article: Still today, more than 30 years after the beginning of the process of visceral leishmaniasis' urbanization, there is little knowledge about the risk factors for its occurrence, despite their relevance to the control and understanding of disease dynamics. The present study is the first systematic review with meta-analysis about factors associated with Leishmania infantum infection in humans in the Americas. After searching different databases, consultations to the reference lists of articles and to experts in the field, 51 studies were reviewed. Theoretical discussions or meta-analysis of p-values or of effect sizes were used to pool information about each variable. The Q test and the I2 statistic were used to assess heterogeneities among the studies. Male sex was associated with visceral leishmaniasis in studies which used the leishmanin skin test for diagnosis and in those where the outcome was the clinical disease; the opposite occurred when serological diagnosis was applied. Younger individuals were less frequently infected than adults, but were more prone to illness. Although with different levels of evidence and of heterogeneity, the presence of dogs at home, higher dog seropositivity in nearby areas, lower socioeconomic status and highly vegetated areas were associated with L. infantum infection. This was not noticed for the presence of chickens in the house and with nutritional status. Susceptibilities to bias and limitations in the analysis and in the description of results were often identified in the studies analyzed. Results showed the existence of consistent patterns for some of the factors analyzed and should be taken into account in developing more effective and well-targeted control measures. Studies must be conducted in new areas of the continent, with improved methodological quality and prioritizing the investigation of the patterns identified and their causes, as well as variables for which knowledge is poor.
  In the Americas, visceral leishmaniasis (VL) is a zoonosis caused by the protozoan parasite Leishmania infantum (syn. L. chagasi). The disease is transmitted to humans by the bite of female sandflies of the genus Lutzomyia [1]. L. longipalpis is the main vector species in the New World [2] but transmission can also occur through Lu. cruzi and Lu. evansi [3], [4]. Other forms of transmission have already been recorded: congenital, drug injection and blood transfusion [5], [6]. Potential wild disease reservoirs are the fox (Lycalopex vetulus and Cerdocyon thous) and the opossum (Didelphis albiventris) [7]. Dogs (Canis familiaris) are considered the main reservoir in the urban setting [8]. Clinical manifestations of the disease can range from asymptomatic forms to severe visceral involvement [9]. The incubation period lasts around two to six months [10]. The fatal outcome occurs predominantly by co-infection or bleeding complications [11]. Studies in Brazil show a variation in case-fatality rates between 4. 2% and 10. 2% in treated patients [12]. For the diagnosis of human visceral leishmaniasis, delayed hypersensitivity tests, such as the Montenegro skin test (leishmanin skin test) [13], parasitological, serological (e. g. , the Indirect fluorescent antibody test - IFAT, the enzyme-linked immunosorbent assay - ELISA, the direct agglutination test - DAT) and molecular methods are commonly used [14]. Immunological techniques are more often used in epidemiological studies since they are easier to implement. The burden of the disease is not exactly known in the Americas, since there is a lack of effective surveillance systems [15]. Although the Brazilian surveillance system is considered better than in other Latin American countries, VL is also underreported in this country [16]. Previously known as a rural endemic disease, visceral leishmaniasis has become endemic and epidemic in large Brazilian cities since the 1980s [17]. The country accounts for 90% of reported cases in the Americas and is the third largest VL focus globally [15]. In Brazil, the recommended control actions against the disease are the use of insecticides against phlebotomines and culling of seropositive dogs, both strategies with little evidence of effectiveness and operationally costly [18], together with the identification and early treatment of human cases [19]. However, they have not produced effective results, given that neither incidence nor fatality rates were reduced in recent years. Moreover, the disease has spread to all regions of the country, and continues to expand into formerly disease-free cities [20]. Identification and quantification of risk factors for VL, besides being useful to understand the determinants of infection acquisition and disease development, can help develop more effective and well-targeted control measures [21]. There are several factors which may be associated with L. infantum infection in humans and many are considered controversial or only partially understood [18]. In this context of heterogeneity in study results, careful evaluation of its potential causes may, in meta-analysis procedures, be more useful than the mechanical calculation of summary measures [22]. Therefore, when summarizing studies that have addressed factors associated with VL, issues such as the quality of studies, design used, methods of data analysis, control for confounders, the way results were reported, and diagnostic tests employed must be considered in order to obtain the best evidence available. In a systematic review of risk factors for visceral leishmaniasis in South Asia [23], consistent patterns have been described for some variables, such as: spatial clustering of cases, conditions of households, areas of vegetation near homes and poverty. However, due to the peculiar characteristics of the disease in that context (the etiological agent is Leishmania donovani; vector is Phlebotomus argentipes; and infected humans are the only reservoir [23]), results cannot be used to make predictions about other contexts. Specifically with respect to human American visceral leishmaniasis, there is no literature record of systematic reviews and meta-analysis regarding factors associated with infection by L. infantum. Thus, through the analysis of observational studies that investigated such factors, this study aimed to obtain more accurate data about each of the factors studied as well as to analyze the quality of publications and to identify gaps in existing knowledge.
