Changing demographics of visceral leishmaniasis in northeast Brazil: Lessons for the future

Background Visceral leishmaniasis (VL) caused by Leishmania infantum became a disease of urban areas in Brazil in the last 30 years and there has been an increase in asymptomatic L. infantum infection with these areas. Methodology/Principal findings A retrospective study of human VL was performed in the state of Rio Grande do Norte, Brazil, for the period of 1990–2014. The data were divided into five-time periods. For all VL cases, data on sex, age, nutritional status and childhood vaccination were collected. Geographic information system tools and statistical models were used to analyze the dispersion of human VL. The mean annual incidence of VL was 4.6 cases/100,000 inhabitants, with total 3,252 cases reported. The lethality rate was 6.4%. Over time the annual incidence of VL decreased in the 0–4 years (p<0.0001) and 5–9 (p <0.0001) age groups, but increased in ages 20–39 (p<0.001) and >40 years (p<0.0001). VL occurred more often in males (β2 = 2.5; p<0.0001). The decreased incidence of VL in children was associated with improved nutritional status and childhood immunizations including measles, poliomyelitis, BCG, and hepatitis B. Human VL correlated temporally and geographically with canine L. infantum infection (p = 0.002, R2 = 0.438), with rainfall and with Lutzomyia longipalpis density (r = 0.762). Overall, the incidence of VL decreased, while VL-AIDS increased, especially between 2010–2014. VL was more frequently found in areas that lacked urban infrastructure, detected by lack of garbage collection and sewers, whereas HIV infection was associated with higher levels of schooling and evidence of higher socioeconomic status. Conclusion/Significance The demographics of VL in northeastern Brazil have changed. Disease incidence has decreased in children and increased in adults. They were associated with improvements in nutrition, socioeconomic status and immunization rates. Concurrent VL-AIDS poses a serious challenge for the future.


Introduction
Visceral leishmaniasis (VL) is a life-threatening disease caused by L. infantum [1], which was first recognized in Brazil in 1932 [2][3][4]. It is likely the parasite initially arrived in northeastern Brazil with people and/or dogs previously infected with L. infantum in southern Europe or North Africa [5;6]. Lu. longipalpis is a competent vector for L. infantum, and it is found in most countries in Latin America. Dogs are considered the major reservoirs of L. infantum. At first, cases of VL in Brazil occurred sporadically in semi-arid, rural areas of the northeast region. Most cases occurred in children under 10 years of age [7;8]. However, in the late 1980's and early 1990's, urban outbreaks occurred in large cities in the northeast and other regions of Brazil [9][10][11][12]. Massive migration of the population to urban regions, adaptation of Lu. longipalpis to peridomestic environments, and transport of L. infantum infected dogs to urban areas occurred during this period. The temporal occurrence of human VL has assumed a variable pattern that correlates with environmental forces including el Niño/la Niña [13;14], which influence rainfall and humidity and thus the density of sand flies.
Several studies in endemic areas of Brazil have shown that most people infected with L. infantum remain asymptomatic, if they are not immunosuppressed [15][16][17][18][19]. It is not clear whether people with VL or with asymptomatic L. infantum infection serve as reservoirs and contribute to the long-term maintenance of this pathogen in endemic areas. In the past, the ratio of symptomatic VL to asymptomatic L. infantum infection among children was approximately one in every six, and for adults the ratio was 1 in 18. Risk factors for developing symptomatic VL in children included malnutrition [20][21][22], neoplastic disorders [19;23] and viral co-infection [24]. Brazil has had freely available vaccination for all age groups since early 1980 and this has had a coverage of over 90% for routine child immunizations. This improved vaccination rate has led to decreased and/or elimination of some of the common and potentially fatal diseases of children. For instance, it is known that measles infection predisposes to opportunistic infection for years [25;26] and measles has been eliminated from Brazil for almost a decade, although there was an outbreak in 2013, which was contained [27;28].
HIV/AIDS is known to increase the risk of developing symptomatic, rather than asymptomatic VL [29][30][31]. HIV co-infection with VL was first noted to contribute to the increased incidence of VL in adults in Europe in the mid-1980's [32]. VL/AIDS was recognized in Brazil in early 90's [32][33][34]. Thus, a new epidemiological pattern of VL is emerging because of spread of HIV to all regions of the country. Subjects with VL-AIDS have an increased risk of VL relapse and death. The goal of the current study was to identify demographic, spatial and socioeconomic factors associated with VL in northeastern Brazil between 1990 and 2014, using VL cases reported in the state of Rio Grande do Norte. We assessed the geographic distribution of VL and its association with the spatial distribution of HIV/AIDS, and with socioeconomic factors that could influence the outcome of L. infantum infection. A better understanding of the epidemiological dynamics of L. infantum and HIV co-infections, and their determinants is essential to guide new health policies.

