Monitoring the Efficacy of Chloroquine-Primaquine Therapy for Uncomplicated Plasmodium vivax Malaria in the Main Transmission Hot Spot of Brazil.

Emerging Plasmodium vivax resistance to chloroquine (CQ) may undermine malaria elimination efforts in South America. CQ-resistant P. vivax has been found in the major port city of Manaus but not in the main malaria hot spots across the Amazon Basin of Brazil, where CQ is routinely coadministered with primaquine (PQ) for radical cure of vivax malaria. Here we randomly assigned 204 uncomplicated vivax malaria patients from Juruá Valley, northwestern Brazil, to receive either sequential (arm 1) or concomitant (arm 2) CQ-PQ treatment. Because PQ may synergize the blood schizontocidal effect of CQ and mask low-level CQ resistance, we monitored CQ-only efficacy in arm 1 subjects, who had PQ administered only at the end of the 28-day follow-up. We found adequate clinical and parasitological responses in all subjects assigned to arm 2. However, 2.2% of arm 1 patients had microscopy-detected parasite recrudescences at day 28. When PCR-detected parasitemias at day 28 were considered, response rates decreased to 92.1% and 98.8% in arms 1 and 2, respectively. Therapeutic CQ levels were documented in 6 of 8 recurrences, consistent with true CQ resistance in vivo In contrast, ex vivo assays provided no evidence of CQ resistance in 49 local P. vivax isolates analyzed. CQ-PQ coadministration was not found to potentiate the antirelapse efficacy of PQ over 180 days of surveillance; however, we suggest that larger studies are needed to examine whether and how CQ-PQ interactions, e.g., CQ-mediated inhibition of PQ metabolism, modulate radical cure efficacy in different P. vivax-infected populations. (This study has been registered at ClinicalTrials.gov under identifier NCT02691910.).

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays.

Plasmodium vivax chloroquine resistance has been documented in nearly every region where this malaria-causing parasite is endemic. Unfortunately, P. vivax resistance surveillance and drug discovery are challenging due to the low parasitemias of patient isolates and poor parasite survival through ex vivo maturation that reduce the sensitivity and scalability of current P. vivax antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for P. vivax parasites. We next performed a medium screen for formulations that enhance ex vivo survival. Finally, we optimized an isotopic metabolic labeling assay for measuring P. vivax maturation and its sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume ex vivo isolates by an average of >40-fold. The use of Iscove's modified Dulbecco's medium for P. vivax ex vivo culture approximately doubled the parasite survival through maturation. Coupling these with [3H]hypoxanthine metabolic labeling permitted sensitive and robust measurements of parasite maturation, which was used to measure the sensitivities of Brazilian P. vivax isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the P. vivax isolate sensitivities to antimalarials for resistance surveillance and drug discovery.

Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model.

Visceral leishmaniasis (VL) is a fatal disease for humans, and no vaccine is currently available. Sand fly salivary proteins have been associated with protection against cutaneous leishmaniasis. To test whether vector salivary proteins can protect against VL, a hamster model was developed involving intradermal inoculation in the ears of 100,000 Leishmania infantum chagasi parasites together with Lutzomyia longipalpis saliva to mimic natural transmission by sand flies. Hamsters developed classical signs of VL rapidly, culminating in a fatal outcome 5-6 months postinfection. Saliva had no effect on the course of infection in this model. Immunization with 16 DNA plasmids coding for salivary proteins of Lu. longipalpis resulted in the identification of LJM19, a novel 11-kDa protein, that protected hamsters against the fatal outcome of VL. LJM19-immunized hamsters maintained a low parasite load that correlated with an overall high IFN-gamma/TGF-beta ratio and inducible NOS expression in the spleen and liver up to 5 months postinfection. Importantly, a delayed-type hypersensitivity response with high expression of IFN-gamma was also noted in the skin of LJM19-immunized hamsters 48 h after exposure to uninfected sand fly bites. Induction of IFN-gamma at the site of bite could partly explain the protection observed in the viscera of LJM19-immunized hamsters through direct parasite killing and/or priming of anti-Leishmania immunity. We have shown that immunity to a defined salivary protein (LJM19) confers powerful protection against the fatal outcome of a parasitic disease, which reinforces the concept of using components of arthropod saliva in vaccine strategies against vector-borne diseases.

