Effectiveness of the fourth dose of COVID-19 vaccines against severe COVID-19 among adults 40 years or older in Brazil: a population-based cohort study.

Background: The emergence of COVID-19 variants with immune scape and the waning of primary vaccine schemes effectiveness have prompted many countries to indicate first and second booster COVID-19 vaccine doses to prevent severe COVID-19. However, current available evidence on second booster dose effectiveness are mostly limited to high-income countries, older adults, and mRNA-based vaccination schemes scenarios. We aimed to investigate the relative vaccine effectiveness (rVE) of the fourth dose compared to three doses for severe COVID-19 outcomes in Brazil; and compare the rVE of a fourth dose with an mRNA vaccine compared to adenovirus-based product in the same settings. Methods: We performed a target emulated trial using a population-based cohort of individuals aged 40 years or older who have received a homologous primary scheme of CoronaVac, ChAdOx1, or BNT162b2, and any third dose product and were eligible for the fourth dose in Brazil. The primary outcome was COVID-19 associated hospitalization or death. We built Cohort A matching individuals vaccinated with a fourth dose to individuals who received three doses to estimate the rVE of the fourth dose. We built Cohort B, a subset of Cohort A, matching mRNA-based (mRNA) to adenovirus-based fourth dose vaccinated individuals to compare their relative hazards for severe COVID-19. Findings: 46,693,484 individuals were included in Cohort A and 6,763,016 in Cohort B. 45% of them were aged between 40 and 60 years old, and 48% between 60 and 79 years old. In Cohort A, the most common previous series was a ChAdOx1 two-dose followed by BNT162b2 (44%), and a CoronaVac two-dose followed by a BNT162b2 (36%). Among those fourth dose vaccinated, 36.9% received ChAdOx1, 32.7% Ad26.COV2.S, 25.8% BNT162b2, and 4.7% CoronaVac. In Cohort B, among those who received an adenovirus fourth dose, 53.7% received ChAdOx1 and 46.3% received Ad26.COV2.S. The estimated rVE for the primary outcome of four doses compared to three doses was 44.1% (95% CI 42.3-46.0), with some waning during follow-up (rVE 7-60 days 46.8% [95% CI 44.4-49.1], rVE after 120 days 33.8% [95% CI 18.0-46.6]). Among fourth dose vaccinated individuals, mRNA-based vaccinated individuals had lower hazards for hospitalization or death compared to adenovirus-vaccinated individuals (HR 0.81, 95% CI 0.75-0.87). After 120 days, no difference in hazards between groups was observed (HR 1.35, 95% CI 0.93-1.97). Similar findings were observed for hospitalization and death separately, except no evidence for differences between fourth dose brands for death in Cohort B. Interpretation: In a heterogeneous scenario of primary and first booster vaccination combinations, a fourth dose provided meaningful and durable protection against severe COVID-19 outcomes. Compared to adenovirus-based booster, a fourth dose wild-type mRNA vaccine was associated with immediate lower hazards of hospitalization or death unsustained after 120 days. Funding: None.

No evidence of fetal defects or anti-syncytin-1 antibody induction following COVID-19 mRNA vaccination.

