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1.  Active and latent tuberculosis in Brazilian correctional facilities: a cross-sectional study 
Tuberculosis (TB) rates among prisoners are more than 20 times that of the general population in Brazil, yet there are limited data available to facilitate the development of effective interventions in this high-transmission setting. We aimed to assess risk factors for TB infection and evaluate the yield of mass screening for active disease among inmates.
We administered a questionnaire and tuberculin skin test (TST) to a population-based sample of inmates from 12 prisons in Central-West Brazil and collected sera for HIV testing and two sputum samples for smear microscopy and culture from participants reporting a cough of any duration. Hierarchical Poisson regression models were used to evaluate factors associated with latent tuberculosis infection (LTBI).
We recruited 3,380 inmates, of which 2,861 (84.6%) were males from 8 prisons, and 519 (15.4%) were females from 4 prisons. Among the 1,020 (30%) subjects who reported a cough, we obtained sputum from 691 (68%) and identified 31 cases of active TB for a point prevalence of 917 (95% CI, 623–1302) per 100,000 prisoners. Evaluation of the two sputum smear samples failed to identify 74% of the TB cases, and 29% of the cases reported less than 2 weeks of symptoms. Obtaining a second culture identified an additional 7 (24%) cases. The prevalences of LTBI were 22.5% and 11.7% for male and female prisoners, respectively and duration of incarceration (in years) was associated with LTBI in male and female in the multivariable model (1.04, 95% CI, 1.01-1.07 and 1.34, 95% CI, 1.06-1.70, respectively). The prevalence of LTBI is 8.6% among newly incarcerated inmates, among whom LTBI prevalence significantly increased by 5% with each year of incarceration.
Although the overall LTBI prevalence among inmates in Central-West Brazil is low, tuberculosis incidence is high (>1,800/100,00), likely due to the high force of infection among a largely susceptible inmate population. Efforts to reduce transmission in prisons may require mass screening for active TB, utilizing sputum culture in case-detection protocols.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-015-0764-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4307675  PMID: 25608746
Tuberculosis; Prisoners; TST; Infection; Active case detection; Screening cross-sectional study; Brazil; Epidemiology
2.  Infections by Leptospira interrogans, Seoul Virus, and Bartonella spp. Among Norway Rats (Rattus norvegicus) from the Urban Slum Environment in Brazil 
Norway rats (Rattus norvegicus) are reservoir hosts for zoonotic pathogens that cause significant morbidity and mortality in humans. Studies evaluating the prevalence of zoonotic pathogens in tropical Norway rat populations are rare, and data on co-infection with multiple pathogens are nonexistent. Herein, we describe the prevalence of leptospiral carriage, Seoul virus (SEOV), and Bartonella spp. infection independently, in addition to the rates of co-infection among urban, slum-dwelling Norway rats in Salvador, Brazil, trapped during the rainy season from June to August of 2010. These data were complemented with previously unpublished Leptospira and SEOV prevalence information collected in 1998. Immunofluorescence staining of kidney impressions was used to identify Leptospira interrogans in 2010, whereas isolation was used in 1998, and western blotting was used to detect SEOV antibodies in 2010, whereas enzyme-linked immunosorbent assay (ELISA) was used in 1998: in 2010, Bartonella spp. were isolated from a subsample of rats. The most common pathogen in both years was Leptospira spp. (83%, n=142 in 1998, 63%, n=84 in 2010). SEOV was detected in 18% of individuals in both 1998 and 2010 (n=78 in 1998; n=73 in 2010), and two species of Bartonella were isolated from 5 of 26 rats (19%) tested in 2010. The prevalence of all agents increased significantly with rat mass/age. Acquisition of Leptospira spp. occurred at a younger mass/age than SEOV and Bartonella spp. infection, suggesting differences in the transmission dynamics of these pathogens. These data indicate that Norway rats in Salvador serve as reservoir hosts for all three of these zoonotic pathogens and that the high prevalence of leptospiral carriage in Salvador rats poses a high degree of risk to human health.
PMCID: PMC3880909  PMID: 24359425
Leptospira interrogans; Seoul virus; Bartonella spp.; Norway rats (Rattus norvegicus); Brazil
3.  Influence of Household Rat Infestation on Leptospira Transmission in the Urban Slum Environment 
The Norway rat (Rattus norvegicus) is the principal reservoir for leptospirosis in many urban settings. Few studies have identified markers for rat infestation in slum environments while none have evaluated the association between household rat infestation and Leptospira infection in humans or the use of infestation markers as a predictive model to stratify risk for leptospirosis.