Immunomodulation of human monocytes following exposure to Lutzomyia intermedia saliva.

BACKGROUND: Sand fly saliva contains potent and complex pharmacologic molecules that are able to modulate the host's hemostatic, inflammatory, and immune systems. In this study, we evaluated the effects of salivary gland sonicate (SGS) of Lutzomyia intermedia, the natural vector of Leishmania braziliensis, on monocytes obtained from the peripheral blood mononuclear cells (PBMC) of healthy volunteers. We investigated the effects of sand fly saliva on cytokine production and surface molecule expression of LPS-stimulated human monocytes uninfected or infected with L. braziliensis. RESULTS: Pre-treatment of non-infected human monocytes with L. intermedia SGS followed by LPS-stimulation led to a significant decrease in IL-10 production accompanied by a significant increase in CD86, CD80, and HLA-DR expression. Pre-treatment with SGS followed by LPS stimulation and L. braziliensis infection led to a significant increase in TNF-alpha, IL-6, and IL-8 production without significant alterations in co-stimulatory molecule expression. However, pre-treatment with L. intermedia SGS did not result in significant changes in the infection rate of human monocytes. CONCLUSION: Our data indicate that L. intermedia saliva is able to modulate monocyte response, and, although this modulation is dissociated from enhanced infection with L. braziliensis, it may be associated with successful parasitism.

Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax.

BACKGROUND: The Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax. METHODS: We used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences), Peru (PER, n = 23), Colombia (COL, n = 31), and Mexico (MEX, n = 19). PRINCIPAL FINDINGS/CONCLUSIONS: We found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10-4 and 6.2 × 10-4) as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed) in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o) gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092). Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between parasite lineages from geographically diverse sites. Further genome-wide analyses are required to test the demographic scenario suggested by our data.

Infection of laboratory colonies of Anopheles mosquitoes with Plasmodium vivax from cryopreserved clinical isolates.

Plasmodium vivax is the most geographically widespread malaria parasite. Unique features of transmission biology complicate P. vivax control. Interventions targeting transmission are required for malaria eradication. In the absence of an in vitro culture, transmission studies rely on live isolates from non-human primates or endemic regions. Here, we demonstrate P. vivax gametocytes from both India and Brazil are stable during cryopreservation. Importantly, cryopreserved gametocytes from Brazil were capable of infecting three anopheline mosquito species in feedings done in the United States. These findings create new opportunities for transmission studies in diverse locales.

Epidemiology of disappearing Plasmodium vivax malaria: a case study in rural Amazonia.

BACKGROUND: New frontier settlements across the Amazon Basin pose a major challenge for malaria elimination in Brazil. Here we describe the epidemiology of malaria during the early phases of occupation of farming settlements in Remansinho area, Brazilian Amazonia. We examine the relative contribution of low-density and asymptomatic parasitemias to the overall Plasmodium vivax burden over a period of declining transmission and discuss potential hurdles for malaria elimination in Remansinho and similar settings. METHODS: Eight community-wide cross-sectional surveys, involving 584 subjects, were carried out in Remansinho over 3 years and complemented by active and passive surveillance of febrile illnesses between the surveys. We used quantitative PCR to detect low-density asexual parasitemias and gametocytemias missed by conventional microscopy. Mixed-effects multiple logistic regression models were used to characterize independent risk factors for P. vivax infection and disease. PRINCIPAL FINDINGS/CONCLUSIONS: P. vivax prevalence decreased from 23.8% (March-April 2010) to 3.0% (April-May 2013), with no P. falciparum infections diagnosed after March-April 2011. Although migrants from malaria-free areas were at increased risk of malaria, their odds of having P. vivax infection and disease decreased by 2-3% with each year of residence in Amazonia. Several findings indicate that low-density and asymptomatic P. vivax parasitemias may complicate residual malaria elimination in Remansinho: (a) the proportion of subpatent infections (i.e. missed by microscopy) increased from 43.8% to 73.1% as P. vivax transmission declined; (b) most (56.6%) P. vivax infections were asymptomatic and 32.8% of them were both subpatent and asymptomatic; (c) asymptomatic parasite carriers accounted for 54.4% of the total P. vivax biomass in the host population; (d) over 90% subpatent and asymptomatic P. vivax had PCR-detectable gametocytemias; and (e) few (17.0%) asymptomatic and subpatent P. vivax infections that were left untreated progressed to clinical disease over 6 weeks of follow-up and became detectable by routine malaria surveillance.