The impact of Coronavirus Disease 2019 (COVID-19) mRNA vaccination on pregnancy and fertility has become a major topic of public interest. We investigated 2 of the most widely propagated claims to determine (1) whether COVID-19 mRNA vaccination of mice during early pregnancy is associated with an increased incidence of birth defects or growth abnormalities; and (2) whether COVID-19 mRNA-vaccinated human volunteers exhibit elevated levels of antibodies to the human placental protein syncytin-1. Using a mouse model, we found that intramuscular COVID-19 mRNA vaccination during early pregnancy at gestational age E7.5 did not lead to differences in fetal size by crown-rump length or weight at term, nor did we observe any gross birth defects. In contrast, injection of the TLR3 agonist and double-stranded RNA mimic polyinosinic-polycytidylic acid, or poly(I:C), impacted growth in utero leading to reduced fetal size. No overt maternal illness following either vaccination or poly(I:C) exposure was observed. We also found that term fetuses from these murine pregnancies vaccinated prior to the formation of the definitive placenta exhibit high circulating levels of anti-spike and anti-receptor-binding domain (anti-RBD) antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) consistent with maternal antibody status, indicating transplacental transfer in the later stages of pregnancy after early immunization. Finally, we did not detect increased levels of circulating anti-syncytin-1 antibodies in a cohort of COVID-19 vaccinated adults compared to unvaccinated adults by ELISA. Our findings contradict popular claims associating COVID-19 mRNA vaccination with infertility and adverse neonatal outcomes.

Association of renalase with clinical outcomes in hospitalized patients with COVID-19.

Renalase is a secreted flavoprotein with anti-inflammatory and pro-cell survival properties. COVID-19 is associated with disordered inflammation and apoptosis. We hypothesized that blood renalase levels would correspond to severe COVID-19 and survival. In this retrospective cohort study, clinicopathologic data and blood samples were collected from hospitalized COVID-19 subjects (March-June 2020) at a single institution tertiary hospital. Plasma renalase and cytokine levels were measured and clinical data abstracted from health records. Of 3,450 COVID-19 patients, 458 patients were enrolled. Patients were excluded if <18 years, or opted out of research. The primary composite outcome was intubation or death within 180 days. Secondary outcomes included mortality alone, intensive care unit admission, use of vasopressors, and CPR. Enrolled patients had mean age 64 years (SD±17), were 53% males, and 48% non-whites. Mean renalase levels was 14,108·4 ng/ml (SD±8,137 ng/ml). Compared to patients with high renalase, those with low renalase (< 8,922 ng/ml) were more likely to present with hypoxia, increased ICU admission (54% vs. 33%, p < 0.001), and cardiopulmonary resuscitation (10% vs. 4%, p = 0·023). In Cox proportional hazard model, every 1000 ng/ml increase in renalase decreased the risk of death or intubation by 5% (HR 0·95; 95% CI 0·91-0·98) and increased survival alone by 6% (HR 0·95; CI 0·90-0·98), after adjusting for socio-demographics, initial disease severity, comorbidities and inflammation. Patients with high renalase-low IL-6 levels had the best survival compared to other groups (p = 0·04). Renalase was independently associated with reduced intubation and mortality in hospitalized COVID-19 patients. Future studies should assess the pathophysiological relevance of renalase in COVID-19 disease.

De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: a case report.

SARS-CoV-2 remdesivir resistance mutations have been generated in vitro but have not been reported in patients receiving treatment with the antiviral agent. We present a case of an immunocompromised patient with acquired B-cell deficiency who developed an indolent, protracted course of SARS-CoV-2 infection. Remdesivir therapy alleviated symptoms and produced a transient virologic response, but her course was complicated by recrudescence of high-grade viral shedding. Whole genome sequencing identified a mutation, E802D, in the nsp12 RNA-dependent RNA polymerase, which was not present in pre-treatment specimens. In vitro experiments demonstrated that the mutation conferred a ~6-fold increase in remdesivir IC50 but resulted in a fitness cost in the absence of remdesivir. Sustained clinical and virologic response was achieved after treatment with casirivimab-imdevimab. Although the fitness cost observed in vitro may limit the risk posed by E802D, this case illustrates the importance of monitoring for remdesivir resistance and the potential benefit of combinatorial therapies in immunocompromised patients with SARS-CoV-2 infection.

Evidence for SARS-CoV-2 Spike Protein in the Urine of COVID-19 Patients.