Study area
The study was conducted in the state of Rio Grande do Norte in northeastern Brazil. The state has an area of 52,811,126 square kilometers, with a population of 3,408,510, 77.8% of whom now live in urban areas. Most of the state has a semi-arid climate, with rainfall less than 800 mm per year and an average temperature of 27˚C. A more humid climate is found along the east coast of the state, which borders the Atlantic Ocean, where the rainfall indices are greater than 1,400 mm per year. The state is grouped in 19 micro-regions (MR), each with distinct climate, topography, hydrography, population density and economy. These micro regions served as units for the current analysis.

Study design
A retrospective study of VL cases diagnosed in the state of Rio Grande do Norte was performed, and correlated with demographic and epidemiologic factors in the corresponding regions of the state. The study was divided into five time periods: (1) 1990-1994, (2)  The association between spatial patterns of VL and HIV-AIDS was assessed using quantitative data available from 1991, 2000 and the 2010 censuses. The incidence of VL, AIDS and VL-AIDS co-infection per 100,000 inhabitants was extracted from the state records listed below. Additional variables that were collected included: (1) dates of new cases of VL, AIDS and VL-/AIDS, (2) sex, (3) age, (4) nutritional status of children under 5 years, (5) vaccination rates for measles, poliomyelitis, BCG and hepatitis B in children under 5 years. The spatial and temporal correlation between human VL and canine infection was also assessed. Environmental or socioeconomic variables considered were: (1) the association of annual rainfall and the density of Lu. longipalpis, (2) socioeconomic data from the censuses included literacy rate, education, income, city water supply, waste disposal, septic tank and presence of sewage.

Data collection
Data on human VL were obtained from the Notifiable Diseases Information System (SINAN). This federal government system catalogs the reported cases and coordinates investigations of diseases for which reporting is mandated by the Brazilian government, as defined by specific legislation. The data are captured in the Health Post Centers and/or hospitals and are sent to state Secretary of Health, whose office uploads the information in SINAN. The list of notifiable disease is updated as new outbreaks occur. For example, the recent epidemic of Zika virus infections led this virus to be added to the list of mandatory reportable diseases. Data on Lu. longipalpis density and rates of infection in domestic dogs were obtained from the Surveillance and Leishmaniasis Control Program, Secretary of Health. Data on HIV or AIDS were obtained by crossing information from SINAN with the Brazilian Mortality Information System database and release of medications for HIV. Nutritional data on children younger than 5 years were obtained from the Brazilian Minister of Health. Those data were available for all 19 micro-regions of the state of Rio Grande do Norte.
Data about immunization coverage were obtained from the Brazilian National Program of Immunization Information System. The vaccination coverage percentage was calculated per estimated population at the age group targeted to be vaccinated and the doses of vaccines administered in each municipality, and grouped into the 19 micro regions for this analysis.
Data variables gathered from censuses included education level, income, local health facility, piped water supply, garbage collection, street cleaning, sewer system, septic tank, urbanization and population density. Those data were collected from the Instituto Brasileiro de Geografia e Estatística (IBGE) website (http://www.ibge.gov.br/home/). Annual rainfall data in the municipalities were obtained from the State of Rio Grande do Norte Agricultural Research Company, EMPARN, (http://www.emparn.rn.gov.br/).

