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dcyphr | Spatial and temporal dynamics of pathogenic Leptospira in surface waters from the urban slum environment

Abstract

Leptospirosis, a widespread zoonotic disease, is an important health problem in slums. But, there is little understanding of the dynamics of Leptospira transmission in slums. In this study, the researchers sampled water in a Brazilian urban slum to study the dynamics of Leptospira. They collected surface water during dry, intermediate, and rainy seasons and quantified the number of Leptospira in these samples using qPCR. They also identified factors that explained differences in the presence and amount of Leptospira in different seasons. In 335 sewage and 250 standing water samples, Leptospira was detected in 36% and 34% of the samples respectively. The probability of finding Leptospira was higher in sewage samples in the rainy season than the dry season (47.2% and 12.5% respectively). Predictors of Leptospira being found in these samples included type of water, elevation, time of day, and season. These findings show that Leptospira are prevalent in slum communities. Seasonal increases in Leptospira may explain the timing of leptospirosis outbreaks. Public health interventions should consider these dynamics of Leptospira in water to decrease the prevalence of the disease.

Aims

In this study, the researchers wanted to understand what kinds of factors influenced the presence of Leptospira in different kinds of water in a Brazilian urban slum.

Introduction

Leptospirosis is a zoonotic disease that causes over 1 million cases and 50,000 deaths each year. Symptoms range from mild and flu-like to severe complications that can cause death. Leptospira, the bacteria that cause leptospirosis, infect the kidneys of several kinds of mammals and are discharged into the environment through urine. Leptospirosis is transmitted through the environment. Human infection happens through contact between contaminated water and open wounds or mucous membranes in the eyes and mouth. Little is understood about the abundance and distribution of Leptospira in surface waters that are a source of transmission. Environmental factors that influence the risk of infection are also not well-understood.
Leptospirosis is an emerging public health problem in tropical and subtropical impoverished slum communities. Inadequate sanitation promotes the presence of rodents which are major carriers of Leptospira. 865 million people reside in urban slums, a number which will increase and expose more people to leptospirosis.
Exposure to contaminated water is a major risk factor for leptospirosis. Changes in the climate leading to more exposure to water is an important factor for leptospirosis transmission. Seasonal rainfall and flooding, and extreme weather cause an increase in leptospirosis outbreaks. Another factor is how close households are to open sewers, which increases contact with sewage, flooding water, and runoff which increases risk of infection.
The abundance and distribution of Leptospira in surface waters is not well-understood. In this study, the researchers try to provide an understanding on the presence and concentration in a Brazilian slum at high-risk for leptospirosis, as well as factors influencing transmission. The researchers sampled surface waters from this Brazilian slum with high infection rates, especially during the rainy season. 585 samples were collected in dry, intermediate, and rainy seasons. Leptospira presence was quantified using qPCR. Factors that explained differences in the presence and location of Leptospira were also identified.

Methods

Study Site
The study was conducted in Pau da Lima in Salvador, Brazil. This slum community has inadequate sanitation infrastructure that leads to frequent flooding during the rainy season. Salvador has a tropical rainforest climate. April to July is considered as the rainy season.
Sampling design and collection
One of the valleys in the Pau da Lima community was selected for a survey of surface waters. The sampling strategy was designed to collect 672 water samples from three categories of sample sites. These sampling sites varied based on elevation (valley top, middle, and bottom) and season (rainy, intermediate, and dry). Fourteen paired sampling sites (a total of 28 sites) were selected along a section of an open sewer that flows from the top to the bottom of the valley. At each of the 28 sites, samples were collected from both the sewer and standing water close to the sewer.
Samples were collected in July 2011, November 2011, and January 2012. These months were selected based on average monthly rainfall. Within each sampling period, samples were collected at each sampling site 3 days per week in the morning and afternoon.
Quantification of Leptospira DNA in surface water
DNA was extracted from the samples using centrifugation and an extraction kit. Leptospira were quantified using qPCR targeting the lipl32 gene.
Data treatment
Positive qPCR samples were included in the analysis.
Statistical analysis
Logistic and linear mathematical models were used to analyze the occurrence of a positive qPCR and the concentration of Leptospira respectively. Factors including sampling location, week, day within week, surface water type, season, period of the day, and elevation were accounted for in the models. Interactions between these factors were tested.
Leptospirosis incidence
Severe leptospirosis infections were identified from a surveillance program at the state infectious diseases hospital.

