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1.  Correction: Collaborative Teaching and Learning: A Model for Building Capacity and Partnerships to Address NTDs 
PLoS Neglected Tropical Diseases  2011;5(8):10.1371/annotation/d33a8e45-4a41-49c7-81b1-1355a4c28a81.
doi:10.1371/annotation/d33a8e45-4a41-49c7-81b1-1355a4c28a81
PMCID: PMC3155473
2.  Prospective Study of Leptospirosis Transmission in an Urban Slum Community: Role of Poor Environment in Repeated Exposures to the Leptospira Agent 
Background
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.
Conclusions/Significance
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.
doi:10.1371/journal.pntd.0002927
PMCID: PMC4038618  PMID: 24875389
3.  Distribution of superantigens in group A streptococcal isolates from Salvador, Brazil 
BMC Infectious Diseases  2014;14:294.
Background
Group A streptococcus (GAS) causes invasive disease, superficial disease, and can asymptomatically colonize humans. Superantigens are one virulence factor found in GAS. Previous studies found associations between the genes that encode superantigens and emm type of GAS. It is unknown if these associations are due to underlying biological factors that limit the distribution of superantigens or, alternatively, if these associations are due to the expansion of local GAS linages where these studies took place. To further address this question we screened GAS isolates collected from Salvador, Brazil for 11 known superantigen genes.
Methods
Seventy-seven GAS isolates were screened by PCR for superantigen genes. These superantigen genes were speA, speC, speG, speH, speI, speJ, speK, speL, speM, ssa, and smeZ. We used Fisher’s two-sided exact test to identify associations between superantigens and GAS emm type. We then compared our results to previous reports of superantigen prevalence and superantigen association with emm type.
Results
In our collection we found several emm type and superantigen genotype combinations that have previously been reported in isolates from Europe and Australia. We also found that speA was significantly associated with emm type 1, and that speC was significantly associated with emm type 12.
Conclusions
Our study reports superantigen genotypes of GAS from a region of the world that is lacking this information. We found evidence of common GAS superantigen genotypes that are spread worldwide as well as novel superantigen genotypes that, so far, are unique to Brazil.
doi:10.1186/1471-2334-14-294
PMCID: PMC4045995  PMID: 24885209
Streptococcus pyogenes; Streptococcal superantigens; Group A streptococcus; Emm types
4.  Longitudinal surveillance for meningitis by Acinetobacter in a large urban setting in Brazil 
The study aim was to describe the emergency of carbapenem resistance and clonal complexes (CC), defined by multilocus sequence typing (MLST), in Acinetobacter baumannii in a surveillance system for meningitis. Starting in 1996 at an urban setting of Brazil, surveillance detected meningitis by Acinetobacter sp for the first time in 2002. Until 2008, 35 isolates were saved. Carbapenem resistance emerged in 2006, reaching 70% of A. baumannii isolates in 2008, including one colistin-resistant. A. baumannii belonged to CC113/79 (University of Oxford/ Institute Pasteur schemes), CC235/162 and CC103/15. Dissemination of infections resistant to all antimicrobial agents may occur in the future.
doi:10.1111/1469-0691.12145
PMCID: PMC3625502  PMID: 23398654
Acinetobacter baumannii; bacterial meningitis; carbapenem-resistance; multilocus sequence typing; clonal complexes
5.  The impact of Brazil’s Bolsa Família conditional cash transfer program on children’s health care utilization and health outcomes 
Background
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1186/1472-698X-14-10
PMCID: PMC4021270  PMID: 24690131
6.  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.
doi:10.4269/ajtmh.2012.12-0245
PMCID: PMC3583330  PMID: 23269657
7.  Characteristics of the Human Host Have Little Influence on Which Local Schistosoma mansoni Populations Are Acquired 
Background
Brazil remains the country in the Americas with the highest prevalence of schistosomiasis. A combination of control efforts and development, however, has sharply reduced its intensity and distribution. The acquisition of specific schistosome populations may be dependent on host characteristics such as sex, age, geography, work, habits and culture. How these and other host characteristics align with parasite subpopulations may guide approaches to improve control.