  Epidemiological cross-sectional, cohort, case-control and ecological studies were included in the review. These should have described associations between socioeconomic, environmental, family and individual variables and the occurrence of any outcome related to the acquisition of infection by L. infantum. This outcome could be the infection itself (as measured primarily in the study, regardless of the diagnostic assay or of the presence or absence of symptoms in the subject), the identification of active patients who had manifested the clinical disease (in the case of case-control studies) or the notification of cases by health services (in the case of ecological studies using secondary data). There was no restriction for age, sex or language. We excluded studies conducted before 1980 or with populations other than those of the American continent, as well as reviews and papers published in scientific conferences or that were purely descriptive with no possibility of obtaining measures of association. With respect to the variables, we excluded those which referred to: a) control actions (not the focus of this review); b) subject' s genetic characteristics (more related to differences in clinical prognosis); c) for how long a subject had been living in a household (relevant only in local contexts); d) the categorization of the subject' s domicile as urban or rural (due to the lack of standardization in this definition). We maintained, however, those related to the environmental characteristics surrounding the households. We also excluded studies and variables whenever what was being described in the text was impossible to understand or when there were inconsistencies in quantitative data presented or flaws that could invalidate the association described. All search strategies were conducted independently by two researchers (Belo, VS & Barbosa, DS) between March and June 2011. The terms used and databases consulted are described in Text S1 in the order they were searched. In addition, Brazilian research experts were consulted and searches through the reference lists of each article selected at the initial stage were conducted. All titles and abstracts of identified articles were analyzed, being initially excluded those deemed irrelevant. When the information provided was not sufficient for the decision, or when studies were considered relevant for at least one of the researchers, the full text was analyzed. At this stage, papers were selected to be part of the present review. The extraction of information contained in the publications was conducted by a researcher (Belo, VS) and reviewed by all authors of this article. We contacted authors of primary studies to obtain data necessary to calculate measures of association (and possible inclusion in meta-analysis) when they were not described in the publications. Individual patient data were not requested. To identify potential risks of bias in studies and limitations in data analysis and in the way results were reported we used the STROBE statement. It issues recommendations that have been developed to improve the presentation of results from observational studies [24] including several items related to the existence of susceptibility to bias [25]. Additionally, questions from the Newcastle-Ottawa Quality Assessment Scale [26] (for assessing quality in observational case-control and cohort studies) and the book of Fischer and Getis [27] (for assessing quality in ecological studies) were consulted. We have not used scales that result in a numerical quality threshold because this procedure involves arbitrary weighting of items, some of which may not to be directly related to the study' s validity [25]. Thus, we decided to approach the problem more generally, pointing out the major limitations and susceptibility to bias of the revised studies. Study quality and susceptibility to bias were used to discuss the limitations of existing knowledge, without