Leptospirosis is a bacterial disease that has emerged as a major health problem in the developing world [1]. The disease is caused by a spirochete from the genus Leptospira, which colonizes the kidney of a wide range of mammals [2]. Human infection occurs after direct contact with an infected animal reservoir, or water and soil contaminated with their urine [1].Infection produces a broad spectrum of clinical manifestations, which may lead from an asymptomatic and mild self-limiting febrile illness to severe disease forms with high case fatality [3]. Leptospirosis has traditionally been a sporadic rural-based disease associated with occupational risk groups such as subsistence farmers [3]. However, changes in human demography during the last 50 years have raised awareness of the emergence of leptospirosis as an urban health problem [4].

Rapid urbanization and urban poverty have led to the dramatic growth of slum settlements throughout low and middle-income countries [5]. To date one billion of the world's population reside in urban slums; this population continues to expand at rates of 10% per year [5]. As a consequence of poor sanitation in these communities, slum residents are increasingly exposed and are at risk of acquiring water and animal-borne diseases [6], [7] such as leptospirosis [1], [4], [8]. In slum settings, endemic transmission of leptospirosis is largely due to circulation of a single serogroup, L. interrogans serogroup Icterohaemorrhagiae, [4], [7], [9], [10] for which the domestic rat is the maintenance host [4], [11]. In tropical urban environments, increased transmission and seasonal outbreaks occur during periods of heavy rainfall and flooding [4], [12], [13]. Furthermore extreme climatic events such as monsoons, typhoons and hurricanes have precipitated urban epidemics, as exemplified by the Mumbai outbreak in 2005 [14] and more recently, in the Philippines in 2009 [15] and Australia in 2011 [16].

Moreover, leptospirosis imparts a large disease burden as the cause of life-threatening infection among slum dwellers. In Brazil, more than 10,000 cases of leptospirosis are reported each year [17], the large majority of whom are residents of urban slums and require hospitalization [18] for severe complications of Weil's disease and leptospirosis-associated pulmonary hemorrhage syndrome (LPHS) [1], [7], [19]. Overall case fatality is >10% among reported cases from Brazil [4] and >50–70% for cases that develop LPHS [7], [19]. However, severe disease represents a small fraction of the overall disease burden [20], [21] and to date, prospective studies have not been performed to identify the risk of leptospirosis among slum dwellers.

Investigations of urban leptospirosis, which have used ecological [4], [12], [22], cross-sectional [9] and case-control study designs [23], [24], have identified infrastructure deficiencies in the environment where slum dwellers reside as risk factors for acquiring leptospirosis and anti-Leptospira antibodies. For example, high risk of Leptospira transmission has been found to be associated with proximity of residence to open sewers and accumulated refuse, flood-risk areas, and areas with high rat infestation [9], [12], [22]–[26]. In addition to environmental features, low socioeconomic status among slum residents contributes to the risk of leptospirosis [12], [22] and anti-Leptospira antibodies [9]. However these investigations are limited by the ecological study design, or in the case of cross-sectional surveys, the use of anti-Leptospira antibodies, which are detected in individuals up to four or more years after infection [27], [28]. To date, there are no studies that have attempted to evaluate prospectively the risk factors for leptospirosis among urban slum populations.

We previously reported the findings of a large seroprevalence survey [9] of a slum community in Salvador, a city in Northeast Brazil where 33% of the population reside in slum settlements [29] and seasonal rainfall-associated epidemics of leptospirosis occur each year [4], [23]. This study found that a large proportion (15.4%) of slum inhabitants had anti-Leptospira antibodies, suggesting that in addition to high rates of infection, repeated exposures with the Leptospira agent may be occurring in this high-risk population. Herein, we report findings from a prospective investigation of this urban slum population to determine the risk of Leptospira infection and identify risk associations for infection.

The findings of this large prospective investigation identified high rates of Leptospira infection among the study urban slum population, with more than 3% of the residents demonstrating serologic evidence of infection over a mean follow-up period of approximately one year. A single L. interrogans serogroup, serogroup Icterohaemorrhagiae, was the presumptive infecting agent, since highest agglutinating antibody titers were observed in 98% of participants with serologically-confirmed infection. Slum residents had an overall high risk for a repeat exposure and infection with the same agent. Furthermore, we identified that there are distinct risk factors for acquiring primary and secondary infection, suggesting that there exist sub-populations among slum residents who are repeatedly infected with leptospirosis.

