Temporal , spatial and spatiotemporal analysis of the occurrence of visceral leishmaniasis in humans in the City of Birigui , State of São Paulo , from 1999 to 2012

Introduction: In 1999, Birigui and Araçatuba were the fi rst municipalities in the State of São Paulo to present autochthonous cases of visceral leishmaniasis in humans (VLH). The aim of this study was to describe the temporal, spatial and spatiotemporal behaviors of VLH in Birigui. Methods: Secondary data were obtained from the Notifi able Diseases Information System from 1999 to 2012. The incidence, mortality and case fatality rates by sex and age were calculated. The cases of VLH were geocoded and grouped according to census tracts. Local empirical Bayesian incidence rates were calculated. The existence of spatial and spatiotemporal clusters was investigated using SaTScan software. Results: There were 156 confi rmed cases of autochthonous VLH. The incidence rate was higher in the 0-4-year-old children, and the mortality and case fatality rates were higher in people aged 60 years and older. The peaks of incidence occurred in 2006 and 2011. The Bayesian rates identifi ed the presence of VLH in all of the census tracts in the municipality; however, spatial and spatiotemporal clusters were found in the central area of the municipality. Conclusions: Birigui, located in the Araçatuba region, has recently experienced increasing numbers of VLH cases; this increase is contrary to the behavior observed over the entire region, which has shown a decreasing trend in the number of VLH cases. The observations that the highest incidence is in children 0-4 years old and the highest mortality is in people 60 years and older are in agreement with the expected patterns of VLH.

Visceral leishmaniasis (VL), or kala-azar, is a parasitic disease that is widely distributed worldwide and is mainly found in tropical and subtropical Asia, the Middle East, Africa, and the Americas 1 .According to the Pan American Health Organization (PAHO) 2 , 38,808 cases of visceral leishmaniasis in humans (VLH) were reported in the Americas from 2001 to 2011; 96.6% of those cases occurred in Brazil.
In Brazil, VL is present in 26 states of the fi ve regions of the Brazilian territory [2][3][4][5] .Since 1980, there has been a change in the epidemiological pattern of VL, which was previously characterized as a rural disease.With the urbanization of Lutzomyia longipalpis (Lutz & Neiva, 1912), the main vector of the parasite in the Americas 6,7 , VL is now present in urban areas.
The vector Lu.longipalpis was fi rst identifi ed in 1997 in the western region of the State of São Paulo in the municipality of Araçatuba 8 .However, the fi rst autochthonous cases of VLH were recorded in 1999 in the municipalities of Araçatuba and Birigui 9 .Thereafter, VLH spread to other municipalities, particularly in the western region of the state; between 1999 and 2011, 73 municipalities of São Paulo had human cases 10 and 125 municipalities documented the presence of the vector 11 .
Due to its high incidence of VLH over recent years, Birigui is considered a municipality with intense VLH transmission and is a priority area for the surveillance and control of this disease in the state of São Paulo 12 .Thus, the objective of this study was to describe the temporal and spatial occurrence of VLH in this municipality, to identify spatial and spatiotemporal clusters of VLH cases and to pinpoint the areas of greatest risk for its occurrence.

METHODS
This descriptive study was performed in the City of Birigui (21°15'49" South, 50°21'10" West), which had an average annual temperature of 23°C, an annual accumulated rainfall of 1229.5 mm, and 108,728 inhabitants in 2010.Birigui is located