Background: SARS-CoV-2 infection has, as of April 2021, affected >133 million people worldwide, causing >2.5 million deaths. Because the large majority of individuals infected with SARS-CoV-2 are asymptomatic, major concerns have been raised about possible long-term consequences of the infection. Methods: Wedeveloped an antigen capture assay to detect SARS-CoV-2 spike protein in urine samples from patients with COVID-19whose diagnosis was confirmed by positive PCR results from nasopharyngeal swabs (NP-PCR+) forSARS-CoV-2. We used a collection of 233 urine samples from 132 participants from Yale New Haven Hospital and the Children's Hospital of Philadelphia that were obtained during the pandemic (106 NP-PCR+ and 26 NP-PCR-), and a collection of 20 urine samples from 20 individuals collected before the pandemic. Results: Our analysis identified 23 out of 91 (25%) NP-PCR+ adult participants with SARS-CoV-2 spike S1 protein in urine (Ur-S+). Interestingly, although all NP-PCR+ children were Ur-S-, one child who was NP-PCR- was found to be positive for spike protein in their urine. Of the 23 adults who were Ur-S+, only one individual showed detectable viral RNA in urine. Our analysis further showed that 24% and 21% of adults who were NP-PCR+ had high levels of albumin and cystatin C, respectively, in their urine. Among individuals with albuminuria (>0.3 mg/mg of creatinine), statistical correlation could be found between albumin and spike protein in urine. Conclusions: Together, our data showed that one of four individuals infected with SARS-CoV-2 develop renal abnormalities, such as albuminuria. Awareness about the long-term effect of these findings is warranted.

Feasibility and acceptability of a pilot, peer-led HIV self-testing intervention in a hyperendemic fishing community in rural Uganda.

BACKGROUND: Novel interventions are needed to reach young people and adult men with HIV services given the low HIV testing rates in these population sub-groups. We assessed the feasibility and acceptability of a peer-led oral HIV self-testing (HIVST) intervention in Kasensero, a hyperendemic fishing community (HIV prevalence: 37-41%) in Rakai, Uganda. METHODS: This study was conducted among young people (15-24 years) and adult men (25+ years) between May and August 2019. The study entailed distribution of HIVST kits by trained "peer-leaders," who were selected from existing social networks and trained in HIVST distribution processes. Peer-leaders received up to 10 kits to distribute to eligible social network members (i.e. aged 15-24 years if young people or 25+ years if adult man, not tested in the past 3 months, and HIV-negative or of unknown HIV status at enrolment). The intervention was evaluated against the feasibility benchmark of 70% of peer-leaders distributing up to 70% of the kits that they received; and the acceptability benchmark of >80% of the respondents self-testing for HIV. RESULTS: Of 298 enrolled into the study at baseline, 56.4% (n = 168) were young people (15-24 years) and 43.6% (n = 130) were adult males (25+ years). Peer-leaders received 298 kits and distributed 296 (99.3%) kits to their social network members. Of the 282 interviewed at follow-up, 98.2% (n = 277) reported that they used the HIVST kits. HIV prevalence was 7.4% (n = 21). Of the 57.1% (n = 12) first-time HIV-positives, 100% sought confirmatory HIV testing and nine of the ten (90%) respondents who were confirmed as HIV-positive were linked to HIV care within 1 week of HIV diagnosis. CONCLUSION: Our findings show that a social network-based, peer-led HIVST intervention in a hyperendemic fishing community is highly feasible and acceptable, and achieves high linkage to HIV care among newly diagnosed HIV-positive individuals.

Serum vitamin D levels and risk of prevalent tuberculosis, incident tuberculosis and tuberculin skin test conversion among prisoners.