Noninvasive ventilation coupled with nebulization during asthma crises: a randomized controlled trial.

BACKGROUND: Despite the clinical improvements attributed to noninvasive ventilation (NIV) during asthma crises, and the well established effects of nebulization, there are few studies on the effects of these interventions together. We hypothesized that nebulization coupled to NIV should raise radio-aerosol pulmonary deposition in asthmatics. The aims of this study were to assess the effects of coupling ß-agonist nebulization and NIV during asthma exacerbations on radio-aerosol pulmonary deposition, using scintigraphy and cardiopulmonary parameters, to correlate pulmonary function with radio-aerosol deposition index, radio-aerosol penetration index, and pulmonary clearance. METHODS: In this controlled trial, 21 adults with moderate to severe asthma attack were randomized to a control group (n = 11) or experimental group (NIV + nebulizer group, n = 10). All subjects inhaled bronchodilators for 9 minutes, and after particles were counted with a gamma camera to analyze regions of interest and pulmonary clearance at 0, 15, 30, 45, and 60 min. RESULTS: Breathing frequency (P = < .001) and minute ventilation (P = .01) were reduced, and tidal volume was increased (P = .01) in the NIV + nebulizer group, compared with the control group. The NIV + nebulizer group had improvement from baseline values, compared to the control group in the following parameters: FEV(1) 46.7 ± 0.5% of predicted vs 29.8 ± 8.9% of predicted, P = .02), FVC (41.2 ± 1.5% of predicted vs 23.2 ± 7.1% of predicted, P = .02), peak expiratory flow (67.3 ± 38.3% of predicted vs 26.9 ± 12.1% of predicted, P = .01), and inspiratory capacity (54.9 ± 28.8% of predicted vs 31.2 ± 9.1% of predicted, P = .01). No differences were observed between groups regarding radio-aerosol deposition index or pulmonary clearance. Negative correlations were found between FEV1, forced expiratory flow during the middle half of the FVC maneuver (FEF(25-75%)), inspiratory capacity, and radio-aerosol penetration index. CONCLUSIONS: Coupling nebulization and NIV during asthma exacerbation did not improve radio-aerosol pulmonary deposition, but we observed clinical improvement of pulmonary function in these subjects. (ClinicalTrials.gov registration NCT01012050).

Single-nucleotide polymorphism and copy number variation of the multidrug resistance-1 locus of Plasmodium vivax: local and global patterns.

Emerging resistance to chloroquine (CQ) poses a major challenge for Plasmodium vivax malaria control, and nucleotide substitutions and copy number variation in the P. vivax multidrug resistance 1 (pvmdr-1) locus, which encodes a digestive vacuole membrane transporter, may modulate this phenotype. We describe patterns of genetic variation in pvmdr-1 alleles from Acre and Amazonas in northwestern Brazil, and compare then with those reported in other malaria-endemic regions. The pvmdr-1 mutation Y976F, which is associated with CQ resistance in Southeast Asia and Oceania, remains rare in northwestern Brazil (1.8%) and its prevalence mirrors that of CQ resistance worldwide. Gene amplification of pvmdr-1, which is associated with mefloquine resistance but increased susceptibility to CQ, remains relatively rare in northwestern Brazil (0.9%) and globally (< 4%), but became common (> 10%) in Tak Province, Thailand, possibly because of drug-mediated selection. The global database we have assembled provides a baseline for further studies of genetic variation in pvmdr-1 and drug resistance in P. vivax malaria.