Methodology/Principal Findings
We enrolled a cohort of 2,003 urban slum residents from Salvador, Brazil in 2004, and followed the cohort during four annual serosurveys to identify serologic evidence for Leptospira infection. In 2007, we performed rodent infestation and environmental surveys of 80 case households, in which resided at least one individual with Leptospira infection, and 109 control households. In the case-control study, signs of rodent infestation were identified in 78% and 42% of the households, respectively. Regression modeling identified the presence of R. norvegicus feces (OR, 4.95; 95% CI, 2.13–11.47), rodent burrows (2.80; 1.06–7.36), access to water (2.79; 1.28–6.09), and un-plastered walls (2.71; 1.21–6.04) as independent risk factors associated with Leptospira infection in a household. We developed a predictive model for infection, based on assigning scores to each of the rodent infestation risk factors. Receiver operating characteristic curve analysis found that the prediction score produced a good/excellent fit based on an area under the curve of 0.78 (0.71–0.84).
Our study found that a high proportion of slum households were infested with R. norvegicus and that rat infestation was significantly associated with the risk of Leptospira infection, indicating that high level transmission occurs among slum households. We developed an easily applicable prediction score based on rat infestation markers, which identified households with highest infection risk. The use of the prediction score in community-based screening may therefore be an effective risk stratification strategy for targeting control measures in slum settings of high leptospirosis transmission.
Author Summary
The Norway rat is an important reservoir for urban leptospirosis, a life-threatening zoonotic disease. In urban settings, leptospirosis transmission occurs primarily in the peri-domiciliary environment of the slums. Rodent control is one of the most frequent strategies to prevent leptospirosis, but the identification of domiciles at higher risk of transmission is challenging. We compared households where an individual with evidence of recent leptospirosis infection resided and households where none of the residents had evidence for infection. Houses with evidence of leptospirosis transmission had higher levels of rodent infestation and environmental conditions related to rodents. We propose a new methodology to easily characterize slum households, based on environmental characteristics, at different levels of risk for leptospirosis transmission. The findings of this study indicate that evaluation for rodent infestation intensity and environmental features may be a feasible strategy for targeting augmented control measures for leptospirosis.
PMCID: PMC4256176  PMID: 25474580
4.  A Putative Regulatory Genetic Locus Modulates Virulence in the Pathogen Leptospira interrogans 
Infection and Immunity  2014;82(6):2542-2552.
Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139− mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.
PMCID: PMC4019197  PMID: 24686063
5.  Post-translational Modification of LipL32 during Leptospira interrogans Infection 
Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin.
Methodology/Principal Findings
Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32.
The exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although definitive determination of the role of these PTMs must await further investigations, the reduced immune recognition of a modified LipL32 epitope suggests the intriguing possibility that LipL32 modification represents a novel mechanism of immune evasion within Leptospira.
Author Summary
Leptospirosis, caused by pathogenic Leptospira spp., constitutes an increasing global public health threat. Humans are accidental hosts, and acquire the disease primarily from contact with water sources that have been contaminated with urine from infected animals. Rats are asymptomatic carriers of infection and are critical for disease transmission to humans, particularly in urban slum environments. In this study, investigation of Leptospira directly isolated from the urine of infected rats showed acetylation or tri-methylation of the highly abundant leptospiral lipoprotein, LipL32. In comparison, Leptospira grown in culture did not result in any LipL32 lysine modifications. A synthetic peptide derived from LipL32 that incorporated a tri-methylated lysine modification exhibited less reactivity with serum from leptospirosis patients compared to an unmodified version of the peptide, suggesting LipL32 modifications may alter protein recognition by the immune response. This study reports, for the first time, modification of a Leptospira protein during infection, and suggests these modifications may have a functional consequence that contributes to bacterial persistence during infection.
PMCID: PMC4214626  PMID: 25356675
6.  Forecasting Temporal Dynamics of Cutaneous Leishmaniasis in Northeast Brazil 
Cutaneous leishmaniasis (CL) is a vector-borne disease of increasing importance in northeastern Brazil. It is known that sandflies, which spread the causative parasites, have weather-dependent population dynamics. Routinely-gathered weather data may be useful for anticipating disease risk and planning interventions.
Methodology/Principal Findings
We fit time series models using meteorological covariates to predict CL cases in a rural region of Bahía, Brazil from 1994 to 2004. We used the models to forecast CL cases for the period 2005 to 2008. Models accounting for meteorological predictors reduced mean squared error in one, two, and three month-ahead forecasts by up to 16% relative to forecasts from a null model accounting only for temporal autocorrelation.
These outcomes suggest CL risk in northeastern Brazil might be partially dependent on weather. Responses to forecasted CL epidemics may include bolstering clinical capacity and disease surveillance in at-risk areas. Ecological mechanisms by which weather influences CL risk merit future research attention as public health intervention targets.