Methodology
A cohort of more than 90% of the residents in two rural communities in Brazil participated in an epidemiologic survey of demographic, socio-economic and behavioral characteristics. The variables sex, age, intensity of infection, socio-economic index, % lifetime spent on site, previous infection, and trips outside the district were used to group parasites infecting individuals. Schistosoma mansoni infection status was determined by examination of stools submitted on 3 different days. The aggregate of eggs collected from the whole stool was used to determine degree of population differentiation from allele frequencies for 15 microsatellites.
Conclusions/Significance
Infection prevalence was 41% for these communities, and the epidemiologic characteristics were similar to many of the endemic areas of Brazil and the world. Parasite population structuring was observed between the two communities (Jost's D 0.046, CI95% 0.042–0.051), although separated by only 8 km and connected by a highway. No structuring was observed when infected individuals were stratified by host's biologic, demographic or epidemiologic characteristics. Those most heavily infected best reflected the communities' overall parasite diversity. The lack of differentiation within villages suggests that individuals are likely to get infected at the same sites or that the same parasite multilocus genotypes can be found at most sites. The geographic structuring between villages and the lack of structuring by age of the host further supports the impression of a population little affected by migration or drift.
Author Summary
Schistosomiasis is one of the world's most important parasitic infections. Its elimination has proved difficult even in countries such as Brazil where access to treatment is readily available. Infection is the result of human contact with surface water where there are infected snails, so that human biology and habits may bring different individuals in contact with different groups of parasites. Identification of schistosome subpopulations may assist understanding transmission patterns and guide control efforts. We compared microsatellite allele frequencies from all of the infections in 2 small villages and determined that the movement of parasites between them was limited. Individual infections were distinct composites of parasites, but if infected humans were grouped by demographic and epidemiologic characteristics, there was no evidence that specific parasite subpopulations were being selected in these types of hosts. Infections were also not differentiated when stratified by host's age indicating that the populations were stable over time. Since the infection cycle requires human fecal contamination of water, local human behavior can to some degree be inferred from the patterns of schistosome subpopulation distribution.
doi:10.1371/journal.pntd.0002572
PMCID: PMC3854954  PMID: 24340115
8.  Genetic Population Structure of Cercariae from an Urban Foci of Schistosoma mansoni, Brazil 
Rapid urbanization in Brazil has meant that many persons from rural areas where Schistosoma mansoni is endemic have migrated to cities. Discovery of a focus of active transmission in the city of Salvador prompted a citywide survey for active and potential transmission sites. Cercariae shed from infected snails collected from four locations were used to determine how these samples were related and if they were representative of the parasite population infecting humans. Each cercarial collection was greatly differentiated from the others, and diversity was significantly lower when compared with eggs from natural human infections in one site. Egg samples collected 7 years apart in one neighborhood showed little differentiation (Jost's D = 0.01–0.03). Given the clonal nature of parasite reproduction in the snail host and the short-term acquisition of parasites, cercariae from collections at one time point are unlikely to be representative of the diversity in the human population.
doi:10.4269/ajtmh.2012.11-0641
PMCID: PMC3516259  PMID: 22949518
9.  Urban slum structure: integrating socioeconomic and land cover data to model slum evolution in Salvador, Brazil 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1476-072X-12-45
PMCID: PMC3924348  PMID: 24138776
10.  Identification of Seroreactive Proteins of Leptospira interrogans Serovar Copenhageni Using a High-Density Protein Microarray Approach 
Background
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.
Methodology
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.
Conclusions
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.
doi:10.1371/journal.pntd.0002499
PMCID: PMC3798601  PMID: 24147173
11.  Cytokine Response Signatures in Disease Progression and Development of Severe Clinical Outcomes for Leptospirosis 
Background
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.