Avaliação do Sistema de Vigilância Epidemiológica da Esquistossomose em dois municípios do estado da Bahia, Brasil / Evaluation of the Schistosomiasis Epidemiologic Surveillance System in two municipalities of the state of Bahia, Brazil

Objetivo: avaliar o Sistema de Vigilância Epidemiológica (SVE) da esquistossomose nos municípios de Antônio Cardoso e Catu, estado da Bahia, Brasil. Métodos: avaliação realizada em 2004, contemplou os determinantes contextuais para a implementação da intervenção, estrutura e processo do SVE da esquistossomose, utilizando, como estratégia de pesquisa, o estudo de caso; para coleta dos dados, foram empregados os roteiros para avaliação de sistemas de vigilância dos Centros de Controle e Prevenção de Doenças dos Estados Unidos da América, além de questionários para análise do contexto, estrutura e processo. Resultados: os resultados indicam um SVE fragmentado, individualizado e não priorizado pelos gestores locais. Conclusão: é necessário um trabalho integrado entre os funcionários da Vigilância Epidemiológica e outros setores da Saúde, especialmente a Atenção Básica, de retroalimentação do SVE e reestruturação do Programa de Vigilância e Controle da Esquistossomose.

Objective: to evaluate the Schistosomiasis Epidemiologic Surveillance System (SESS) in the municipalities of Antônio Cardoso and Catu, state of Bahia, Brazil. Methods: evaluation carried out in 2004, considered the contextual determinants for the implementation of the intervention, structure and process of the SESS; it was adopted the research strategy of case study; for data collection, it was used the US Atlanta-based Centers for Disease Control and Prevention Guidelines for Evaluating Surveillance Systems, and questionnaires to analyse context, structure and process. Results: the study results indicate an SESS fragmented, individualized, and not prioritized by local managers. Conclusion: it is necessary an integrate work between Epidemiologic Surveillance professionals and other Health sectors, specially Primary Health Care, as well asfeedback of generated information by the SESS and restructuring of the Schistosomiasis Surveillance and Control Program.

Influence of light-activated and auto- and dual-polymerizing adhesive systems on bond strength of indirect composite resin to dentin.

STATEMENT OF PROBLEM: Clinicians must be aware of the bonding effectiveness of auto- and dual- polymerizing adhesive systems before choosing the material and technique of cementing inlay/onlays to dentin. An inadequate choice may compromise the success of indirect restorations. PURPOSE: This study compared the microtensile bond strength (MTBS) of indirect composite resin bonded to dentin by light-activated, autopolymerizing, and dual-polymerizing adhesive systems. MATERIAL AND METHODS: Occlusal dentin surfaces of 36 human third molars were exposed and flattened. Teeth were assigned to 1 of the following 6 groups (n=6) of adhesive luting systems: 2 dual-polymerizing systems (Scotchbond Multipurpose Plus/Rely X [SBMP] and Prime & Bond NT Dual Cure/Enforce [PBDC]); 1 autopolymerizing system (ED Primer/Panavia F [EDP]); and 3 light-activated systems (control groups) (Adper Single Bond/Rely X [SB], Prime & Bond NT/Enforce [PB], and Clearfil SE Bond/Panavia F [CF]). The restorative materials were applied according to manufacturer's directions. A 2-mm-thick prepolymerized composite resin (Clearfil APX) disc was cemented with the resin cements on the bonded dentin. Teeth were stored in water at 37 degrees C for 24 hours. Afterwards, teeth were sectioned both mesial-distally and buccal-lingually to obtain multiple bonded beam specimens with 0.8 mm(2) of cross-sectional area. Each specimen was tested in tension at a crosshead speed of 0.5 mm/min until failure. Data (MPa) were analyzed by 1-way analysis of variance and the Tukey post hoc test (alpha=.05). Failure patterns of tested specimens were analyzed using scanning electron microscopy. RESULTS: Mean MTBS values (MPa) for experimental groups were as follows: SBMP, 32.89 +/- 3.26(a); SB, 26.74 +/- 7.45(ab); PB, 26.11 +/- 4.48(ab); CF, 25.30 +/- 6.42(ab); EDP, 16.82 +/- 5.53(bc); PBDC, 11.20 +/- 5.89(c) (P<.001). Groups with similar lowercase letters were not significantly different. Failure pattern of fractured specimens varied according to the polymerization mode. CONCLUSION: The autopolymerizing system and one of the dual-polymerizing systems were as effective as the light-activated systems in bonding indirect composite restorations to dentin.