Author Summary
Cutaneous leishmaniasis (CL) is a disease resulting from infection by the Leishmania parasites, which humans may acquire when bitten by an infected sandfly. From a public health standpoint, it is important to identify cases early and monitor patients' clinical outcomes because unsuccessfully-treated patients are at risk for severe complications. Since weather conditions affect survival and reproduction of sandflies that transmit Leishmania, routinely-gathered weather and climate data may be useful for anticipating CL outbreaks, bolstering clinical capacity for high-risk periods, and initiating interventions such as active case-finding during these periods to limit disease burden. Here we assessed whether the number of CL cases occurring per month in a rural region of Bahía, Brazil was associated temperature, humidity, precipitation, and El Niño sea surface temperature oscillation patterns observed during preceding seasons. We formulated models that improved accuracy of one, two, and three month-ahead CL predictions by accounting for weather. Forecasts of this nature can contribute to reducing CL burden by informing resource allocation and intervention planning in preparation for epidemics.
PMCID: PMC4214672  PMID: 25356734
7.  Identification of Cell-Binding Adhesins of Leptospira interrogans 
Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic Leptospira species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate L. interrogans sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its L. biflexa homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for L. interrogans. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in L. interrogans infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and vaccines.
Author Summary
Leptospirosis, caused by pathogenic species of the genus Leptospira, is an infectious disease that has emerged as a globally important health problem. Infection can either lead to mild illness or can progress to a severe disease form manifested by jaundice, kidney and liver dysfunction, and widespread blood vessel damage. It is thought that the ability of the bacteria to recognize and bind to human and animal cells is important for Leptospira spp. to cause the disease. Using phage display, we were able to identify bacterial proteins that mediate the binding of the bacteria to host cells. One of the identified proteins, LIC11574, attaches to different types of host cells, and to VE-cadherin, a cell surface protein previously identified as receptor for disease-causing L. interrogans. All bacterial proteins identified were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals. Our findings may be of value in leptospirosis control and prevention, with these bacterial surface proteins as new targets for serodiagnosis and vaccine development.
PMCID: PMC4183468  PMID: 25275630
8.  Urban population genetics of slum-dwelling rats (Rattus norvegicus) in Salvador, Brazil 
Molecular ecology  2013;22(20):10.1111/mec.12455.
Throughout the developing world, urban centers with sprawling slum settlements are rapidly expanding and invading previously forested ecosystems. Slum communities are characterized by untended refuse, open sewers, and overgrown vegetation, which promote rodent infestation. Norway rats (Rattus norvegicus), are reservoirs for epidemic transmission of many zoonotic pathogens of public health importance. Understanding the population ecology of R. norvegicus is essential to formulate effective rodent control strategies, as this knowledge aids estimation of the temporal stability and spatial connectivity of populations. We screened for genetic variation, characterized the population genetic structure, and evaluated the extent and patterns of gene flow in the urban landscape using 17 microsatellite loci in 146 rats from 9 sites in the city of Salvador, Brazil. These sites were divided between three neighborhoods within the city spaced an average of 2.7 km apart. Surprisingly, we detected very little relatedness among animals trapped at the same site and found high levels of genetic diversity, as well as structuring across small geographic distances. Most FST comparisons among sites were statistically significant, including sites <400 m apart. Bayesian analyses grouped the samples in three genetic clusters, each associated with distinct sampling sites from different neighborhoods or valleys within neighborhoods. These data indicate the existence of complex genetic structure in R. norvegicus in Salvador, linked to the heterogeneous urban landscape. Future rodent control measures need to take into account the spatial and temporal linkage of rat populations in Salvador, as revealed by genetic data, to develop informed eradication strategies.
PMCID: PMC3864905  PMID: 24118116
population genetics; urban ecology; Rattus norvegicus; rodent control
9.  Unsuspected Leptospirosis Is a Cause of Acute Febrile Illness in Nicaragua 
Epidemic severe leptospirosis was recognized in Nicaragua in 1995, but unrecognized epidemic and endemic disease remains unstudied.
Methodology/Principal Findings
To determine the burden of and risk factors associated with symptomatic leptospirosis in Nicaragua, we prospectively studied patients presenting with fever at a large teaching hospital. Epidemiologic and clinical features were systematically recorded, and paired sera tested by IgM-ELISA to identify patients with probable and possible acute leptospirosis. Microscopic Agglutination Test and PCR were used to confirm acute leptospirosis. Among 704 patients with paired sera tested by MAT, 44 had acute leptospirosis. Patients with acute leptospirosis were more likely to present during rainy months and to report rural residence and fresh water exposure. The sensitivity of clinical impression and acute-phase IgM detected by ELISA were poor.
Leptospirosis is a common (6.3%) but unrecognized cause of acute febrile illness in Nicaragua. Rapid point-of-care tests to support early diagnosis and treatment as well as tests to support population-based studies to delineate the epidemiology, incidence, and clinical spectrum of leptospirosis, both ideally pathogen-based, are needed.