Poor vitamin D status has been associated with tuberculosis (TB); whether poor status is cause or consequence of disease is uncertain. We conducted a case-control study and two nested case-control studies to determine whether vitamin D levels were associated with active TB, tuberculin skin test (TST) conversion, and risk of progression to the active TB in prisoners in Brazil. In multivariable conditional logistic regression, subnormal vitamin D levels (OR, 3.77; 95% CI, 1.04-13.64) were more likely in prisoners with active TB. In contrast, vitamin D was not found to be a risk factor for either TST conversion (OR, 2.49; 95% CI, 0.64-9.66) or progression to active disease (OR, 0.59; 95% CI, 0.13-2.62). Black race (OR, 11.52; 95% CI, 2.01-63.36), less than 4 years of schooling (OR, 2.70; 95% CI, 0.90-8.16), cigarette smoking (OR, 0.23; 95% CI, 0.06-0.79) were identified as risk factors for TST conversion. Risk of progression to active TB was found to be associated with cigarette smoking (OR, 7.42; 95% CI, 1.23-44.70). Our findings in the prison population show that poor vitamin D status is more common in individuals with active TB, but is not a risk factor for acquisition of latent TB or progression to active TB.

Differences in the Prevalence of Non-Communicable Disease between Slum Dwellers and the General Population in a Large Urban Area in Brazil.

Residents of urban slums are at greater risk for disease than their non-slum dwelling urban counterparts. We sought to contrast the prevalences of selected non-communicable diseases (NCDs) between Brazilian adults living in a slum and the general population of the same city, by comparing the age and sex-standardized prevalences of selected NCDs from a 2010 survey in Pau da Lima, Salvador Brazil, with a 2010 national population-based telephone survey. NCD prevalences in both populations were similar for hypertension (23.6% (95% CI 20.9⁻26.4) and 22.9% (21.2⁻24.6), respectively) and for dyslipidemia (22.7% (19.8⁻25.5) and 21.5% (19.7⁻23.4)). Slum residents had higher prevalences of diabetes mellitus (10.1% (7.9⁻12.3)) and of overweight/obesity (46.5% (43.1⁻49.9)), compared to 5.2% (4.2⁻6.1) and 40.6% (38.5⁻42.8) of the general population in Salvador. Fourteen percent (14.5% (12.1⁻17.0)) of slum residents smoked cigarettes compared to 8.3% (7.1⁻9.5) of the general population in Salvador. The national telephone survey underestimated the prevalence of diabetes mellitus, overweight/obesity, and smoking in the slum population, likely in part due to differential sampling inside and outside of slums. Further research and targeted policies are needed to mitigate these inequalities, which could have significant economic and social impacts on slum residents and their communities.

Rat infestation associated with environmental deficiencies in an urban slum community with high risk of leptospirosis transmission / Infestação por roedores associada a fatores ambientais numa comunidade carente com alto risco de transmissão da leptospirose / Infestación por roedores asociada a factores ambientales en una comunidad sin recursos con alto riesgo de transmisión de la leptospirosis

Abstract: We analyzed environmental factors that provide food, water and harborage to rodents and the risk of household rodent infestation in a slum community with a high risk of leptospirosis transmission. Detailed environmental surveys were performed in 221 households. Multivariate regression models evaluated the association between rodent infestation and socioeconomic status and environmental attributes obtained from Geographical Information System surveys. The general household infestation rate was 45.9%. Rattus norvegicus signs were the most prevalent, present in 74% of the infested households. The risk for rodent infestation was associated with environmental factors supporting harborage for rats, such as dilapidated fences/walls (OR: 8.95; 95%CI: 2.42-33.12) and households built on an earthen slope (OR: 4.68; 95%CI: 2.23-9.81). An increase of 1 meter from the nearest sewer was associated with a 3% (95%CI: 1%-5%) decrease in the risk of rodent infestation. A lack of sanitation where poor people live provides factors for rat infestation and could the target of educational interventions.