Avaliação do sistema de vigilância epidemiológica da esquistossomose no estado da Bahia / Evaluation of the schistosomiasis epidemiological surveillance system in the state of Bahia

Este estudo se propôs a avaliar o Sistema de Vigilância Epidemiológica (SVE) da Esquistossomose no Estado da Bahia, considerando tanto a esfera estadual como a municipal, esta restrita a dois municípios situados na área endêmica para a esquistossomose no Estado. Trata-se de um estudo avaliativo que contempla os determinantes contextuais da implantação, da intervenção e da avaliação da estrutura e do processo da vigilância epidemiológica da esquistossomose, e utiliza como estratégia de pesquisa o estudo de caso (municípios). Para a coleta dos dados, foram utilizados osroteiros para avaliação de sistemas de vigilância dos Centros de Controle de Doenças - CDC/Atlanta - Estados Unidos, e questionários específicos para análise do contexto, estrutura, processo e atributos do sistema: oportunidade, aceitabilidade, simplicidade, flexibilidade, representatividade, sensibilidade, valor preditivo positivo e utilidade. Na série histórica do percentual de positividade para Schistosoma mansoni, no Estado daBahia, considerando a implantação do Programa de Controle da Esquistossomose (PCE), observou-se uma redução das taxas de positividade. Em 1979, o percentual médio foi de 25,1%, enquanto que entre 2001 e 2003, as taxas mantiveram-se na faixade 5,6%. O mesmo observou-se nos dois municípios, em Antônio Cardoso, o percentual foi 31,0% em 1983 e de 5,6% em 2003; em Catu, foi de 23,0% em 1990 e de 8,0% em 2003. Constatou-se, igualmente, que no Estado da Bahia o Programa de Controle daEsquistossomose foi descentralizado para os municípios e que em cada um deles existe um subsistema de vigilância epidemiológica como parte do SVE municipal, não tendo ocorrido interrupção das atividades, de modo a manter-se o número de examesrealizados nos anos de estabilização do programa. Os resultados baseados na percepção dos entrevistados indicam a existência de um sistema de vigilância de...

Efeitos da saliva de lutzomyia intermédia sobre monócitos humanos / Effects of the saliva of intermediate lutzomyia on human monocytes

A saliva dos flebotomíneos contém moléculas farmacológicas potentes e complexas que, quando inoculadas na pele do vertebrado, são capazes de modular os sistemas hemostático, inflamatório e imune do hospedeiro. No presente estudo, nós avaliamos os efeitos do conteúdo da glândula salivar de Lutzomyia intermedia, o vetor natural da Leishmania braziliensis, na produção de citocinas e na expressão de moléculas de superfície em monócitos estimulados com LPS, infectados ou não por L. braziliensis. Avaliamos também a carga parasitária em células infectadas e expressão de quimiocinas em monócitos pré-sensibilizados com a saliva de L. intermedia e com a saliva de L. longipalpis. Monócitos selecionados de células mononucleares do sangue periférico (CMSP) de doadores saudáveis foram pré-sensibilizados com SGS de L. intermedia, estimulados com LPS e, em seguida, infectados ou não com L. braziliensis. A produção de citocinas foi avaliada por ELlSA em sobrenadante de cultura; expressão de moléculas de superfície foi determinada por citometria de fluxo e a expressão de quimiocinas foi analisada por PCR em Tempo Real em ensaios de Quantificação Relativa (QR). Monócitos humanos pré-tratados com SGS de L. intermedia mostraram uma redução significante na produção de IL-10 e um aumento significante na expressão de CD86. A infecção com L. braziliensis aumentou significativamente a produção de TNF-a, porém não alterou a expressão de CD86, nem a taxa de infecção em monócitos pré-sensibilizados com SGS. A expressão de IL-8, MIP-1a, MIP-1f3 e Rantes aumentou após estímulo de monócitos humanos com SGS de L. intermedia e L. longipalpis. Nossos dados indicam que a saliva de L. intermedia é capaz de modificar a produção de citocinas, a expressão de moléculas de superfície e a expressão de quimiocinas em monócitos humanos e estas alterações podem afetar a evolução da leishmaniose em infecções naturais.