Seroprevalence of and risk factors for Leishmania seropositivity in a sample population of Western Sicily ( Italy )

Background: Leishmania is a vector-borne parasite responsible for significant morbidity and mortality worldwide. The aim of this study was to assess the prevalence of and risk factors for Leishmania infantum seropositivity in a sample of Sicilian population. MeThodS: a total of 260 subjects were interviewed using a standardized questionnaire and requested for an venous blood sample. reSulTS: overall, 36 subjects (13.8%) were seropositive against L. infantum with a statistically significant higher prevalence of positivity in older subjects (p=0.04). after adjustment for age, a higher risk for leishmania seropositivity was found in subjects who had pets living outdoors and untreated with anti-pests, and in those who were current smokers (adj-or = 2.95 and adj-or = 3.11, respectively; p < 0.05). concluSionS: our data confirm that Leishmania infections among Sicilian citizens can be considered relatively frequent, suggesting that a percentage of Leishmania seropositivity can be probably attributed to exposure to both old and new risk factors.


inTroducTion
Leishmania is a vector-borne protozoan parasite responsible for significant morbidity and mortality worldwide, being endemic in 88 countries, with approximately 0.9 to 1.6 million new cases per year [1].Leishmania infantum is the only species present in Sicily and is transmitted through a zoonotic mechanism which involves dogs as the main reservoir of infection [2].The protozoan is spread through the bite of an infected female phlebotomine sandfly that are widely distributed in Mediterranean Countries with a LEISHmANIA SEROPOSITIVITy IN SICILy peak of activity during summertime [3].Most human infections by L. infantum are subclinical or asymptomatic whereas only a minority, especially in immunosuppressed subjects, can cause obvious disease including cutaneous (CL) and visceral (VL) manifestations [4].
Following World War II, the widespread application of dichlorodiphenyltrichloroethane (DDT) in much of southern Europe was associated with a considerable decrease of several arthropod vectors, including phlebotomine sandflies [5].The banning of DDT could be responsible for an inversion of this trend as observed in Brazil where the discontinuation of the insecticide has been associated with a marked increase in abundance of the Anopheles vector and increased malaria cases [6].If this association should be confirmed also in Italy, where DDT use has been banned for at least three decades, a variable number of endemic arthropod-borne infections could be insufficiently controlled [7].According to this hypothesis, an active circulation of Toscana virus, an arthropod-borne virus transmitted by the same vector of Leishmania, has been found in the general Sicilian population in 2009 [8].Therefore, a high number of subjects could be also exposed to L. infantum infection in an area as endemic such as Sicily.In this paper, we have assessed the seroprevalence of L. infantum-specific antibodies in a sample of the general Sicilian population identifying potential environmental and individual factors associated with Leishmania antigen seropositivity.

MeThodS
A cross-sectional study was carried out from September 2009 to July 2010 in Calatafimi-Segesta, which is a small city located in the province of Trapani, western Sicily, Italy.Calatafimi-Segesta has a resident population of about 7,144 inhabitants [9] and is situated at an altitude of 338 m above sea level.
A sample of subjects was randomly selected from the National Health roster for Calatafimi, after which, patients were stratified in terms of gender and age according to the whole population structure.Criteria for exclusion of subjects from the study were: residence other than in Calatafimi-Segesta, age <5 years, severe cognitive dysfunction and inability of the general practitioner to reach each subject within the recruitment period.Randomly selected participants who had died before recruitment were also excluded.
As reported in detail elsewhere a random sample of 271 eligible subjects were successfully recruited and enrolled [10].Each subject provided a venous blood sample and was interviewed using a standardized questionnaire including information on: • sociodemographics, occupation, educational level, ownership of an extra-urban holiday residence; • anthropometry; • lifestyle habits and exposure to animals.Prior to 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.Unfortunately, for 11 subjects the blood sample was not enough to allow laboratory analyses therefore, only the data provided by the 260 individuals was finally included and discussed in our study.
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.
The presence of anti-Leishmania antibodies was measured by an indirect immunofluorescence antibody test (IFAT) performed in conformity of recommendation of the World Organisation for Animal Health using MHOM/TN/80/IPT1 MON 1 as whole parasite antigen fixed on multi-spot slides (BioMerieux) and a fluorescent-labeled, antihuman gamma globulin (SIGMA) as conjugate [11].Positive sera were serially diluted and tested to establish the maximum reaction titre, starting from dilution at concentration of 1:40.Positive and negative controls were included in each slide.All samples showing positivity at a dilution ≥1:40 were considered to be reactive.
The data was analyzed using the R statistical software package [12].The significance level for all analyses was chosen to be 0.05, two-tailed.Absolute and relative frequencies were calculated for qualitative variables.Continuous normally distributed variables were summarized as mean ± standard deviation.Categorical and categorized (age and BMI) variables were analyzed using Chi-square test (Mantel-Haenszel), Chi-square for linear trend test or logistic regression analysis as indicated.Odds ratio (OR) and 95% LEISHmANIA SEROPOSITIVITy IN SICILy confidence interval (95% CI) were calculated.Age adjustment was evaluated by performing a logistic regression analysis.