These findings highlight the potential large and unrecognized burden of leptospirosis in urban slum settlements. There have been no comparable studies performed in slum settings that have followed large numbers of community-based participants and prospectively ascertained outcomes with standard serologic methods. A longitudinal study had been performed in Iquitos, Peru and found that annual incidence of Leptospira seroconversion was 288 per 1,000 persons based on IgM ELISA seroconversion during follow-up of 158 urban slum residents [33]. The much higher incidence observed in Iquitos may be due to the use of the IgM ELISA rather than the standard MAT method [33], which is more specific in detecting exposure to pathogenic leptospires [1], [3]. Alternatively, the difference may reflect differences in the frequency of infection among slum settlements that have distinct ecological and socio-economic characteristics.

A key knowledge gap in leptospirosis centers on the natural history of the disease and specifically, the proportion of infections that progress to develop disease and severe outcomes in the setting of high endemic transmission [1], [3]. Leptospirosis cases were not identified among the sample of 2,003 participants who participated in the cohort study to obtain direct estimates of the infection-to-severe-disease ratio. However, active hospital-based surveillance of the 12,651 community members that were identified during the baseline census found that the annual incidence of severe leptospirosis was 19.8 cases per 100,000 population at the study site during the cohort follow-up period. Comparison of this estimate of the severe disease incidence and the infection rate among cohort participants suggests that the infection-to-disease ratio may be as high as 191∶1 (95% CI, 82–542∶1). Although cohort participants with documented seroconversion did not report being hospitalized or visiting an ambulatory clinic for leptospirosis during follow-up, it is plausible that a significant proportion developed sub-clinical illness or clinical disease, which would not be identified and diagnosed as leptospirosis unless cases developed classic severe manifestations [1]. The high infection-to-disease ratio suggests that like dengue and other causes of acute fever in tropical urban environments, the burden of leptospirosis is under-recognized and significantly greater than reflected by reporting of severe cases.

The study findings indicate that the same L. interrogans serovar causes asymptomatic and sub-clinical infections as well as severe disease in this urban slum population. Among cohort participants with documented seroconversion, 98% had highest MAT titers that were directed against L. interrogans serovar Copenhageni, indicating that the Icterohaemorrhagiae serogroup was the infecting agent. We have observed that during long-term hospital-based surveillance in Salvador [4], [34], [35] and the study site community, >95% of the severe leptospirosis cases had highest MAT titers directed exclusively against the same serovar. L. interrogans serovar Copenhageni has been the sole serovar from serogroup Icterohaemorrhagiae to be isolated from this patient population during long-term surveillance [4], [7], as well as rat populations from the study community [36] and the city of Salvador [10], [37]. Together these findings indicate that transmission of leptospirosis is due to a single circulating serovar and provides additional evidence that Rattus norvegicus, the most common host of L. interrogans serovar Copenhageni [3], [10], is the principal reservoir in this urban slum setting. Moreover, our findings raise an important question with respect to what specific factors influence disease progression and the diverse range of clinical outcomes after infection with a single serovar agent. These factors may relate to strain-specific differences within the serovar that contribute to the strain's virulence, or alternatively, host-specific susceptibility or resistance factors and types of environmental exposures that contribute to the inoculum dose during infection [1], [3].

Re-infection was a frequent event among cohort participants during follow-up and raises the issue of whether natural infection confers immunity to a subsequent infection with a homologous serovar. Although there is clear evidence that immunization with live attenuated [38] and killed leptospires protects experimental animals against lethal infection [3], naturally-acquired immunity to re-infection in humans is poorly understood due to the limited number of prospective studies in well-characterized populations. A study from rural Andaman Islands used similar serologic criteria as employed in this study to prospectively identify Leptospira infection among school children and found that that primary infection rates were higher than secondary re-infection rates [39]. Additionally, the study observed a non-significant association between increased morbidity during follow-up and seronegative status during the baseline survey [39]. Although those findings are suggestive that a previous infection may protect against a subsequent infection, the conclusions were limited by the small numbers of clinical cases identified and potential confounding due to multiple circulating serovars.

Our study found that in an urban setting of transmission of a single serovar agent, prior exposure and infection did not confer complete protection against a subsequent serologically-ascertained, asymptomatic or sub-clinical infection with the same serovar. However, we could not determine whether prior infection protects an individual against developing clinical disease during subsequent re-infection since leptospirosis cases were not identified among cohort participants, nor could we evaluate the temporal relationship between initial infection and subsequent re-infection events due to the short follow-up period. Further prospective investigation is therefore needed to elucidate this question, which has major implications for development of an effective vaccine for leptospirosis in humans.