interference, however, in meta-analysis procedures. There was no exclusion of studies due to those factors. Each association between a given exposure variable and a specific outcome was considered a separate and independent meta-analysis. Results of primary studies - except ecological - were described by Odds ratios (OR) and confidence intervals (CI). In cases in which there was information about the probability of significance (p-value), the direction of the association and the sample size of the study, the OR (or correlation coefficient) was estimated by reverse computation. To make decisions about whether to combine or not effect measures, the agreement among the issues added by each study and the differences on their characteristics were evaluated. Whenever a summary measure of effect was obtained, the random effects model was used to pool the data. Under this model it is allowed that the true effect size varies from study to study, thus the overall variability includes the within-studies variance as well as the estimate of the between-studies variance. However, in cases where the number of studies was less than four, due to the lack of precision of the measure of variability [28], we preferred to use the fixed effects model. The Q-Statistical test was used to analyze the occurrence of heterogeneity in effect sizes across studies. The I2 statistic was calculated to determine what proportion of the variance observed represented a real dispersion in the effect sizes (variation between studies), i. e. not due to random error (intra-study variation). Due to the sufficient number of studies and the significant heterogeneity among them, subgroup analyses were carried out for gender, age, presence of dogs in the household and presence of chickens/birds. The following groups were considered: type of study (i. cross-sectional; ii. cohort; iii. case-control), method for measuring the outcome (i. leishmanin skin test (LST); ii. serological; iii. serological and LST; iv. other; v. clinical case), age group of participants (i. children; ii. adults; iii, all ages) and adjustment for confounding (i. yes; ii. no). The Q test was used for comparison of effect sizes and of heterogeneity between subgroups. Those which explain heterogeneity best (higher between subgroups and lower within subgroups) were described. For variables on which subgroup analyses were performed, studies were combined only within the subgroups and an overall measure was not calculated. With regard to studies in which more than one diagnostic method had been performed, results of serological tests (ELISA firstly) were kept in the meta-analysis and results of LST were excluded. However, in analyses where studies were grouped by the method of measuring the outcome, the result of each technique was maintained and used for analyzing the subgroup to which it belonged. The weighted Z-method for combining p-values [29] was applied when the diversity of studies in terms of design, populations or other characteristics became inadequate to obtain a summary-measure based on effect sizes, provided that the question analyzed was similar. We investigated the existence of publication bias among the studies selected for each targeted association (provided that the number of publications is large enough) using the funnel plot, the Egger statistical test and the statistic of “Durval and Tweedie' s Trim and Fill”. The last procedure re-computes the effect size imputing studies until the funnel plot is symmetric about the new effect size. Thus the best estimate of the unbiased effect size is calculated [28]. Theoretical discussions analyzing not only statistical significance, but also the strength and the direction of associations were conducted for variables on which it was not possible to use the methods presented. To perform the meta-analysis of p-values, we used the software Meta-P and for remaining analyzes we used the software CMA, version 2. 0. 057.