reSulTS
The characteristics of the study of the general population was reported in Table 1 Overall, 36 subjects (13.8%) resulted seropositives against Leishmania with a statistically significant higher prevalence of positivity in older subjects (p=0.04).A higher, but not statistically significant, percentage of Leishmania seropositivity was observed in males (16.8%), overweight (17.2%), widows (20.0%), farmers (33.3%), and subjects with low/intermediate education levels (15.2%).

diScuSSion
The results of the present study confirm that asymptomatic Leishmania infections among Sicilian citizens can be considered relatively frequent, showing a prevalence similar to those found in some south-European countries such as Spain (ranging from 4.9% to 24%) and France (ranging from 13% to 32%) [13][14][15][16].Diversely, a lower seroprevalence was observed in Greece (ranging from 0.5% to 2.8%) and other Italian regions as well as Piedmont (7.4%) and Sicily, where other authors have reported very low positivity rates among healthy blood donors (0% in 2005 and 0.75% in 2008) [17][18][19][20][21].These latter results, carried out in the same regional macroarea, could apparently generate some confusion, however, it should be noted that all of these comparisons should be considered with precautions for two main reasons.Firstly, in absence of gold-standards, the choice of different laboratory methods and cut-off values could affect the prevalence of positive subjects.Considering that the intention of our study was to evaluate the exposure to infection rather than assess a diagnostic significance, in our setting we have adopted a serum antibody titers 1:40 as an indicator of previous Leishmania infection as also suggested by other authors [14].Secondly, some highly selected group as healthy blood donors could not be fully representative of the base population in sex and age distribution, being furthermore of different geographic districts.
Despite this heterogeneity however, the previous comparisons could suggest an active circulation of L. infantum in Sicily after a period of relatively good control as a consequence of DDT campaigns.This latter consideration could be supported by about 6% of seropositives found among younger subjects ( < 30 years old) born after DDT discontinuation and, intriguingly, our seroprevalence is very close to the prevalence of leishmanin skin test positivity (16.6%) documented in Sicily before DDT discontinuation [22].
Furthermore, a positive association with age has been well documented by others, being probably attributable to a higher cumulative probability of having been infected over the years [13,19].
Of particular interest in terms of public health, the strong association between Leishmania seropositivity and daily contact with animals living outdoors and not treated by antipests.This observation, confirmed also after adjustment for age, suggests that insecticidal use on animals appears to represent an effective way of reducing Leishmania transmission to humans and appears to be consistent with a reduced risk of infection among dogs found by others [23].Leishmania seropositivity has been also significantly associated with current smoking habits.To the best of our knowledge a similar finding is not present in the international literature, probably because it has never been investigated.Although this result needs to be LEISHmANIA SEROPOSITIVITy IN SICILy cautiously interpreted and further investigated, it must be stressed that smokers could be more exposed to sandfly bites due to their habit of smoking in outdoor environments as also requested by the Italian law for public places and, furthermore, smoking could attract sandflies by releasing carbon dioxide, that is recognized to be a nearly universal mosquito activator and attractant [24].
Finally, it should be pointed out that our study may have some limitations due to its relatively small sample size and restriction to the general population of a small city in western Sicily.Moreover, the use of a serodiagnostic Epidemiology Biostatistics and Public Health -2014,Volume 11, Number 2 Epidemiology Biostatistics and Public Health -2014,Volume 11, Number 2

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Torina A, Sole M, Reale S, Vitale F, Caracappa S. Use of phlebotomine sand flies as indicator of leishmania e 9 0 2 4 -5 . Of the 260 recruited subjects, 141 were females (54.2%) with an average age of 49.2 ± 19.4 years, and 119 were males (45.8%) with an average age of 50.4 ± 21.7 years.Low or intermediate level of education was represented in 64% of respondents, 45.5% of respondents were overweight and 66.5% of participants were married/free union.