Resumo: O estudo analisou fatores ambientais que facilitam a presença de alimento, água e abrigo para roedores e risco de infestação por roedores numa comunidade com alto risco de transmissão da leptospirose. Foram realizados inquéritos ambientais detalhados em 221 domicílios. Modelos de regressão multivariada avaliaram a associação entre infestação por roedores e nível socioeconômico e atributos ambientais obtidos através de inquéritos com Sistemas de Informação Geográfica. O estudo mostrou uma taxa global de 45,9% de infestação domiciliar. Sinais de Rattus norvegicus eram os mais prevalentes, presentes em 74% dos domicílios infestados. O risco de infestação por roedores esteve associado a fatores ambientais que forneciam abrigo aos ratos, tais como cercas e muros dilapidados (OR: 8,95; IC95%: 2,42-33,12) e domicílios construídos diretamente sobre encostas (OR: 4,68; IC95%: 2,23-9,81). Cada incremento de um metro de distância a partir do esgoto mais próximo esteve associado a uma diminuição de 3% (IC95%: 1%-5%) no risco de infestação por roedores. A falta de saneamento básico nos locais de moradia das famílias pobres facilita a infestação por ratos e é alvo prioritário para intervenções educativas.

Resumen: El estudio analizó factores ambientales que facilitan la presencia de alimento, agua y abrigo para roedores y el riesgo de infestación por roedores en una comunidad sin recursos con alto riesgo de transmisión de la leptospirosis. Se realizaron encuestas ambientales detalladas en 221 domicilios. Los modelos de regresión multivariada evaluaron la asociación entre infestación por roedores, nivel socioeconómico y características ambientales, obtenidos a través de encuestas con Sistemas de Información Geográfica. El estudio mostró una tasa global de un 45,9% de infestación domiciliaria. Los indicios de Rattus norvegicus eran los más prevalentes, presentes en un 74% de los domicilios infestados. El riesgo de infestación por roedores estuvo asociado a factores ambientales que proporcionaban abrigo a las ratas, tales como cercas y muros derruidos (OR: 8,95; IC95%: 2,42-33,12) y domicilios construidos directamente sobre pendientes (OR: 4,68; IC95%: 2,23-9,81). Cada incremento de un metro de distancia, a partir del alcantarillado más próximo, estuvo asociado a una disminución de un 3% (IC95%: 1%-5%) en el riesgo de infestación por roedores. La falta de saneamiento básico en las viviendas de las familias pobres facilita la infestación por ratas y es el objetivo prioritario para intervenciones educativas.

A Cross-Sectional Survey of HIV Testing and Prevalence in Twelve Brazilian Correctional Facilities.

BACKGROUND: Prior studies have reported higher HIV prevalence among prisoners than the general population in Brazil, but data have been derived from single prisons. The aim of this study was to evaluate HIV testing practices, prevalence and linkage to care among inmates in a network of 12 prisons. METHODS: We administered a questionnaire to a population-based sample of inmates from 12 prisons in Central-West Brazil and collected sera for HIV and syphilis testing from January to December 2013. We evaluated factors associated with HIV testing and infection using multivariable logistic regression models. Six months after HIV testing, we assessed whether each HIV-infected prisoner was engaged in clinical care and whether they had started antiretroviral therapy. RESULTS: We recruited 3,362 inmates, of whom 2,843 (85%) were men from 8 prisons, and 519 (15%) were women from 4 prisons. Forty-five percent of participants reported never having been tested for HIV previously. In multivariable analysis, the variables associated with previous HIV testing were lack of a stable partner (adjusted odds ratio [AOR]: 1.38; 95% CI: 1.18-1.60), completed more than four years of schooling (AOR 1.40; 95% CI: 1.20-1.64), history of previous incarceration (AOR: 1.68; 95% CI: 1.43-1.98), history of mental illness (AOR 1.52; 95% CI: 1.31-1.78) and previous surgery (AOR 1.31; 95% CI: 1.12-1.52). Fifty-four (1.6%) of all participants tested positive for HIV; this included 44 (1.54%) men and 10 (1.92%) women. Among male inmates, HIV infection was associated with homosexuality (AOR 6.20, 95% CI: 1.73-22.22), self-report of mental illness (AOR 2.18, 95% CI: 1.13-4.18), history of sexually transmitted infections (AOR 3.28, 95% CI: 1.64-6.56), and syphilis sero-positivity (AOR 2.54, 95% CI: 1.20-5.39). Among HIV-infected individuals, 34 (63%) were unaware of their HIV status; only 23 of these 34 (68%) newly diagnosed participants could be reached at six month follow-up, and 21 of 23 (91%) were engaged in HIV care. CONCLUSIONS: HIV testing rates among prison inmates are low, and the majority of HIV-infected inmates were unaware of their HIV diagnosis. Incarceration can be an opportunity for diagnosis and treatment of HIV among vulnerable populations who have poor access to health services, but further work is needed on transitional HIV care for released inmates.