  Forty-seven publications (51 studies) were included in the review. Figure 1 shows a flowchart of the search strategy and study selection, along with the reasons for exclusions. Regarding the type of study, 24 were cross-sectional, 17 ecological, 7 case-control and 3 were cohorts. Three studies were conducted in Venezuela, two in Colombia, one in Honduras and forty-five in Brazil. In five studies the population consisted of children only. In studies where the infection was measured directly by investigators, the diagnostic test most often used was the LST, followed by ELISA and IFAT. In all case-control studies, cases were patients with clinical disease. The main potential sources of bias in cross-sectional and cohort studies were the eligibility criteria employed and losses due to refusal to participate and during follow-up. In eight studies, the process of selection of participants was not clearly explained. In nine no reference was made to refusals or losses and in sixteen studies, although they have been quantified, there was no discussion about those profiles and the way they could have affected the results. With respect to case-control studies, there were also limitations on the description of the selection of participants and refusals in three studies. In four there was a clear possibility of recall bias. In ecological studies, in addition to limitations inherent to the design and use of secondary data, we detected the possibility of ecological fallacy in five of them and no verification of addresses of cases in six studies. Regarding the limitations of the analyses, only thirteen studies did some kind of control of confounding. Except for two, none of the others described the criteria used for categorizing continuous variables. In ten the strength of the associations was not considered, and only statistical significance was analyzed. In six studies the normality of the outcome variable was not checked, although this was necessary for the statistical tests applied. Specifically for ecological studies, seven did not take into account the possible spatial dependence of data. The main limitations of data presentation were related to the non-description of results for variables that were not statistically significant. In addition, several variables had to be excluded because it was not possible to understand the text description or the analyses performed. In general, there were no differences in the quality of published as compared to unpublished studies. The limitations and susceptibilities to bias identified in both groups were of the same types and occurred at a similar frequency. Table S1 describes the characteristics, susceptibilities to bias and limitations of each study reviewed. The presence of publication bias was analyzed only for four variables since there were not enough publications to discuss its possible influence on the other associations reported. According to the methodology used, no evidence of publication bias was identified for associations regarding gender. In Egger' s test, the p-value (one-tailed) was 0. 34 and the funnel plot was apparently symmetrical (Figure 9a). The same occurred for age (p = 0. 16 in Egger' s test) (Figure 9b). For the presence of dogs in the household, however, Egger' s test identified the possibility of publication bias (p = 0. 042). The analysis of the funnel plot showed that it was slightly asymmetrical (Figure 9c); it was then necessary to impute three studies using the “Durval and Tweedie' s Trim and Fill” method to obtain symmetry. The new OR estimated was slightly lower (1. 17; 1. 01–1. 36) but remained statistically significant. Finally, the Egger' s test was not significant for the presence of birds (p = 0. 06), but the funnel plot was not symmetrical; three studies under “Durval and Tweedie' s Trim and Fill” method were imputed in order to obtain symmetry. The recomputed OR was stronger but the association was on the same direction (0. 87; 0. 65–1. 16) and non-significant (Figure 9d).
  This review has brought to light several aspects relating to factors associated with L. infantum infection in humans in the Americas, as well as limitations in the literature consulted and gaps in the existing knowledge. For the variable gender we verified that in studies with clinical cases and in studies that used LST, male gender was positively associated with VL. However, with serological tests we verified an association in the opposite direction. Despite the fact that only the first two were statistically significant, the observation of the measures of association and the statistically significant difference in the Q test show that the difference between groups was consistent. In five out of six studies with serological tests, LST were also performed, that is, subjects were the same. Previous studies argued that hormonal or immune issues could affect the progress of infection [76], [77], and male subjects would be more likely to become symptomatic. Such assumptions could justify the results of studies with symptomatic cases; however this would not explain the difference identified between types of diagnostic methods for detecting asymptomatic infection. In a study in Teresina, Costa et al. [78] suggested that infection with L. infantum more often affects men because they are more exposed to vector bites, since they commonly do not wear shirts due to the high temperatures in that region of Brazil. This can lead to greater exposure of the body surface to the vector. Another postulated explanation for the association between male sex and infection is that men engage more frequently in household tasks or leisure-time activities outdoors, especially after normal working hours, during the peak period of vector activity [40]. For age, it became clear that children are less likely to receive a positive diagnosis of asymptomatic infection. Since LST are indicative of infections that have occurred throughout a subject' s entire life [79] and serological tests may remain positive even long after the infection [14], results are within what would be expected. One