Author Summary
Leptospirosis, transmitted by pathogenic species of the bacterium Leptospira, is distributed worldwide but infections due to unrecognized epidemic or endemic disease are under-appreciated. We prospectively studied patients ≥2 years of age who presented with acute febrile illness in Nicaragua and systematically collected detailed information about exposures and features of the illness as well as serum and blood to confirm acute infections. Among 704 patients with paired sera tested by MAT, we found acute leptospirosis in 6.3% (44/704). Patients with acute leptospirosis were more likely to present during rainy months and to report rural residence and fresh water exposure. Leptospirosis is a common (6.3%) but unrecognized cause of acute febrile illness in Nicaragua.
PMCID: PMC4109853  PMID: 25058149
10.  Prospective Study of Leptospirosis Transmission in an Urban Slum Community: Role of Poor Environment in Repeated Exposures to the Leptospira Agent 
Leptospirosis has emerged as an urban health problem as slum settlements have rapidly spread worldwide and created conditions for rat-borne transmission. Prospective studies have not been performed to determine the disease burden, identify risk factors for infection and provide information needed to guide interventions in these marginalized communities.
Methodology/Principal Findings
We enrolled and followed a cohort of 2,003 residents from a slum community in the city of Salvador, Brazil. Baseline and one-year serosurveys were performed to identify primary and secondary Leptospira infections, defined as respectively, seroconversion and four-fold rise in microscopic agglutination titers. We used multinomial logistic regression models to evaluate risk exposures for acquiring primary and secondary infection. A total of 51 Leptospira infections were identified among 1,585 (79%) participants who completed the one-year follow-up protocol. The crude infection rate was 37.8 per 1,000 person-years. The secondary infection rate was 2.3 times higher than that of primary infection rate (71.7 and 31.1 infections per 1,000 person-years, respectively). Male gender (OR 2.88; 95% CI 1.40–5.91) and lower per capita household income (OR 0.54; 95% CI, 0.30–0.98 for an increase of $1 per person per day) were independent risk factors for primary infection. In contrast, the 15–34 year age group (OR 10.82, 95% CI 1.38–85.08), and proximity of residence to an open sewer (OR 0.95; 0.91–0.99 for an increase of 1 m distance) were significant risk factors for secondary infection.
This study found that slum residents had high risk (>3% per year) for acquiring a Leptospira infection. Re-infection is a frequent event and occurs in regions of slum settlements that are in proximity to open sewers. Effective prevention of leptospirosis will therefore require interventions that address the infrastructure deficiencies that contribute to repeated exposures among slum inhabitants.
Author Summary
Leptospirosis is a disease that is transmitted by human contact with an environment contaminated with urine from animals, such as rodents, infected by the Leptospira bacteria. Human illness due to these bacteria can be mild, or can have very severe complications. Residents of urban slum settlements are at high risk for this disease, but the specific risk factors for transmission in these settlements are not understood because of the lack of prospective studies in this epidemiological setting. We performed a prospective study in a Brazilian slum community to measure the risk of infection, identify the environmental and social factors that place slum residents at risk for infection, and determine whether some individuals are at risk of repeated infections. We identified a burden of infection with leptospirosis among slum residents, and found that male gender and low income both increase the risk for infection. In addition, a significant proportion of slum residents had a second exposure to leptospirosis and re-infection occurred most frequently among young adults and the poorest members of the slum community who reside in proximity of open sewers. These risk factors are amenable to interventions aimed to reduce the burden that leptospirosis imparts in this high-risk setting.
PMCID: PMC4038618  PMID: 24875389
11.  El Niño Southern Oscillation and Leptospirosis Outbreaks in New Caledonia 
Leptospirosis is an important cause of seasonal outbreaks in New Caledonia and the tropics. Using time series derived from high-quality laboratory-based surveillance from 2000–2012, we evaluated whether climatic factors, including El Niño Southern Oscillation (ENSO) and meteorological conditions allow for the prediction of leptospirosis outbreaks in New Caledonia. We found that La Niña periods are associated with high rainfall, and both of these factors were in turn, temporally associated with outbreaks of leptospirosis. The sea surface temperature in El Niño Box 4 allowed forecasting of leptospirosis outbreaks four months into the future, a time lag allowing public health authorities to increase preparedness. To our knowledge, our observations in New Caledonia are the first demonstration that ENSO has a strong association with leptospirosis. This association should be tested in other regions in the South Pacific, Asia or Latin America where ENSO may drive climate variability and the risk for leptospirosis outbreaks.