Statistical analysis
The effect of sex and micro-region (MR) on the temporal variation in the incidence VL from 1990 to 2014 were evaluated by a general linear model with categorical explanatory variables [35] according to the following formula: Y ti = β 0 + β 1 t + β 2 I(Sex) + θ i MR(i|19) + error, (model 1) where the dependent variable was the incidence of VL (Y ti ) per 100,000 inhabitants, in the year t and in the micro-region i. The independent variables were the time (t) in years, the categorical variables were sex (1 if male) and micro-region (1-19) MR (i |19). The β1 coefficient measured the average annual increase in the incidence of VL, whereas β2 measured the differential incidence of VL between male and female. Micro-region 19 was considered the reference, since it was the site of the first VL outbreak in the state of Rio Grande do Norte. The micro-region parameter was considered when evaluating the existence of spatial aggregation. The temporal incidence of VL considering the patient's age was analyzed by linear regression using the following statistical model: Z t = β 0 + β 1 t + error (model 2). The Z t is the rate of cases per 100,000 inhabitants in year t, and β1 is the slope of the adjusted line that defined the secular trend of the incidence of VL for the subsequent year. A rising trend in cases/unit of time unit was observed when β1>0, whereas a downward trend in case rates/time was observed when β1<0. The case rate over time was stationary if β1 = 0. The model was adjusted in each of the following age groups: 0-4 years, 5-9 years, 10-19 years; 20-39 years and !40 years. The incidence/time unit was calculated within each age group.

The impact of vaccination coverage on the incidence of VL
The impact of routine vaccination of the population, including vaccines for measles, poliomyelitis, BCG and hepatitis B, on the incidence of VL was analyzed in children under 5 years of age between 2000 and 2014. We evaluated these data for each micro-region. Vaccination rate data were superimposed on micro-region VL incidence data to determine whether vaccination coverage correlated with VL, particularly in children under age 5. We used an adjusted linear regression model as defined by the formula : Y ti = β 0 + β 1 t + β 2 X ti + θ i MR(i|19) + error, (model 3). Similar to model 1, the independent variable Xti was the vaccination coverage in micro-region i during year t. Because they were strongly correlated, an analysis of principal components was made and the vaccination coverage was represented by the score of the first component.

The impact of nutritional status on the incidence of VL in children
The nutritional status was considered by the proportion p i of children whose weight for age was categorized as very low(p 1 ), low(p 2 ), appropriate(p 3 ) and high(p 4 ). From those, a Nutritional Status Index-NSI was built considering a weighted proportion with zero sum weights w = (-1, -

Spatial relationships between canine and human VL, and between HIV and human VL
The relationship between the incidence of human VL with canine VL in micro-regions/time was performed by adjusting a simple linear regression model defined by the formula: Y t = β 0 + β 1 X t + error, (model 5). The dependent variable, Y t was set to log (rate human VL+ 0.5) in the year t in which there was canine examination, and the independent variable was X t the corresponding level of canine infection (LCI). The level of canine infection was defined as a weighted proportion of infected dogs, using weights ranging from zero to 100 depending on the total number of dogs examined versus the number that had Leishmania infection, model 6, as follows: The spatial dependence and the association between response variables was performed by modeling the distribution of human VL case events with the spatial distribution of canine VL and social predictive factors, by adjusting mixed autoregressive spatial linear models (Spatial Lag Model Autoregressive-SAR) that captured the self-spatial correlation through a single ρ parameter (rho) added to the regression model; this was chosen to model factors in the same test: i.e., temporal variation in VL, effect of sex, and geographic micro-region. The equation was expressed as: y = ρXy + XBeta + error, (Model 7), where y is incidence of VL per 100,000 inhabitants at micro-region level. ρ (rho) measures the spatial dependence the VL incidence, W is the weight matrix modeling the spatial structure, X is the matrix of predictor variables, β is the regression coefficient vector which evaluated the association between Y and X, and error represents the residuals. The log transformation was applied to normalize the response distribution. The same analytical approach was applied to the spatial distribution of HIV/AIDS. The predictor variable data (X) was collected in the censuses 2000 and 2010. All statistical models tested herein are shown in S1 Supporting Information.

Software used
We used Excel 2013 in the construction of the database, Statistica StatSoft version 7.0, in the estimation of linear models (Models 1 to 7) and Quantum GI version 2.12.e-Lyon (http:// www.gnu.org.licenses) in the construction of maps and R System version 3.2.2 (https://www.rproject.org) for the mixed autoregressive linear models.

Ethical considerations
This study was reviewed and approved by the Universidade Federal do Rio Grande do Norte Ethical review board CAAE12584513.1.0000.5537. Data were anonymous records and exempted from signed consent.