Active and latent tuberculosis in Brazilian correctional facilities: a cross-sectional study.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Oxidative stress markers correlate with renal dysfunction and thrombocytopenia in severe leptospirosis.

Leptospirosis is a zoonotic disease that causes severe manifestations such as Weil's disease and pulmonary hemorrhage syndrome. The aim of this study was to evaluate whether reactive oxygen species (ROS) production and antioxidant reduced glutathione (GSH) levels are related to complications in patients hospitalized with leptospirosis. The ROS production and GSH levels were measured in blood samples of 12 patients and nine healthy controls using chemiluminescence and absorbance assays. We found that ROS production was higher and GSH levels were lower in leptospirosis patients compared with healthy individuals. Among patients, GSH depletion was correlated with thrombocytopenia and elevated serum creatinine, whereas a strong positive correlation was observed between ROS production and elevated serum potassium. Additional investigation of the biological significance of ROS production and GSH levels is warranted as they may guide the development of novel adjuvant therapies for leptospirosis targeting oxidative stress.

Surgery for valvular heart disease: a population-based study in a Brazilian urban center.

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.

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin.

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.

Genetic diversity of the Leptospiral immunoglobulin-like (Lig) genes in pathogenic Leptospira spp.

Recent serologic, immunoprotection, and pathogenesis studies identified the Lig proteins as key virulence determinants in interactions of leptospiral pathogens with the mammalian host. We examined the sequence variation and recombination patterns of ligA, ligB, and ligC among 10 pathogenic strains from five Leptospira species. All strains were found to have intact ligB genes and genetic drift accounting for most of the ligB genetic diversity observed. The ligA gene was found exclusively in L. interrogans and L. kirschneri strains, and was created from ligB by a two-step partial gene duplication process. The aminoterminal domain of LigB and the LigA paralog were essentially identical (98.5+/-0.8% mean identity) in strains with both genes. Like ligB, ligC gene variation also followed phylogenetic patterns, suggesting an early gene duplication event. However, ligC is a pseudogene in several strains, suggesting that LigC is not essential for virulence. Two ligB genes and one ligC gene had mosaic compositions and evidence for recombination events between related Leptospira species was also found for some ligA genes. In conclusion, the results presented here indicate that Lig diversity has important ramifications for the selection of Lig polypeptides for use in diagnosis and as vaccine candidates. This sequence information will aid the identification of highly conserved regions within the Lig proteins and improve upon the performance characteristics of the Lig proteins in diagnostic assays and in subunit vaccine formulations with the potential to confer heterologous protection.

Reversal of renal tubule transporter downregulation during severe leptospirosis with antimicrobial therapy.

Tubular dysfunction is a hallmark of severe leptospirosis. Antimicrobial therapy is thought to interfere on renal involvement. We evaluated the expression of a proximal tubule type-3 Na+/H+ exchanger (NHE3) and a thick ascending limb Na+-K+-2Cl(-) cotransporter (NKCC2) in controls and treated hamsters. Animals infected by a serovar Copenhageni isolate, were treated or not with ampicillin (AMP) and/or N-acetylcysteine (NAC). Leptospiral antigen(s) and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen(s) in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. NAC effect was restricted to lowering TBARS. Early and late AMP treatment rescued tubular defects in severe leptospirosis disease, and there was no evidence of benefit from antioxidant therapy.