Author Summary
The El Niño Southern Oscillation is a major ocean – atmosphere phenomenon that strongly contributes to the timing and intensity of rainfall in the tropical Pacific islands and beyond. As a consequence, it also has a major effect on the number of cases of leptospirosis. By incorporating oceanographic parameters in models, we have been able to predict leptospirosis outbreaks in New Caledonia 4 months in advance. We discuss that this forecasting delay might be used to implement timely interventions prior to the occurrence of outbreaks. Possible interventions include controlling rodent reservoir populations before population growth and maintaining sewage networks or river banks. The major impact of El Niño on the climate of Pacific Islands Countries and Territories suggests that other countries might be able to build similar predictive models.
PMCID: PMC3990495  PMID: 24743322
12.  The impact of Brazil’s Bolsa Família conditional cash transfer program on children’s health care utilization and health outcomes 
Conditional cash transfer (CCT) programs provide poor families with cash conditional on investments in health and education. Brazil’s Bolsa Família program began in 2003 and is currently the largest CCT program in the world. This community-based study examines the impact of Bolsa Família on child health in a slum community in a large urban center.
In 2010, detailed household surveys were conducted with randomly selected Bolsa Família beneficiaries and non-beneficiaries in a Brazilian slum community of approximately 14,000 inhabitants in a large urban center. 567 families (with 1,266 children) were interviewed. Propensity score methods were used to control for differences between beneficiary and non-beneficiary children to estimate program impacts on health care utilization and health outcomes.
Bolsa Família has increased the odds of children’s visits to the health post for preventive services. In children under age seven, Bolsa Família was associated with increased odds for growth monitoring (OR = 3.1; 95% CI 1.9-5.1), vaccinations (OR = 2.8; 95% CI 1.4-5.4), and checkups (OR = 1.6; 95% CI 0.98-2.5), and with the number of growth monitoring visits (β = 0.6; p = 0.049) and checkups (β = 0.2; p = 0.068). There were positive spillover effects on older siblings (ages 7-17) no longer required to meet the health conditionalities. Bolsa Família increased their odds for growth monitoring (OR = 2.5; 95% CI 1.3-4.9) and checkups (OR = 1.7; 95% CI 0.9-3.2) and improved psychosocial health (β = 2.6; p = 0.007).
Bolsa Família has improved health care utilization, especially for services related to the health conditionalites, and there were positive spillover effects on older siblings. The findings of this study are promising, but they also suggest that further improvements in health may depend on the quality of health care services provided, the scope of services linked to the health conditionalities, and coordination with other social safety net programs.
PMCID: PMC4021270  PMID: 24690131
13.  Leptospirosis in a subsistence farming community in Brazil 
Leptospirosis has been reported in rural areas of Brazil. However, there is limited information about the exposure risk or the risk of Leptospira infection for rural-based populations. A cross-sectional study was carried out in order to determine the prevalence and risk factors for prior Leptospira infection in a rural subsistence farming region of the state of Rio Grande do Norte, an area in which outbreaks of leptospirosis have occurred. Among 290 individuals enrolled, 44 (15.2%) had anti-Leptospira IgM antibodies as determined by IgM ELISA. Infection tended to occur with activities related to the rice fields (P = 0.08). Our findings indicate that Leptospira infection occurs even in years of low rainfall, and may have an important impact among poor rural-based subsistence farmers in Brazil. Additional studies are needed to characterize the mode of transmission in this region.
PMCID: PMC3970206  PMID: 18599101
Leptospirosis; Disease outbreaks; Surveillance; Prevalence; Rural population; Brazil
The Journal of infection  2009;60(3):218-223.
To identify prediction factors for the development of leptospirosis-associated pulmonary hemorrhage syndrome (LPHS).
The study comprised of 203 patients, aged ≥14 years, admitted with complications of the severe form of leptospirosis at the Emílio Ribas Institute of Infectology (Sao Paulo, Brazil) between 1998 to 2004. Laboratory and demographic data were obtained and the severity of illness score and involvement of the lungs and others organs were determined. Logistic regression was performed to identify independent predictors of LPHS. A validation cohort of 97 subjects with severe form of leptospirosis admitted at the same hospital between 2004-2006 was used to independently evaluate the predictive value of the model.
The overall mortality rate was 7.9%. Multivariate logistic regression revealed that five factors were independently associated with the development of LPHS: serum potassium (mmol/L) (OR=2.6; 95%CI=1.1-5.9); serum creatinine (μmol/L) (OR=1.2; 95%CI=1.1-1.4); respiratory rate (breaths/min) (OR=1.1; 95%CI=1.1-1.2); presenting shock (OR=69.9; 95%CI=20.1-236.4), and Glasgow Coma Scale Score (GCS)<15 (OR=7.7; 95%CI = 1.3-23.0). We used these findings to calculate the risk of LPHS by the use of a spreadsheet. In the validation cohort, the equation classified correctly 92% of patients (Kappa statistic = 0.80).