Conclusion/Significance
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.
doi:10.1371/journal.pntd.0002457
PMCID: PMC3777885  PMID: 24069500
12.  Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis 
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2334-13-26
PMCID: PMC3558362  PMID: 23339355
Bacterial meningitis; Diagnostic test evaluation; Real-time PCR; Streptococcus pneumoniae; Neisseria meningitidis; Haemophilus influenzae
13.  Accuracy of a Dual Path Platform (DPP) Assay for the Rapid Point-of-Care Diagnosis of Human Leptospirosis 
Background
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.
Methodology
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.
Results
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).
Conclusions
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.
doi:10.1371/journal.pntd.0001878
PMCID: PMC3486890  PMID: 23133686
14.  Schistosoma mansoni population structure and persistence after praziquantel treatment in two villages of Bahia, Brazil 
International journal for parasitology  2011;41(10):1093-1099.
Praziquantel has been used to treat schistosome infections since 1979 and currently is the only chemotherapeutic agent in production for this purpose, raising concerns about the potential for the emergence of drug resistance. In practice, 10–20% of infected patients will continue to excrete eggs after treatment. It is not understood to what degree this represents selection of a resistant population or incomplete elimination due to the presence of immature worms at the time of treatment. We used a population genetics approach to test whether or not persistent Schistosoma mansoni parasites were drawn from the same population as susceptible parasites. In this study, stool samples were collected from 96% of individuals in two small Brazilian communities (populations 482 and 367) and examined for S. mansoni eggs. The combined prevalence of S. mansoni infections in the villages was 41%. Total egg DNA was extracted from each sample and was genotyped at 15 microsatellite markers. Day-to-day variation of the infrapopulation from an individual human host was low (median differentiation using Jost’s D = 0.010), so that a single stool was representative of the genotypes present in stool eggs, at least in the short term. Average pairwise analysis of D among all pre-treatment infrapopulations suggested moderate differentiation (mean D = 0.082 and 0.122 for the two villages), whereas the pre-treatment component population differentiation between the two communities was 0.047. The differentiation of the component population remaining after treatment from the fully susceptible component population was low (mean D = 0.007 and 0.020 for the two villages), suggesting that the persistent parasites were not selected by praziquantel treatment. We will continue to follow these communities for evidence of selection or changes in population structure.
doi:10.1016/j.ijpara.2011.06.002
PMCID: PMC3155667  PMID: 21784077
Praziquantel; Resistance; Microsatellite; Population genetics; Sampling; Genetic differentiation; Selection
15.  Surgery for Valvular Heart Disease: A Population-Based Study in a Brazilian Urban Center 
PLoS ONE  2012;7(5):e37855.
Background
In middle income countries, the burden of rheumatic heart disease (RHD) remains high, but the prevalence of other heart valve diseases may rise as the population life expectancy increases. Here, we compared population-based data on surgical procedures to assess the relative importance of causes of heart valve disease in Salvador, Brazil.
Methodology/Principal Findings
Medical charts of patients who underwent surgery for valvular heart disease from January 2002–December 2005 were reviewed. Incidence of surgery for valvular heart disease was calculated. Logistic regression was used to identify factors associated with in-hospital death following surgery. The most common etiologies for valvular dysfunction in 491 valvular heart surgery patients were RHD (60.3%), degenerative valve disease (15.3%), and endocarditis (4.5%). Mean annual incidence for surgeries due to any valvular heart diseases, RHD, and degenerative valvular disease were 5.02, 3.03, and 0.77 per 100,000 population, respectively. Incidence of surgery due to RHD was highest in young adults; procedures were predominantly paid by the public health sector. In contrast, the incidence of surgery due to degenerative valvular disease was highest among those older than 60 years of age; procedures were mostly paid by the private sector. The overall in-hospital case-fatality ratio was 11.9%. Independent factors associated with death included increase in age (odds ratio: 1.04 per year of age; 95% confidence interval: 1.02–1.06), endocarditis (6.35; 1.92–21.04), multiple valve operative procedures (4.35; 2.12–8.95), and prior heart valve surgery (2.49; 1.05–5.87).
