and Guarantee of Digital Rights. The entire study was complied with the ethical safeguards of the Declaration of Helsinki and its updates and with Spanish legislation on clinical research in humans and data protection. The study was approved by the Hospital Universitario de Fuenlabrada (HUF) Ethics and Research Committee.
| Results|| |
A total of 516 sera from individuals residing in the southwest of the Autonomous Community of Madrid were analyzed: 260 corresponding to the first period of the study and 256 collected in 2018 and 2019. Of the total sera collected, 43% (225 samples) were from females and 56.4% from males (291 samples). The overall seroprevalence (considering both study periods) of IgG antibodies against TOSV was 34.5%. A total of 60.7% of the positive sera were collected in the first study period (2007) vs. 39.3% in 2018–2019 (p = 0.003). The overall mean age of the donors was 44.7 ± 18.09 years and of seropositive donors was 51.97 ± 19.59 years. The latter was significantly higher (p < 0.001) than the mean age of seronegative donors (40.12 ± 15.29 years). No differences in gender were seen between seropositive and seronegative subjects (p = 0.550) in either of the study periods [Table 1].
|Table 1: Main characteristics in terms of age, gender and IgG against Toscana virus results from the population of the study. 2007 samples: samples obtained from stored surplus diagnostic material at Hospital Universitario de Fuenlabrada serum bank. 2019 samples: samples obtained from healthy volunteers at Hospital Universitario de Fuenlabrada|
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The analysis of the serum samples collected in 2007 yielded a seroprevalence of IgG antibodies against TOSV of 41.5% (108 samples). The mean age of the participants was 49.26 ± 20.65 years. Seropositive subjects were significantly older (58.56 ± 19.51 years) than seronegative subjects (41.20 ± 17.91 years) (p < 0.001). There were no significant differences by gender (p = 0.454) or the month in which the sample was collected (p = 0.518).
In the sera collected in the second study stage (2018–19), the seroprevalence of IgG antibodies against TOSV was 27.3% (70 samples). The mean age of the donors was 39.99 ± 13.50 years. Seropositive and seronegative donors had a similar age (41.51± 14.64 vs. 39.19 ± 12.59; p = 0.226) and gender ratio (p = 0.648) [Table 2].
|Table 2: Differences in seroprevalence for Toscana virus according to age and gender in the global population. 2007 samples: samples obtained from stored surplus diagnostic material at Hospital Universitario de Fuenlabrada serum bank. 2019 samples: samples obtained from healthy volunteers at Hospital Universitario de Fuenlabrada |
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The statistical analysis of the variables collected in the survey of donors who participated in this study in 2018–19 did not show a significant relationship between seropositivity and having resided in the study area before 2009 (p = 1) or a personal or family history of leishmaniasis (p = 0.328). There were also no significant differences in the presence or absence of anti-TOSV IgG antibodies by the storey of the home or workplace, the neighborhood in which these properties were located, or the presence or absence of pets (of any kind) in the home (p > 0.05 in all cases). In the survey, outdoor activities were also included as a variable, and, when there was an affirmative answer, the respondent could tell the average time spent on these activities throughout the week (1–2 hours/week, 3–5 hours/ week, or more than 5 hours/week), the preferred period of the day for these activities (afternoon, dusk, night, or any time), and the green areas or zones where this type of activity was done. There was no significant relationship between seropositivity against TOSV and any of these variables [Table 3].
|Table 3: Relationships between IgG against Toscana virus results and main epidemiological risk factors in the samples analyzed from 2019 Sera|
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| Discussion|| |
Seroprevalence studies are a fundamental tool in understanding the epidemiological and circulation pattern of microorganisms, as well as in monitoring their progression over time. In the case of TOSV, conducting this type of study is essential to accurately determine its epidemiological situation, because, although it is a neurotropic virus that can produce clinical manifestations at the central nervous system level, it seems that most infections with this virus are completely or almost asymptomatic so its underdiagnosis is frequent,,.