We developed and validated a multivariate model for predicting LPHS. This tool should prove useful in identifying LPHS patients, allowing earlier management and thereby reducing mortality.
PMCID: PMC3921886  PMID: 20026189
Leptospirosis; Leptospirosis-associated pulmonary hemorrhage syndrome; ROC curve; Predictive model; Brazil
15.  Knowledge, Attitudes, and Practices Related to Leptospirosis among Urban Slum Residents in Brazil 
Leptospirosis disproportionately affects residents of urban slums. To understand the knowledge, attitudes, and practices regarding leptospirosis, we conducted a cross-sectional study among residents of an urban slum community in Salvador, Brazil. Of the 257 residents who were interviewed, 225 (90%) were aware of leptospirosis and more than two-thirds of respondents correctly identified the modes of disease transmission and ways to reduce exposure. However, study participants who performed risk activities such as cleaning open sewers had limited access to protective clothing such as boots (33%) or gloves (35%). Almost all respondents performed at least one activity to prevent household rat infestation, which often included use of an illegal poison. Our findings support the need for interventions targeted at the individual and household levels to reduce risk of leptospirosis until large-scale structural interventions are available to residents of urban slum communities.
PMCID: PMC3583330  PMID: 23269657
16.  An outbreak of severe leptospirosis in capuchin (Cebus) monkeys 
Veterinary journal (London, England : 1997)  2010;188(2):10.1016/j.tvjl.2010.05.002.
Naturally acquired acute leptospirosis in monkeys is uncommon. This study reports an outbreak of severe leptospirosis among 52 capuchin (Cebus) monkeys that had been rescued from homes and housed in a wildlife rehabilitation center in Colombia in 2007. Case confirmation consisted of Leptospira isolation followed by a polymerase chain reaction targeting the LipL32 gene. The attack and mortality rates were 71% and 27%, respectively. Sixteen cases were confirmed. Necropsy revealed diffuse jaundice and pulmonary hemorrhage. Multi-locus sequence typing identified the agent to be Leptospira interrogans sequence type 17, indicating rats as the source of infection. An environmental survey confirmed rodent infestation as the cause of the outbreak. The extent of Leptospira transmission between humans and monkeys is unknown. Improper husbandry of non-human primates could create new reservoirs and transmission routes for Leptospira threatening conservation efforts and public health.
PMCID: PMC3865605  PMID: 20554228
Leptospirosis; Cebus; Capuchin monkey; Outbreak; Rat
17.  Eschar-associated Spotted Fever Rickettsiosis, Bahia, Brazil 
Emerging Infectious Diseases  2011;17(2):275-278.
In Brazil, Brazilian spotted fever was once considered the only tick-borne rickettsial disease. We report eschar-associated rickettsial disease that occurred after a tick bite. The etiologic agent is most related to Rickettsia parkeri, R. africae, and R. sibirica and probably widely distributed from São Paulo to Bahia in the Atlantic Forest.
PMCID: PMC3204763  PMID: 21291605
Spotted fever group rickettsiosis; rickettsia; eschar; multiple-locus sequence analysis; ticks; molecular diagnosis; dispatch
18.  Urban slum structure: integrating socioeconomic and land cover data to model slum evolution in Salvador, Brazil 
The expansion of urban slums is a key challenge for public and social policy in the 21st century. The heterogeneous and dynamic nature of slum communities limits the use of rigid slum definitions. A systematic and flexible approach to characterize, delineate and model urban slum structure at an operational resolution is essential to plan, deploy, and monitor interventions at the local and national level.
We modeled the multi-dimensional structure of urban slums in the city of Salvador, a city of 3 million inhabitants in Brazil, by integrating census-derived socioeconomic variables and remotely-sensed land cover variables. We assessed the correlation between the two sets of variables using canonical correlation analysis, identified land cover proxies for the socioeconomic variables, and produced an integrated map of deprivation in Salvador at 30 m × 30 m resolution.
The canonical analysis identified three significant ordination axes that described the structure of Salvador census tracts according to land cover and socioeconomic features. The first canonical axis captured a gradient from crowded, low-income communities with corrugated roof housing to higher-income communities. The second canonical axis discriminated among socioeconomic variables characterizing the most marginalized census tracts, those without access to sanitation or piped water. The third canonical axis accounted for the least amount of variation, but discriminated between high-income areas with white-painted or tiled roofs from lower-income areas.
Our approach captures the socioeconomic and land cover heterogeneity within and between slum settlements and identifies the most marginalized communities in a large, complex urban setting. These findings indicate that changes in the canonical scores for slum areas can be used to track their evolution and to monitor the impact of development programs such as slum upgrading.