Conclusions/Significance
RHD remains the main cause for valvular heart surgery in Salvador, which primarily affects young adults without private health insurance. In contrast, surgery due to degenerative valvular disease primarily impacts the elderly with private health insurance. Strategies to reduce the burden of valvular heart disease will need to address the disparate factors that contribute to RHD as well as degenerative valve disease.
doi:10.1371/journal.pone.0037855
PMCID: PMC3362603  PMID: 22666401
16.  Detection and Quantification of Leptospira interrogans in Hamster and Rat Kidney Samples: Immunofluorescent Imprints versus Real-time PCR 
PLoS ONE  2012;7(2):e32712.
A major limitation in the clinical management and experimental research of leptospirosis is the poor performance of the available methods for the direct detection of leptospires. In this study, we compared real-time PCR (qPCR), targeting the lipL32 gene, with the immunofluorescent imprint method (IM) for the detection and quantification of leptospires in kidney samples from the rat and hamster experimental models of leptospirosis. Using a virulent strain of Leptospira interrogans serovar Copenhageni, a chronic infection was established in the rat model, which were euthanized 28 days post-infection, while the hamster model simulated an acute infection and the hamsters were euthanized eight days after inoculation. Leptospires in the kidney samples were detected using culture isolation, qPCR and the IM, and quantified using qPCR and the IM. In both the acute and chronic infection models, the correlation between quantification by qPCR and the IM was found to be positive and statistically significant (P<0.05). Therefore, this study demonstrates that the IM is a viable alternative for not only the detection but also the quantification of leptospires, particularly when the use of qPCR is not feasible.
doi:10.1371/journal.pone.0032712
PMCID: PMC3290571  PMID: 22393440
17.  Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae prior to Introduction of the 10-Valent Pneumococcal Conjugate Vaccine in Brazil, 2000-2007 
Vaccine  2010;29(6):1139-1144.
This study describes the serotype distribution and antibiotic resistance patterns among 397 S. pneumoniae meningitis case isolates recovered in Salvador, Brazil, during the period of 2000-2007, before introduction of the 10-valent pneumococcal conjugate vaccine.
The active hospital-based surveillance showed a decline in the annual incidence rates of pneumococcal meningitis during the period of study, from 1.12 cases to 0.83 cases/100,000 persons for all age groups (P <0.001), with an overall case-fatality rate of 28.6 % (113 of 395) for all patients and 41.9% (57 of 136) for those <5 years of age. Serotypes 14 (n= 55; 13.9%), 3 (n= 32; 8.1 %), 23F (n=32; 8.1 %), 19F (n=31; 7.8%), 6B (n=30; 7.6%), 18C (n=28; 7.1 %), and 6A (n=20; 5%) were the most prevalent serotypes. In patients < 5 years the estimated projected coverage of 7-, 10- and 13-valent conjugate vaccines was 74.3%, 75.7% and 83.1%, respectively. Antimicrobial susceptibility testing revealed that 22.1% (n=88) of isolates were non-susceptible to penicillin, 56% were non-susceptible to trimethoprim/sulfamethoxazole, and 29.6% were non-susceptible to tetracycline. Nonsusceptibility to penicillin and cefotaxime was detected solely among serotype 14 isolates (n=4; 1%). This study provides an important baseline to assess the impact of conjugate vaccine implantation on the epidemiology of meningitis due to Streptococcus pneumoniae in Salvador, Brazil.
doi:10.1016/j.vaccine.2010.12.021
PMCID: PMC3026083  PMID: 21182994
Streptococcus pneumoniae; Vaccine; Antimicrobial resistance; Meningitis; Children
18.  Attenuated Nephritis in Inducible Nitric Oxide Synthase Knockout C57BL/6 Mice and Pulmonary Hemorrhage in CB17 SCID and Recombination Activating Gene 1 Knockout C57BL/6 Mice Infected with Leptospira interrogans ▿ 
Infection and Immunity  2011;79(7):2936-2940.