should also take into account whether epidemiological or behavioral factors could be associated with identified patterns in addition to such characteristics of the tests. Results in opposite directions and with strong odds ratios verified in both case-control studies analyzed reveal that children are more prone to develop clinical disease, despite being less likely to get infected. This fact may be verified when one observes the number of symptomatic cases routinely notified by healthcare services [62]. The presence of dogs in the household was positively associated with VL. Among the variables subjected to meta-analysis, this was the one with the lowest heterogeneity. Besides the fact that the dog is considered the most important reservoir of the infection in urban settings [8], its presence is positively correlated with vector abundance, potentially increasing the risk of transmission [80]. Furthermore, even without the possibility of performing meta-analysis, ecological studies showed consistent evidence that dog seropositivity is associated with human VL. This emphasizes the relevance of dogs at least as a marker of the occurrence of visceral leishmaniasis among humans. Regarding the presence of chickens and birds in households, although there is evidence that they might have a role in attracting Phlebotomine sand flies [81], [82], according to the available evidence their presence could not be incriminated as a risk factor for VL. Unlike dogs, chickens and birds are not reservoirs of L. infantum [83] and they might even divert sandflies attention. Indeed the summary measure obtained shows that a protective role of the presence of such animals cannot be discarded, as already discussed in the literature [84]. With respect to the variables for which no subgroup analysis was performed, malnutrition decreased the likelihood of positive results, possibly due to the immunological techniques employed for the diagnosis. It should be noted, however, that these results do not exclude the role of malnutrition in increasing the risk of developing clinical disease, as already shown in other publications [85], [86]. As for the prior occurrence of VL in relatives or neighbors and its relationship with an increase in positivity, the association was stronger for the first one, the same pattern as shown in a study conducted by D' Oliveira et al. [50]. Since most studies considered as relatives only those who lived in the same household, the focal nature of the disease could explain the data presented. However, genetic factors that may be associated with the infection should not be disregarded. With respect to socioeconomic issues, although the strength of evidence was variable depending on the factor analyzed and the number of studies being small, the set of data presented shows that there is a close relationship between visceral leishmaniasis and poor living conditions, lack of basic urban infrastructure services and low levels of education. Such relationship between low socioeconomic status and the risk of VL could be explained to the extent that poor economic conditions are associated with greater abundance of vectors, with the lack of responsible pet ownership practices and with the location of houses in peripheral areas of the cities where vegetation density is favorable for the presence of vectors and, perhaps, of sylvatic reservoirs [61], [80], [87], [88]. Finally, albeit in a small number of studies, variables relating to features of the environment and vegetation (evaluated through the NDVI) demonstrated that visceral leishmaniasis occurs more frequently in areas where the level of urbanization is lower and where vegetation is more abundant, probably creating adequate habitats for breeding of vector population [89]. Although quality analysis performed in this review also has limitations arising from the fact that there is no single standardized tool recommended to evaluate observational studies [25], in particular regarding specific issues of ecological studies, several limitations and susceptibility to bias could be identified in the studies analyzed. Even though some of those might be considered inevitable, such as the lack of quality of secondary data used in ecological studies, many other limitations and biases could have been avoided if researchers had adopted the correct procedures for selecting participants, measuring variables and analyzing data, and also had they used more standardized, appropriate and transparent ways of reporting their studies. Based on data presented, the following should be considered in future studies: a) strategies to minimize refusals or losses should be strengthened, as well as other possible design-specific biases identified; b) standardized procedures to select participants should be adopted, considering the need for this selection to be random and to take into account a sample size that is appropriate so that the study has enough statistical power; c) combinations of diagnostic tests should be used so that results may be more valid and reliable; d) control for confounding should be performed and interactions between variables should be analyzed; e) analyses with continuous data in their original form should be performed, considering their advantages [90] and in cases where stratifications prove to be necessary, these should be explained and justified in terms of analysis and practical applicability and not only aiming to achieve statistical significance; f) studies should use correct statistical procedures which extend beyond the p-value, also discussing the strength and direction of associations; g) studies should use the STROBE statement so that results are better described, clearer and more standardized. A number of limitations in the present study deserve mention. Considering that control for confounding was performed in few studies and considering different factors, there may be a large amount of residual confounding in the data summarized in this review. This could mean that the strength of associations might be overestimated. The same overestimation