PMCID: PMC3924348  PMID: 24138776
19.  Serological trail of Brucella infection in an urban slum population in Brazil 
Brucellosis is a re-emerging zoonosis with new cases reported each year in many Latin American countries, but it is mostly under-recognized. This study presents a serological investigation of infection with Brucella abortus and Brucella canis in a poor urban community in the city of Salvador, Brazil.
Human sera (n = 180) were randomly selected from 3,171 samples taken from healthy individuals during 2003-2004 and tested with C-ELISA for B. abortus and I-ELISA for B. canis.
Thirteen percent (24/180) of the individuals were positive for B. abortus and 4.6 % (8/174) were positive for B. canis. Among the variables studied only age (older than 45 years) appeared to be a risk factor for the detection of Brucella antibodies.
These results indicate the presence of Brucella infection in this settlement and highlight the need to understand the epidemiology of infection under these circumstances to establish the necessary measures for surveillance and control.
PMCID: PMC3800144  PMID: 23000868
serology; Brucella abortus; Brucella canis; zoonosis; urban slum
20.  Identification of Seroreactive Proteins of Leptospira interrogans Serovar Copenhageni Using a High-Density Protein Microarray Approach 
Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease.
In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results.
Principal findings
We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients.
Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for diagnosing this neglected tropical disease. Further research is needed to assess the utility of these antigens in more deployable diagnostic platforms.
Author Summary
Leptospirosis is an infectious zoonotic disease that causes non-specific signs and symptoms in humans, which hampers the clinical diagnosis and treatment by physicians. Complications can occur if the proper treatment is not initiated early in the course of illness. Although the early diagnosis is critical for preventing unnecessary complications, currently available tests do not exhibit sufficient diagnostic sensitivity in the beginning of disease. We took advantage of high throughput techniques to perform an embracing study of the humoral immune response to the bacteria in order to identify antigens that could be used in a new test for the diagnosis of leptospirosis. A protein microarray chip containing 2,241 leptospiral proteins was constructed and probed with serum samples from patients and healthy individuals. We identified 24 proteins that are recognized by patients' sera but not by healthy individuals. These proteins are potential diagnostic markers, especially the ones identified for acute-phase patients, which can discriminate between a positive and a negative leptospirosis case within a few days after onset of symptoms. This work establishes the protein microarray approach for improving our understanding of the serological response to leptospirosis. Further research is needed to assess the performance of these antigens in the clinical setting.
PMCID: PMC3798601  PMID: 24147173
21.  Cytokine Response Signatures in Disease Progression and Development of Severe Clinical Outcomes for Leptospirosis 
The role of the immune response in influencing leptospirosis clinical outcomes is not yet well understood. We hypothesized that acute-phase serum cytokine responses may play a role in disease progression, risk for death, and severe pulmonary hemorrhage syndrome (SPHS).
Methodology/Principal Findings
We performed a case-control study design to compare cytokine profiles in patients with mild and severe forms of leptospirosis. Among patients hospitalized with severe disease, we compared those with fatal and nonfatal outcomes. During active outpatient and hospital-based surveillance we prospectively enrolled 172 patients, 23 with mild disease (outpatient) and 149 with severe leptospirosis (hospitalized). Circulating concentrations of pro- and anti-inflammatory cytokines at the time of patient presentation were measured using a multiplex bead array assay. Concentrations of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, and TNF-α were significantly higher (P<0.05) in severe disease compared to mild disease. Among severe patients, levels of IL-6 (P<0.001), IL-8 (P = 0.0049) and IL-10 (P<0.001), were higher in fatal compared to non-fatal cases. High levels of IL-6 and IL-10 were independently associated (P<0.05) with case fatality after adjustment for age and days of symptoms. IL-6 levels were higher (P = 0.0519) among fatal cases who developed SPHS than among who did not.
This study shows that severe cases of leptospirosis are differentiated from mild disease by a “cytokine storm” process, and that IL-6 and IL-10 may play an immunopathogenic role in the development of life-threatening outcomes in human leptospirosis.
Author Summary
Leptospirosis is a tropical bacterial disease that is transmitted to humans from infected animals. Leptospirosis symptoms can range from mild fever to fatal disease forms, such as massive bleeding into the lungs, called Severe Pulmonary Hemorrhage Syndrome (SPHS). It is not known what determines the severity of leptospirosis, but we hypothesized that it may be influenced by differences in the type and concentration of signaling proteins called cytokines that are produced by the immune system in response to infection. We collected blood from patients with mild and severe leptospirosis, and compared the concentration of eight different cytokines circulating in the blood. We found that patients with severe leptospirosis had higher levels of most cytokines. Among patients who had severe forms, higher levels of specific cytokines called IL-6 and IL-8 were predictive of death even after statistical adjustment for age and number of days of symptoms prior to hospitalization. IL-6 was higher in patients who died from SPHS compared to those who died of other leptospirosis complications. This knowledge suggests that severe forms of leptospirosis may be due to a specific kind of immune response, which may lead to targeted therapies to reduce the impact of this disease.