Incidence and spatial distributions of human VL and AIDS in the state of Rio Grande do Norte, Brazil
A total of 3,252 cases of VL were reported in the state of Rio Grande do Norte, northeast Brazil, between the years of 1990 and 2014. The mean annual incidence was 4.6 VL cases/100,000 inhabitants. Fig 1A shows the incidence of VL, AIDS and VL/AIDS by 5-year period. The overall VL lethality rate was 6.4%, with a total of 210 deaths (210/3,252). However, the highest lethality rate was 8.2% in the period 1990-1994 and was associated with the highest incidence of VL (Fig 1B).
There were 5,777 cases of AIDS with an average incidence of 8.1 cases/100,000 inhabitants ( Fig 1A) between 1990 and 2014. During the study period, the average incidence of concurrent VL/AIDS was 0.16 per 100,000 inhabitants. However, on the 5 th period (2010-2014) it reached 0.46/100,000 (Fig 1A).
VL cases were predominantly found between 1990-1994 in the eastern coastal region of Rio Grande do Norte, but the disease subsequently spread to the Northeastern Coast (θ 16 = 5.983; p<0.0001) and to other areas (θ 17 = 6.256; p<0.0001, model 1), Table 1 and Fig 2. Although there has been an increase in areas reporting VL, there was a mean decrement of 0.135 VL cases/per year (β 1 = -0.135, p<0.0001) ( Table 1).

Factors influencing human visceral leishmaniasis: Age, sex, nutrition, vaccination and canine visceral leishmaniasis
Over time there was a decreasing trend in the incidence of VL in both sexes. However, the male incidence was uniformly higher by approximately 2.5 per 100,000 (β2 = 2.498; p< 0.0001), (Table 1; Fig 3A). There were two major peaks of VL, the first in 1991-1992 and the second in the 1999-2000 (Fig 3A).
The score in year t and the micro-region i representing the vaccination coverage, at this time and place was calculated by X ti = 0.0989BCG ti + 0.3993POLIO ti + 0.3181MEASLES ti + 0.3915HEPATITIS ti , obtained from the first principal component. This means that an increase in one unit in the vaccination coverage score was associated with a reduction of 4.8 in the incidence rate of VL in children younger than five years. There is a strong association between the two variables estimated by a third-degree polynomial relationship (Fig 4A).  The association between the Nutritional Status Index-NSI and the incidence rate of VL in children less than 5 years was evaluated by adjusting for other parameters, models 3 and 4. There was a trend toward an inverse correlation between better nutritional status and VL in children, but this did not reach statistical significance, (β 2 = -141.76; p = 0.1136). In contrast, there was a positive linear correlation between the incidence of human VL and canine VL (Human VL = 0.4038 + 0.2889LCI; r = 0.456, p = 0.0758), evaluated by the level of L. infantum canine infection (LCI) per models 5 and 6, as described in the methods session (Fig 4B). An increase of one LCI unit was associated with an increase in the incidence rate of human VL.

Environmental determinants of visceral leishmaniasis
There was a positive correlation between the density of sand flies and the rainfall index (r = 0.762, p<0.0001), according to model 5. In addition, an increase in annual rainfall index correlated with increased annual incidence of VL (r = 0.616, p = 0.005). The fluctuation in rainfall index explained 38% of the variation in the incidence of VL, with a100 mm increment in annual rainfall associated with an increase of 0.6 in VL/100,000 inhabitants (Fig 4C).

Socioeconomic determinants of human visceral leishmaniasis and HIV/ AIDS
Among the socioeconomic variables that correlated with the incidence of VL was the percent-