might also have occurred due to the decision to use odds ratio as a measure of association [91], even if it was necessary so that studies which provided only p-values and sample sizes could be used, as well as for the inclusion of results from logistic regression models. Although results from analyses of publication bias showed no consistent evidence of its occurrence, it should be noted that such analyses were limited considering the existing heterogeneity among the various studies results. Furthermore, they could not be performed for most variables. Despite the fact that an extensive search for theses and dissertations was carried out, we did not include studies published in annals of scientific conferences. In addition, for some of the studies included there was significant loss of information on the association for certain variables because non-significant results were not described. Finally, one should consider the low power of some procedures carried out, as well as the limitations imposed by the fact that it was impossible to perform subgroup analyses for most variables. This caused many summary measures to be obtained for heterogeneous data. Such procedures were followed in order to avoid limited and biased analyses based on statistical significance only, such as ‘vote counting’, which we also tried to avoid when discussing the strength and direction of associations even when data allowed theoretical discussions only [28], [92]. The findings of the present review might influence and improve the design of VL control strategies. For instance, the presence of positive dogs might be a useful indicator for monitoring the force of transmission to humans and thus, areas with high canine prevalence or incidence should be prioritized when delivering interventions. Also concerning the reservoir, it should be stressed the importance of policies to promote responsible dog ownership. Additionally, identification of areas at higher risk for prioritizing intervention should take into account the abundance of green vegetation as well as the prior occurrence of VL cases. Since children are more prone to illness and they have the potential to act as information multipliers [93], they might be the focus of attention in health education initiatives. The results related to socioeconomic conditions showed that inadequate urban infrastructure should also be considered a target for interventions against VL. It is of paramount importance to emphasize the need for the formulation of public policies that will improve living conditions and access to education for the population as a whole, since visceral leishmaniasis is just one among other diseases that are rooted into scarcity and poverty [94]. Our work is the first systematic review about factors associated with visceral leishmaniasis in Latin America. Several relevant aspects that help understand the disease dynamics were clarified and new questions were raised. Patterns identified for all variables, as well as the reason for greater chance of infection among adults and greater likelihood of falling ill among children or reasons why the type of diagnostic tests modified the direction of association between gender and asymptomatic infection should be further evaluated. Future studies should investigate whether these patterns are consistent and how subjects' immune response and those characteristics of the tests employed could explain the observed data. With respect to variables about which there is little or inconsistent knowledge, such as subject' s level of knowledge about the disease, ethnic group, backyard conditions and the presence of other animals, especially synanthropic and chickens, future studies should build upon the available information. Additionally, the following should also be encouraged: expanding the use of high definition satellite imaging, taking several environmental aspects into account, from backyard features to broader levels, such as those studied by Franke et al. [73], as well as the association with abundance of Phlebotomine sand flies and with the incidence of infections in human beings. Finally, it is also crucial to conduct research in other geographic areas in Brazil and in other Latin American countries where the disease is endemic. More cohort studies are required and the quality of projects, analyses and publications needs to improve dramatically.
summary: Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis. In the Americas, VL is a zoonosis caused by the protozoan parasite Leishmania infantum, an intracellular micro-organism transmitted to humans through the bite of female phlebotomine sand flies. The domestic dog is considered the main reservoir and human beings are dead-end hosts. The disease occurs in 12 countries of the New World and Brazil accounts for 90% of the reported cases. The factors associated with infection are only partially understood, making harder understanding the disease dynamics and the development of effective and well-targeted control measures. Our work is the first systematic review with meta-analysis about factors associated with human visceral leishmaniasis in the Americas. It provides an organization of the available information, the description of more accurate data on the role of each of the factors studied, the evaluation of possible causes of heterogeneity in the results of the studies as well as the analysis of the quality of publications and limitations in the existing knowledge. The results of this study might be useful in the definition of which and where target interventions should be delivered, and for guiding researchers towards the development of future studies of better methodological quality.
keywords: medicine
  social epidemiology
  infectious diseases
  survey methods
  infectious disease epidemiology
  epidemiology
  leishmaniasis
  neglected tropical diseases
  spatial epidemiology
  biology
  epidemiological methods
  infectious disease control
  zoology
  parasitology
title: Factors Associated with Visceral Leishmaniasis in the Americas: A Systematic Review and Meta-Analysis