The main vector of TOSV is P. perniciosus, which also transmits other infectious agents, such as Leishmania spp. In 2009, an important outbreak of leishmaniasis associated with an increase in vector density occurred in the southwest of the Autonomous Community of Madrid, with a peak incidence in 2011 when a total of 197 cases were reported. Until mid-2020, more than 750 cases of this disease have been diagnosed, 479 of them as cutaneous leishmaniasis and 303 as visceral disease. In the context of this outbreak, an entomological study was carried out that detected TOSV B-genotype infected Phlebotomus in this geographic area. This situation was related to the creation of large peri-urban green spaces that, together with the construction of railroads and roads in the area, favored both the settlement of Phlebotomus and its access to urban areas with a high human population density,,. These factors explain the increase in overall seroprevalence observed in our study (34.5%) compared to that observed in the only previous study in the region, in which the rate was between 8 and 12%. Similarly, the results obtained when analyzing the two study periods separately (41.7% seropositivity in 2007 and 27.3% in 2018–2019) indicate that, before the leishmaniasis outbreak, there was a very active circulation of the virus in our region and that it persists, although in lower numbers and more in line with that observed in surrounding countries,,,.
One factor that may at least partially explain the decrease in the seroprevalence rates between the two study periods is the implementation of leishmaniasis outbreak control strategies starting in 2014. These included the surveillance and control of the vector and of the lagomorph population, as well as the cleaning and maintenance of the environment, which lowered the rate of leishmaniasis in the region. In all likelihood, these measures have also contributed to the decrease in the number of individuals seropositive for TOSV by decreasing the likelihood of contact between the vector and humans, which highlights the relevance of epidemiological surveillance in the follow-up and control of infections caused by this type of virus.
A particular aspect of the 2009 outbreak was that rabbits and hares were the main reservoir of the parasite as well as the major source of food for the vector,,. Seroprevalence studies carried out on different mammalian species (mainly dogs and cats) have postulated that these animals could play a role in the TOSV persistence cycle and in its transmission to humans,,,,. To date, there has been no study of this type on rabbits or hares, so it has not been possible to determine the role of these animals in the circulation of TOSV. In our study, and in agreement with a previous study on the leishmaniasis outbreak, we did not find a relationship between living with pets or contact with pets and being seropositive against TOSV which suggests that lagomorphs could have played some type of role in the transmission of the virus to humans during these years. It would be necessary to conduct specific seroprevalence studies in these animal species to determine their role in the circulation and persistence of TOSV in our environment.
In line with earlier studies on seroprevalence against TOSV, we found that the seropositivity rate increased with age,,,,,,,,: the mean age of the subjects with anti-TOSV IgG antibodies was 51.97 years in the overall study population and 58.56 years in the first cohort (2007). Although we did observe this trend in the second period of the study (2018–19), the relationship between the two variables was not significant. Most likely, the use of samples from healthy volunteers at this stage may have influenced this result, since sera from individuals older than 70 years were not available.
Again, in line with the literature,,,,,, we did not find an association between gender and the presence of IgG antibodies against TOSV. And, no environmental factors, such as outdoor activities, the places where the outdoor activities were done, and the location of the home within the municipality, were found to be risk factors associated with seropositivity for TOSV, a result also found in a previous study carried out in this same region on the leishmaniasis outbreak.
IgG antibodies against TOSV were detected using the commercial ELISA test Enzywell Toscana IgG/IgM virus (Diesse®, Italy), since, in addition to its high sensitivity and specificity, it has been used in the vast majority of seroprevalence surveys conducted so far, which makes the results more reproducible and comparable,,,,,,. Cross-reactions have been described between TOSV and other members of the genus Phlebovirus,,, mainly those belonging to the species Sand fly fever Naples phlebovirus. A study conducted in 2012–2013 on sand flies captured in the southwest area of the Autonomous Community of Madrid showed the presence of specimens infected by TOSV plus the Arbia virus (species Salehabad phlebovirus) and one specimen infected by the Arrabida-like virus (species Sandfly fever Naples phlebovirus). This could mean that some of the positive results we obtained were not due to previous contact with TOSV but rather due to contact with viruses of the same family. The recent increase in the number of aseptic meningitis of unknown aetiology in our Autonomous Community leads us to believe that the majority of seropositive detected in our study are against TOSV.
| Conclusion|| |
Based on the results obtained in this seroprevalence study, we consider it essential to include, from now on, the detection of TOSV in the routine diagnosis of lymphocytic meningitis that occurs in our region, which will allow us to know more accurately the real numbers of central nervous system infections by this virus. It is also necessary to study the epidemiological behavior of TOSV in our environment to determine if the environmental and vector control measures maintained over time have been effective in reducing its circulation and if this has translated into a decrease in the seroprevalence of the disease and in cases of TOSV-related aseptic meningitis.
Conflict of interest: None
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[Table 1], [Table 2], [Table 3]