Discussion
Transmission of VL occurs in settings where the infected sand fly vector lives in proximity to a mammalian reservoir and susceptible humans [36], or through other means of transmission such as blood transfusion [37]. Despite efforts of health officials to interrupt the routes of transmission of L. infantum, VL continues to be a major health problem in Brazil after Malaria [38]. The demographics have changed substantially since VL was first reported in the 1930's [2]. During early years, the disease occurred predominantly in rural areas of the Northeast region, with most cases of VL occurring in children younger than 10 years [7]. Mass migration of the population to urban areas beginning in the 1980s was accompanied by a change in the pattern of transmission to peri-urban regions of large cities in the Northeast and the southeast regions of the country [39].
The state of Rio Grande do Norte in northeastern Brazil, provides an example of the changing epidemiology of VL. There was a significant increase in the age at disease diagnosis, with an increase in adult VL. The disease decreased in children under age 10 years and increased in  3B). An increase in the average age of VL has also been observed in other Brazilian states [39;40]. Human VL has been associated with poverty and malnutrition in children [20-22;41]. We hypothesized that multiple socioeconomic factors might contribute to the significant reduction in childhood VL in the less than 10 age groups. Since 1999, social programs to decrease poverty and economic measures to control inflation have been successfully implemented in Brazil, with a coincident improvement in many measures of health [42]. Interventions have included supplementation of micronutrients, including iron and vitamin A in pregnant women and children aged 6-18 months, as well as fortification of wheat and corn flours with iron and vitamins [43]. There is an increase in vaccine coverage, with more uniform administration of vaccines as polio, measles, BCG and others. Those measures have been associated with increased average birth weight [22;44], decreased childhood diarrheal diseases [45;46]. The improved health could lead to healthier gut brush border, better absorption of nutrients and protection against opportunistic pathogens such as Leishmania. As an example, studies have shown that the vaccinepreventable disease measles can induce immunosuppression for years [26;47].
In previous studies in the state of Rio Grande do Norte, we found that children and adults were infected at comparable rates with L. infantum, as detected by positive anti-leishmanial serology and/or positive skin test response to Leishmania antigens [15]. Since a majority of L. infantum infections are asymptomatic, it is likely that the above-mentioned health interventions have resulted in enhanced development of protective Type 1 immune responses to Leishmania spp. and other pathogens, with a decreased likelihood that L. infantum infection will progress to VL in young children. In addition, improved socioeconomic status, improved living conditions, and expansion of urban regions may be responsible for decreased sand fly density and transmission in some areas.
In the current study males accounted for most VL cases (67%). Greater susceptibility of males to VL has also observed in other human studies [8,40] and in experimental models of Leishmania infection, hamster and murine models of VL [48]. Higher levels of testosterone have been associated with increased risk of VL caused by L. donovani in India and Sudan [49;50] possibly mediated by increased IL-10 production and down regulation of Th1 responses.
Domestic dogs are thought to be the primary reservoir of L. infantum in Brazil [51]. A correlation between human and canine VL was observed in the current study, and in reports from other areas in Brazil and Northern Africa [52][53][54]. Further studies are needed to better define the roles of dogs and asymptomatically infected humans as reservoirs for L. infantum in the epidemiology of VL in Brazil.
Higher rainfall indices correlated temporally and geographically with a higher incidence of human VL, especially in areas close to the Atlantic Ocean. An association between VL with increased rainfall has been reported in other regions of Brazil [55;56]. However, some cases of VL occurred in areas with lower humidity, higher temperatures and lower rainfall indices. It is likely that variations in the microenvironment provided niches in which sand flies were nonetheless able to thrive in proximity to humans and a dog reservoir.
HIV/AIDS occurred predominantly in males in the initial stage of the pandemic, although recently more women have become infected with HIV in Brazil [57]. The highest incidence of HIV/AIDS occurs in urban regions, coinciding with regions that have a higher incidence of VL. HIV/AIDS has been expanding throughout Brazil since 1990, and has now spread to all areas of Rio Grande do Norte. Coinfection with HIV and Leishmania spp. has contributed to the increased incidence of VL in adults in southern Europe in Spain, France, Italy and Portugal [31;58;59]. Consistently, in this report we document the presence of VL/AIDS in Rio Grande do Norte since 1990, but there was a considerable increase in coinfection in the third period (2000)(2001)(2002)(2003)(2004), presumably because HIV infections spread to areas that were endemic for L. infantum infection. A large number of individuals are asymptomatically infected with L. infantum in the state of Rio Grande do Norte [15;17;60], and people with asymptomatic L. infantum and HIV seem to be at greater risk of developing VL and of death [61]. Therefore, it is imperative that strategies and guidelines be developed to prevent the development of VL during HIV infection. In summary, the demographics of VL in northeastern Brazil have changed substantially over the past 25 years. The incidence has decreased in children in association with improved nutrition, socioeconomic status, childhood immunizations, and overall health. In contrast, the incidence of VL in adults has increased. The latter could be explained in part by failure to develop immunity to the parasite as a child, and the geographic coincidence of HIV infection and VL. The emergence of concurrent VL-AIDS poses a serious health challenge for the future.