Results

Rainfall pattern and leptospirosis incidence
July 2011 was the intermediate season, November 2011 was the rainy season, and Janurary 2012 was the dry season. 101 severe leptospirosis cases were reported during the study period. The number of cases peaked in the rainy season, and less cases were reported in the dry and intermediate seasons.
Specificity of Leptospira qPCR assay
The researchers verified whether the qPCR used in the analysis was detecting infectious Leptospira and nothing else. Samples with positive qPCR were randomly selected, and their DNA was sequenced. All sequenced samples showed high similarity to the targeted gene sequence, indicating that the qPCR specifically detects infectious Leptospira.
Distribution and quantification of Leptospira in surface waters
585 samples (335 sewage and 250 standing water) were collected and tested by qPCR to quantify Leptospira. Among these samples, 236 were positive for Leptospira DNA, with 36% and 46% of sewage and standing water samples being positive respectively. Sewage had the most positive samples in the rainy season and the least positive samples in the dry season. More sewage samples were positive at the bottom of the valley than upper areas of the valley. The number of positive samples in standing water was more stable across seasons and elevations. Standing water was found less in the middle of the valley and during the dry season. Samples collected in the morning and afternoon had similar rates of positivity.
Overall, concentrations of Leptospira in surface water were low and did not differ between types of water, seasons, elevations, and periods of collection.
Spatial and temporal predictors of Leptospira DNA presence and concentration
The probability of finding a positive sample was higher in the bottom of the valley than the middle or the top.
Two significant kinds of interactions existed between factors analyzed for their influence on Leptospira concentration: between season and type of water, and between season and period of collection. Analysis of the interaction between season and type of water showed the sewage samples in the rainy and intermediate seasons were more likely to be positive than in the dry season.  In the rainy seasons, sewage samples had higher probabilities of being positive than standing water samples. In the analysis between season and period of collection, rainy season samples had higher probabilities of being positive in the morning than the afternoon. These results suggest that elevation, season, type of water, and period of collection influence the probability of finding Leptospira in surface waters in the slum.
In the rainy season, positive samples had higher concentrations of Leptospira compared to the dry season. Samples collected in the morning had higher concentrations compared to those in the afternoon. However, these differences were small.

Discussion

The researchers found that Leptospira are prevalent in sewage and standing water, but in low concentrations. The results indicate that Leptospira prevalence differs in different locations and seasons, being more prevalent in lower areas of the valley and in the rainy season.
The probability of finding positive Leptospira samples had a seasonal pattern. More samples were positive in the rainy season than the dry season. This may be due to many factors including the washing away of Leptospira from the soil due to rain, dissolving Leptospira biofilms, or increased survival due to higher oxygen levels and diluted toxic sewage compounds. These factors need further studies. This seasonal pattern is consistent with other studies, and has been reported in other settings with heavy rainfall causing increased contact with contaminated water. This contact has been suggested as a main source of leptospirosis outbreaks.
Both sewage and standing water samples contained Leptospira. The results show that in rainy periods, sewers and overflow are drivers of infection. In the dry season, standing water samples showed more positivity than sewage. These results suggest that sewage and standing water are distinct reservoirs for Leptospira, and have different mechanisms that influence the presence of Leptospira. Previous studies have pointed to puddles, which are abundant in slums, as sources of contact with Leptospira-contamined water. Public health authorities should consider standing water, along with sewage, when designing interventions for reducing the incidence of leptospirosis.
Positivity in samples was higher at the bottom of the valley, which agrees with previous studies. This may result from lower elevations having higher flooding risk during rainfall events. Open sewers and other forms of drainage collect at the bottom of the valley, so surface water may be more contaminated at lower elevations.
The concentration of Leptospira in all samples was relatively low. However, these low concentrations contrasted with high infection rates in the community. The concentrations found in the study are far less than reported concentrations necessary for infection. The researchers suggest that there may be mechanisms where the concentration necessary for infection decreases, such as the disruption of skin barriers. More studies are necessary to confirm this hypothesis.
The study site has similar characteristics to other communities across the world. These results may help to understand geographic and seasonal differences in leptospirosis epidemics. The results of this study are important to implement efficient interventions to reduce leptospirosis worldwide.