Urban slum residents who acquired a primary and secondary infection had some similar risk associations in common, yet also had important differences in infection rates and type of risk factors. Male gender and low socioeconomic status were independent risk factors for primary and secondary infections, although the associations with secondary infection were non-significant. Although male gender has not been identified as a risk factor for Leptospira infection in cohort studies performed in rural or mixed settings [33], [40], [41], males have a significantly higher risk for leptospirosis and anti-Leptospira antibodies in population-based surveillance [4], [23], [24] and seroprevalence surveys [9], [30], respectively, in Salvador and other urban settings [42]–[44]. We found that for each one-dollar increase in the daily per capita household income, the odds of primary and secondary infection decreased by 46% and 48%, respectively. Poverty and low socioeconomic status may contribute to infection risk through diverse mechanisms that include psychosocial processes that promote risky behaviors and exposures with a contaminated environment, the limited use of protective clothing against abrasions that facilitate entry of the Leptospira spirochete [21], lack of access to amenities and social support [45] and inadequate household sanitation conditions. A previous study in the same area demonstrated that adolescents and individuals who did not complete primary school had lower levels of knowledge and practices regarding leptospirosis [46]. Our prospective study confirms the findings of a seroprevalence survey performed in the same community [9] that relatively small differences in socioeconomic level, independent of poor environment, influence the risk for leptospirosis in slum populations characterized by overall high levels of absolute poverty.

The secondary infection rate was more than twice that of the primary infection rate (RR: 2.31; 95% CI: 1.30–4.10) among cohort participants, indicating that seropositive status at the baseline survey was a marker for increased risk for infection. In contrast to primary infection, individuals who had 15–34 years of age had significantly higher risk for acquiring a secondary infection (Table 3 and Figure 1), suggesting that a sub-population of young adults are repeatedly infected with pathogenic Leptospira. Furthermore we found that residence in proximity of an open sewer was significantly associated with an increased risk of primary infection and not secondary infection. It seems plausible that open sewers are a risk factor for primary infection, but the magnitude of this risk association was lower than that for secondary infection and not detected in this study. Together these finding suggest that there are distinct environmental settings and behavioral factors that contribute to repeat exposures.

Seasonal flooding is a frequent occurrence in slum communities in Salvador and especially for households situated on the poorest land quality at the bottom of valleys. Ganoza and colleagues found that environmental surface water from urban slums in Peru contained high concentrations of L. interrogans serovar Icterohaemorrhagiae [47]. Previous cross-sectional and case-control studies found that residence in flood-prone areas and in proximity to open sewer and rainwater drainage systems were associated with increased risk for anti-Leptospira antibodies and leptospirosis [9], [12], [23], [26]. The findings of this prospective study suggest that these infrastructure deficiencies of slum settlements also serve as transmission sources for repeated exposures to the Leptospira pathogen. Furthermore the findings demonstrate that adolescents and young adults are the primary risk group for repeated exposures and re-infection and indicate the need for specific interventions that target this high risk group.

This study has several limitations which need to be considered. The proportion of females and younger participants among study participants was greater than among non-participants. Infection rates that were adjusted for the age and gender distribution of the eligible population did not differ from crude rates, indicating that differences between enrolled and non-enrolled participants may have not introduced a significant bias in rate estimates. Among enrolled participants, 21% did not complete follow up due primarily to out-migration. This sub-group had a higher proportion of males, was better educated and had a higher income in comparison to those who completed follow up. Although we could not fully address the potential for bias, predictive factors for loss-to-follow-up were included in our modeling approach and the estimates for the risk associations may therefore be valid approximations.

In addition, our findings may not be broadly generalizable since the study was performed in a single slum community in Brazil. The incidence of Leptospira infection and risk associations identified in our study is expected to vary given the differences in underlying conditions of social deprivation, environmental degradation and climate where urban slum communities are situated. However the study site is a typical slum community in the city of Salvador and Brazil, where respectively, 33% [29] and 28% [5] of the population inhabit in such settlements. Furthermore, a large proportion of the one billion inhabitants of urban slums, as defined by the UN-HABITAT [5], reside in communities that have the same features of poverty, climate, poor environment and inadequate access to sanitation as found in our study site. The findings of this study may therefore be highly relevant to the situation of leptospirosis in urban slum settlements in developing countries and across tropical regions.

Furthermore, the study offers insights on the approaches needed to effectively address this neglected disease as the world's population of slum dwellers doubles to two billion by 2020 [48]. The study provides the first prospective evidence to support the assertion that defined infrastructure deficiencies in slum communities serve as transmission sources for leptospirosis. Removal of these sources, through implementation of adequate closed sewage and drainage systems, should therefore be a public health priority. Our findings also highlight the importance of adolescents and young adults as a risk group for spill-over infections. Efforts need to be made to identify and target through intervention the risky behaviors in this age group that promote recurring exposures with environmental contamination. Similarly, further work is required to identify the processes by which the social gradient of status influences unequal health outcomes within slum populations living in absolute poverty. By elucidating such mechanisms, we may not only identify effective prevention for leptospirosis, but may also identify common processes and interventions for the large range of communicable and non-communicable diseases that affect marginalized urban communities.