Disseminated leishmaniasis: a new and emerging form of leishmaniasis observed in northeastern Brazil.

During the past decade, there has been an increase in the number of patients with disseminated leishmaniasis (DL), which is characterized by a large number of acneiform and papular skin lesions, with very few or no parasites in the skin tissue. The present report describes 42 cases of DL identified between 1992 and 1998 in an area where Leishmania braziliensis transmission is endemic; 8 of the patients were prospectively diagnosed. In a contrast to localized cutaneous leishmaniasis (LCL), acquisition of DL was associated with age >19 years (P<.05), male sex (P<.05), and agricultural occupation (P<.001). Patients with DL presented with 10-300 lesions that were a mixture of acneiform, papular, nodular, and ulcerated types. Twelve (29%) of 42 patients had mucosal involvement. Patients with DL had lower levels of interferon-gamma (P<.05) and tumor necrosis factor-alpha (P<.05) production, compared with patients with LCL. DL is an emerging clinical distinct form of leishmaniasis associated with agricultural activities and host immunological response.

Rapid translocation of polarized MDCK cell monolayers by Leptospira interrogans, an invasive but nonintracellular pathogen.

Pathogenic spirochetes of the genus Leptospira are a major cause of human zoonotic infectious disease worldwide. After gaining entry through the skin, the organism causes disease by hematogenously disseminating to multiple organs. The mechanism by which it penetrates the mammalian cell barriers to disseminate is not well understood. In this study, we used a low-passage-number isolate of Leptospira interrogans to elucidate the invasive potential of this spirochete. Quantification of bacteria by dark-field microscopy revealed that pathogenic spirochetes were able to translocate through polarized MDCK cell monolayers at a rate significantly greater than that of nonpathogenic Leptospira or a recognized invasive bacterial pathogen, Salmonella: In contrast to Salmonella, L. interrogans did not alter transepithelial electrical resistance during cell translocation. Both transmission and scanning electron microscopy revealed tight association of the extracellular spirochetes with the host cell plasma membrane, without membrane perturbations suggestive of cytoskeletal rearrangement. Spirochetes were not observed within intercellular junctions or membrane-bound compartments inside cells. They were found within the cytoplasm of only 8% of the counted cells. These results indicate that Leptospira is an invasive but not a facultative intracellular organism. We propose that the rapid translocation of mammalian cells by pathogenic Leptospira is a mechanism designed to evade killing by host cells that permits the organism to quickly reach the bloodstream and disseminate to multiple organs.

Characterization of protective and non-protective surface membrane carbohydrate epitopes of Schistosoma mansoni

We have produced a number of monoclonal antibodies, protective and non-protective, which recognize a complex of schistosomula antigens, including the 38 kDa antigen. Eight different protective and non-protective monoclonal antibodies, varying in isotypes, were used in the binding assays. Lectin inhibition studies suggested that the monoclonal antibodies probably recognized carbohydrate epitopes on the antigen(s). Immunoprecipitation studies showed that at least two of the monoclonal antibodies recognized different epitopes on the same molecule. Additionally, we tested for monoclonal antibody binding after the antigens were treated with; 1) proteases, 2) periodate, 3) various exo- and endoglycosidases, 4) mild acid hydrolysis. We also tested for binding of the antibodies to keyhole limpet hemocyanin (KLH). Using the 8 monoclonal antibodies as probes, we were able to define at least 4 different carbohydrate epitopes related to the protective monoclonal antibodies, and at least one epitope which is seen by the non-protective antibodies. The epitope seen by the non-protective antibodies was shown to be cross-reactive with epitopes on KLH. These results demonstrate the importance of epitope mapping studies for any defined vaccine.