PMCID: PMC3777885  PMID: 24069500
22.  The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis 
Vaccine  2007;25(33):6277-6286.
Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund’s adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P <0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis.
PMCID: PMC1994161  PMID: 17629368
Leptospirosis; subunit vaccine; Leptospiral immunoglobulin-like protein; recombinant protein; immunity; antibodies; hamsters
23.  Leptospirosis-associated Severe Pulmonary Hemorrhagic Syndrome, Salvador, Brazil 
Emerging Infectious Diseases  2008;14(3):505-508.
We report the emergence of leptospirosis-associated severe pulmonary hemorrhagic syndrome (SPHS) in slum communities in Salvador, Brazil. Although active surveillance did not identify SPHS before 2003, 47 cases were identified from 2003 through 2005; the case-fatality rate was 74%. By 2005, SPHS caused 55% of the deaths due to leptospirosis.
PMCID: PMC2570821  PMID: 18325275
Leptospirosis; Leptospira; pulmonary hemorrhage; urban epidemics; incidence; epidemiology; serovar Copenhageni; dispatch
24.  Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis 
Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard.
Methods and findings
CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously.
Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF.
Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.
PMCID: PMC3558362  PMID: 23339355
Bacterial meningitis; Diagnostic test evaluation; Real-time PCR; Streptococcus pneumoniae; Neisseria meningitidis; Haemophilus influenzae
25.  Accuracy of a Dual Path Platform (DPP) Assay for the Rapid Point-of-Care Diagnosis of Human Leptospirosis 
Diagnosis of leptospirosis by the gold standard serologic assay, the microscopic agglutination test (MAT), requires paired sera and is not widely available. We developed a rapid assay using immunodominant Leptospira immunoglobulin-like (Lig) proteins in a Dual Path Platform (DPP). This study aimed to evaluate the assay's diagnostic performance in the setting of urban transmission.
We determined test sensitivity using 446 acute and convalescent sera from MAT-confirmed case-patients with severe or mild leptospirosis in Brazil. We assessed test specificity using 677 sera from the following groups: healthy residents of a Brazilian slum with endemic transmission, febrile outpatients from the same slum, healthy blood donors, and patients with dengue, hepatitis A, and syphilis. Three operators independently interpreted visual results without knowing specimen status.
The overall sensitivity for paired sera was 100% and 73% for severe and mild disease, respectively. In the acute phase, the assay achieved a sensitivity of 85% and 64% for severe and mild leptospirosis, respectively. Within seven days of illness onset, the assay achieved a sensitivity of 77% for severe disease and 60% for mild leptospirosis. Sensitivity of the DPP assay was similar to that for IgM-ELISA and increased with both duration of symptoms (chi-square regression P = 0.002) and agglutinating titer (Spearman ρ = 0.24, P<0.001). Specificity was ≥93% for dengue, hepatitis A, syphilis, febrile outpatients, and blood donors, while it was 86% for healthy slum residents. Inter-operator agreement ranged from very good to excellent (kappa: 0.82–0.94) and test-to-test reproducibility was also high (kappa: 0.89).
The DPP assay performed acceptably well for diagnosis of severe acute clinical leptospirosis and can be easily implemented in hospitals and health posts where leptospirosis is a major public health problem. However, test accuracy may need improvement for mild disease and early stage leptospirosis, particularly in regions with high transmission.
Author Summary
Leptospirosis is an important cause of acute fever in the tropics and the mortality rate may exceed 15% in patients with severe disease manifestations. The gold standard serological test for diagnosing leptospirosis, the microagglutination test or MAT, requires significant laboratory resources and results are not timely. Improved diagnostics are therefore critically needed to identify patients and outbreaks earlier and to thereby prevent unnecessary deaths. The need for a rapid diagnostic test is particularly acute in resource-poor settings where leptospirosis is a major public health problem and sophisticated laboratories are unavailable. In this study, we measured the diagnostic accuracy of the novel Dual Path Platform (DPP) for leptospirosis using serum from patients with mild and severe disease. The DPP assay detected up to 85% of severe leptospirosis and 64% of mild leptospirosis patients using the initial clinical specimen collected at hospital presentation and its diagnostic performance was comparable to a commonly used IgM-ELISA. Furthermore, the DPP assay produces a result in 20 minutes and can be more easily implemented in field settings than existing diagnostic technologies. The commercially available DPP kit offers the simple, accurate, and quick diagnosis of leptospirosis and, consequently, more timely clinical and public health decision-making.
PMCID: PMC3486890  PMID: 23133686

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