The aims of this study were to investigate the frequency of pulmonary hemorrhage (PH) in mice unable to produce functional B and T lymphocytes and to explore the effect of an inducible nitric oxide synthase gene (Inos) knockout (KO) on the frequency/severity of interstitial nephritis in vivo. We studied the outcome of infection by the virulent Leptospira interrogans serovar Copenhageni strain Cop. The animals used were Inos KO mice, recombination activating gene 1 (Rag1) KO mice, CB17 severe combined immunodeficiency (SCID) mice, and the respective wild-type (WT) C57BL/6 and BALB/c controls. The Inos KO and WT mice survived with no clinical symptoms of leptospirosis. The frequency and severity of nephritis was significantly lower in the Inos KO mice. All of the Rag1 KO and SCID animals died of acute leptospirosis, whereas all of the WT mice survived. PH was observed in 57 and 94% of Rag1 KO mice and in 83 and 100% of SCID mice, using inoculum doses of 107 and 106 leptospires, respectively. There was no evidence of PH in the WT controls. In conclusion, the loss of the Inos gene had a negligible effect on the outcome of leptospiral infection, although we observed a reduced susceptibility for interstitial nephritis in this group. Of note, the absence of functional B- and T-cell lymphocytes did not preclude the occurrence of PH. These data provide evidence that PH in leptospirosis may not be related only to autoimmune mechanisms.
doi:10.1128/IAI.05099-11
PMCID: PMC3191955  PMID: 21576342
19.  Clinical outcome of pneumococcal meningitis during the emergence of pencillin-resistant Streptococcus pneumoniae: an observational study 
BMC Infectious Diseases  2011;11:323.
Background
Prior to the availability of generic third-generation cephalosporins, penicillins were widely used for treatment of pneumococcal meningitis in developing countries despite concerns about rising levels of penicillin resistance among pneumococcal isolates. We examined the impact of penicillin resistance on outcomes of pneumococcal meningitis over a ten year period in an infectious diseases hospital in Brazil.
Methods
Clinical presentation, antimicrobial therapy and outcomes were reviewed for 548 patients with culture-confirmed pneumococcal meningitis from December, 1995, to November, 2005. Pneumococcal isolates from meningitis patients were defined as penicillin-resistant if Minimum Inhibitory Concentrations for penicillin were greater than 0.06 μg/ml. Proportional hazards regression was used to identify risk factors for fatal outcomes.
Results
During the ten-year period, ceftriaxone replaced ampicillin as first-line therapy for suspected bacterial meningitis. In hospital case-fatality for pneumococcal meningitis was 37%. Of 548 pneumococcal isolates from meningitis cases, 92 (17%) were resistant to penicillin. After controlling for age and severity of disease at admission, penicillin resistance was associated with higher case-fatality (Hazard Ratio [HR], 1.62; 95% Confidence Interval [CI], 1.08-2.43). Penicillin-resistance remained associated with higher case-fatality when initial therapy included ceftriaxone (HR, 1.68; 95% CI 1.02-2.76).
Conclusions
Findings support the use of third generation cephalosporin antibiotics for treatment of suspected pneumococcal meningitis even at low prevalence of pneumococcal resistance to penicillins.
doi:10.1186/1471-2334-11-323
PMCID: PMC3276609  PMID: 22103652
20.  POOR CLINICAL OUTCOME FOR MENINGITIS CAUSED BY H. INFLUENZAE SEROTYPE A STRAINS CONTAINING THE IS1016-BEXA DELETION 
The Journal of infectious diseases  2010;202(10):1577-1584.