RESULTS
in the western region of the São Paulo state and is part of the administrative region (AR) of Araçatuba.
The source of information on the occurrence of autochthonous cases of VLH in Birigui during the period of 1999-2012 was the Notifi able Diseases Information System (SINAN) database, which was provided by the surveillance system of the municipality.The following information was considered for VLH cases: date of onset of symptoms, age, sex and address.The information source for the number of inhabitants, age and sex of the entire municipality, and its census tracts was the Brazilian Institute of Geography and Statistics (IBGE).
The incidence, mortality and case fatality rates were calculated overall and by sex and were age-standardized for the entire study period.These rates were also calculated by age and sex.Additionally, the overall incidence rates and incidence rates by sex were calculated and age-standardized for each individual year of the study.The VLH cases were geocoded based on the address from the database of Birigui addresses and grouped by urban census tracts.The incidence rates were obtained by census tracts and were age-standardized for each year.The incidence and mortality rates by census tracts, standardized by age for the entire study period, were also calculated.Thematic maps were created to represent the distribution of VLH cases in Birigui and to represent the incidence and mortality rates.
Bayesian empirical local rates for each urban census tract were estimated based on the number of cases that occurred throughout the study period and the population at the midpoint (2006) using the empirical Bayesian method available with TerraView 4.2.2 software 13 .The neighborhood criterion considered sectors to be neighbors when they had at least one side or vertex in common.To verify the existence of spatial autocorrelation between the incidence rates of VLH for the entire study period, the global Moran index was calculated 13 .
Three databases were created to identify spatial and spatiotemporal clusters of VLH cases in Birigui from 1999-2012.The fi rst database, which is called the bank of cases, contained each VLH case with the identifi cation of the census tract and the onset date of the symptoms (month and year).The second database was a fi le containing population data for each census tract.The third database had plane Cartesian geographic coordinates of the centroid of each census tract.The three fi les were imported into SaTScan 9.1.1software.
To identify purely spatial clusters, scan statistics were used on SaTScan, and a Poisson discrete model was constructed with the following conditions: time accuracy, in months, during the period of 1999-2012; non-occurrence of geographical overlap of the clusters; and maximum size cluster equal to 50% of the exposed population, with circular clusters and 999 replications 14 .To identify spatiotemporal clusters, a discrete Poisson model was used with the same conditions as the purely spatial analysis; this model considered the maximum size of the temporal cluster to be equal to 50% of the study period 14 .Next, the Birigui areas that were classifi ed as spatial and spatiotemporal clusters were visited to identify their main characteristics in terms of property type; presence of backyards; vegetation and animals; and socioeconomic, demographic and environmental conditions.The fi rst two cases of VLH in Birigui were confi rmed in 1999; these cases were followed by a period of two years (2000  and 2001) in which no cases were documented (Figure 1A).After 2001, there was a rise in the incidence curve of VLH, culminating in a peak in 2006.This peak was followed by a fall in incidence and a new peak in 2011; the incidence rate in 2011 was higher than the incidence rate in 2006 (Figure 1A).From 1999-2012, 156 autochthonous cases were confi rmed in the county, corresponding to a total incidence rate of 13.5 cases per 100,000 inhabitant-years; there were 12.8 cases per 100,000 inhabitant-years in males and 14.4 in females.Considering the entire study period, the VLH incidence rate was higher in children aged 0-4 years old (Figure 1B), and the mortality and case fatality rates were higher in people aged 60 years and older (Figures 1C and D).

Ethical considerations
Although the distribution of VLH between genders was presented in a homogeneous fashion, further analysis of the incidence rates by gender and age revealed that girls aged 0-4 years were more affected than boys, and men aged between 20-59 years were more affected than women (Figure 1B).Furthermore, the case fatality and mortality rates were higher in men aged 60 years and older compared with women of a similar age (Figures 1C and D).
Of the total (156) confirmed cases in the period from 1999-2012, 153 (98.0%) were geocoded.Three cases were not geocoded: one because it was located in a rural area and two because they did not have address information.As shown in Figure 2A, VLH cases occurred in 87 of the 156 census tracts of the urban area.Of all the census tracts with cases, the majority (50) had only one case; there were two or three cases in 30 census tracts, and there were four or fi ve cases in six census tracts.Only one census tract had a higher concentration, with a total of nine cases.According to the census tracts, the incidence rates ranged from 0 to 78.38 cases per 100,000 inhabitant-years (Figure 2B).The global Moran index, which was calculated from these rates, was not statistically signifi cant and showed that the distribution had random behavior.
Figure 2C shows the Bayesian incidence rate of VLH, which identifi ed decreases in the amplitude and in the peak of incidence and also identifi ed the presence of the disease in all of the census tracts.According to the census tracts, the mortality rates ranged from 0 to 31.26 cases per 100,000 inhabitant-years (Figure 2D), and the global Moran index, which was calculated from these rates, was not statistically signifi cant, showing that the distribution had random behavior.
Figure 3 and Figure 4 show the expansion process of VLH cases in the municipality of Birigui during the period  of the study (1999-2012).The first reported cases were identifi ed in the central region of the county.Over the years, the distribution of cases has expanded among the census tracts, reaching the outlying regions of the city and simultaneously occurring in the city center.We observed a variation in the incidence rates of VLH that was expressed from year to year and among the census tracts; there was also an increase in the incidence rates surrounding the years with incidence peaks (2006 and 2011).In 2012, there was a higher incidence rate of VLH in a census tract compared with other years.
From the scan statistics, spatial clusters were identifi ed throughout the study period and encompassed fi ve census tracts with a total of 20 cases of VLH; this was a statistically signifi cant result [relative risk (RR) = 3.59, p<0.05] (Figure 2E).A spatiotemporal cluster was also identifi ed from February 2006 to September 2012, encompassing 44 census tracts and a total of 59 cases of VLH; this was a statistically signifi cant result [RR = 3.58, p<0.05] (Figure 2F).Furthermore, the spatiotemporal cluster included the two annual incidence peaks of VLH.
The Birigui areas that were classified as spatial and spatiotemporal clusters of VLH cases were visited, and these areas had ground-level houses in which low income and average income families live.Several of these houses had shaded backyards due to the presence of bushes and fruit trees (mango, jaboticaba tree, banana, mulberry, acerola tree, etc.).The majority of the homes had dogs, and some homes also had chickens and horses.In some parts of the cluster areas, there were dogs in the streets (Figure 5 -features homes or homes with VLH cases).
The analysis of the occurrence of VLH in Birigui allowed for the visualization of several important characteristics of the disease, including the observation that the population aged 0 to Studies by Silva and Gaioso 16 and Goes et al. 17 compared the incidence of VLH between sexes and found a predominance of male cases, which was not observed in Birigui.This inconsistency might be related to a potentially greater exposure to the vector in the male population because males are more likely to perform activities in rural and sylvatic environments 16,17 .This was not the case in Birigui during the study period because the disease occurred mainly in urban areas; this fi nding might have favored the more equal incidence rates between the sexes.
When the incidence rates of VLH in Birigui were distributed among their respective age groups, it was observed that the sex balance masks other signifi cant differences.The higher incidence observed in girls 0-4 years old compared with boys of the same age group differs from other studies that reported no differences in susceptibility to VLH between the sexes 15,18 .In contrast, Costa 19 reported a higher susceptibility in male children younger than fi ve of age because of the hormonal characteristics of the sex; these differences have not yet been explained.Visceral leishmaniasis in humans has been described as an opportunistic disease in human immunodefi ciency virus (HIV) infected individuals.This coinfection might be related to the higher incidence of the disease in males aged 20-60 years compared with females because this age group has the highest percentage of HIV-positive individuals 20 .
Despite the large number of cases in the 0-4-year-old age group, the case fatality rate in that age group was low.Case fatality rates peaked among persons aged 60 and older perhaps because of the combination of VLH and other diseases previously acquired in this age group.Additionally, adverse reactions caused by medications used to treat VLH occur more frequently in the older age group, and elderly persons are likely to have impaired immunity 3,21 .

FIGURE 1 -
FIGURE 1 -Incidence rates of visceral leishmaniasis in humans (VLH) by sex and year (A); incidence rates (B), mortality rates (C) and case fatality rates of VLH (D) by sex and age for the entire study period, 1999-2012, Birigui, State of São Paulo, Brazil.
had a much higher incidence of VLH than other age groups.According to Xavier-Gomez et al.1 , the immaturity of humoral and cellular immunity makes children more susceptible to infections.
This project was approved by the Ethics Committee of the Faculty of Public Health, University of São Paulo, by the Platform Brasil, CAAE: 14107313.3.3333.5421,Opinion Number: 257 511 on 04/26/2013.