Following introduction of Haemophilus influenzae type b (Hib) conjugate vaccines, meningitis caused by serotypes other than Hib has gained importance. We conducted active hospital-based surveillance for meningitis over an 11-year period in Salvador, Brazil. H. influenzae isolates were serotyped and analyzed by PCR, pulsed-field gel electrophoresis and DNA sequencing to identify strains with a specific deletion (IS1016) in the bexA gene (IS1016-bexA). We identified 43 meningitis cases caused by non-type b H. influenzae: 28 (65%) were caused by type a (Hia), 9 (21%) by non-capsulated strains and 3 (7%) each by types e and f. Hia isolates clustered in two clonal groups; clonal group A strains (n=9) had the IS1016-bexA deletion. Among children <5 years, meningitis caused by Hia from clonal group A had higher case-fatality than clonal group B. Despite small numbers, these results indicate that the presence of IS1016-bexA deletion is associated with enhanced virulence in non-type b H. influenzae.
doi:10.1086/656778
PMCID: PMC2957550  PMID: 20932170
Haemophilus influenzae; non-type b H. influenzae; meningitis; Hib conjugate vaccine; virulence; IS1016-bexA deletion; molecular epidemiology
21.  Dengue hemorrhagic fever is associated with polymorphisms in JAK1 
European Journal of Human Genetics  2010;18(11):1221-1227.
To identify genes associated with the clinical presentation of dengue, 50 cases of probable or possible dengue hemorrhagic fever (DHF), 236 dengue fever (DF), and 236 asymptomatic infections were genotyped for 593 single-nucleotide polymorphisms (SNPs) in 56 genes across the type 1 interferon (IFN) response pathway as well as other important candidate genes. By single locus analysis comparing DHF with DF, 11 of the 51 markers with P<0.05 were in the JAK1 gene. Five markers were significantly associated by false discovery rate criteria (q<0.20 when P<6 × 10−4). The JAK1 SNPs showed differential distribution by ethnicity and ancestry consistent with epidemiologic observations in the Americas. The association remained significant after controlling for ancestry and income. No association was observed with markers in the gene encoding CD209 (DC-SIGN). An association between DHF and JAK1 polymorphisms is in agreement with expression profiles showing generalized decreased type 1 IFN-stimulated gene expression in these patients.
doi:10.1038/ejhg.2010.98
PMCID: PMC2950898  PMID: 20588308
flavivirus; population structure; interferon; genetic association; Brazil
22.  Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin 
BMC Microbiology  2011;11:129.
Background
In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc.
Results
The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion.
Conclusions
This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.
doi:10.1186/1471-2180-11-129
PMCID: PMC3133549  PMID: 21658265
23.  Dengue hemorrhagic fever is associated with polymorphisms in JAK1 
To identify genes associated with the clinical presentation of dengue, 50 cases of probable or possible dengue hemorrhagic fever, 236 dengue fever and 236 asymptomatic infections were genotyped for 593 single nucleotide polymorphisms in 56 genes across the type 1 interferon response pathway as well as other important candidate genes. By single locus analysis comparing dengue hemorrhagic fever with dengue fever, 11 of the 51 markers with p<0.05 were in the JAK1 gene. Five markers were significantly associated by false discovery rate criteria (q<0.20 when p<6 × 10−4). The JAK1 single nucleotide polymorphisms showed differential distribution by ethnicity and ancestry consistent with epidemiologic observations in the Americas. The association remained significant after controlling for ancestry and income. No association was observed with markers in the gene encoding CD209 (DC-SIGN). An association between dengue hemorrhagic fever and JAK1 polymorphisms is in agreement with expression profiles showing generalized decreased type 1 interferon-stimulated gene expression in these patients.
doi:10.1038/ejhg.2010.98
PMCID: PMC2950898  PMID: 20588308
Flavivirus; population structure; interferon; genetic association; Brazil
25.  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.
doi:10.3201/eid1702.100859
PMCID: PMC3204763  PMID: 21291605
Spotted fever group rickettsiosis; rickettsia; eschar; multiple-locus sequence analysis; ticks; molecular diagnosis; dispatch

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