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Zika virus (ZIKV) infections occurred in epidemic form in the Americas in 2014–2016, with some of the earliest isolates in the region coming from Haiti. We isolated ZIKV from 20 children with acute undifferentiated febrile illness who were part of a cohort of children seen at a school clinic in the Gressier region of Haiti. The virus was also isolated from three pools of Aedes aegypti mosquitoes collected at the same location. On phylogenetic analysis, three distinct ZIKV clades were identified. Strains from all three clades were present in Haiti in 2014, making them among the earliest isolates identified in the Western Hemisphere. Strains from all three clades were also isolated in 2016, indicative of their persistence across the time period of the epidemic. Mosquito isolates were collected in 2016 and included representatives from two of the three clades; in one instance, ZIKV was isolated from a pool of male mosquitoes, suggestive of vertical transmission of the virus. The identification of multiple ZIKV clades in Haiti at the beginning of the epidemic suggests that Haiti served as a nidus for transmission within the Caribbean.

To identify factors affecting compliance with follow-up during treatment in confirmed malaria patients at two health centers in Haiti.A prospective observational study of malaria patients undergoing treatment over a six-week period. Patients' return visits (follow-up visits) to the health centers for consultation in accordance with the physicians' requests were recorded and used to determine compliance. Socioeconomic data were obtained from patient enrollment questionnaires and through post-treatment interviews. The management practices and procedures at the health centers to retain patients were also reviewed. Descriptive statistics and Spearman's rank correlation were used to identify significant factors, which were used as variables in a logistic regression model.Sixty-eight percent of the malaria patients completed follow-up, with higher compliance being recorded in the larger, more established health center of Leogane (67%) than Cite Soleil (33%). The patient socioeconomic profiles differed between the two health center locations by level of education, religious diversity, household size, and percentage of married individuals. Crude logistic regression analyses identified health center location (OR = 0.179 [95% CI 0.064, 0.504]) and household size (OR = 1.374 [95% CI 1.056, 1.787]) to be associated with compliance. The adjusted model only identified health center location (OR = 0.226 [95% CI 0.056, 0.918]) as significantly associated with compliance.Although patients' household size may be important according to the crude logistic regression analysis, in the adjusted analysis the site location of the health center where patients receive treatment was identified as the only important factor associated with follow-up compliance in malaria patients during treatment in Haiti. This information might be helpful to improve treatment outcomes and contribute to the monitoring of antimalarial resistance in Haiti.Determinar los factores que afectan el cumplimiento del seguimiento durante el tratamiento de los pacientes con malaria confirmada en dos centros de salud de Haití.Se llevó a cabo un estudio observacional prospectivo de los pacientes con malaria que recibían tratamiento a lo largo de un período de seis semanas. Se registraron las consultas subsiguientes de los pacientes a los centros de salud (consultas de seguimiento) de acuerdo con la solicitud de los médicos, que se emplearon para determinar el cumplimiento. Se obtuvieron los datos socioeconómicos de los cuestionarios del registro de pacientes y mediante entrevistas postratamiento. También se examinaron las prácticas y los procedimientos de gestión del centro de salud para promover la retención de los pacientes. Se emplearon estadísticas descriptivas y la correlación de rangos de Spearman para determinar los factores significativos, que se usaron como variables en un modelo de regresión logística.El 68% de los enfermos de malaria completaron el seguimiento. El mayor cumplimiento se registró en el centro de salud más grande y de mayor prestigio de Léogâne (67%) en comparación con el centro de Cité Soleil (33%). Los perfiles socioeconómicos de los pacientes difirieron entre las dos ubicaciones del centro de salud en lo concerniente al nivel de escolaridad, diversidad religiosa, tamaño del hogar y porcentaje de personas casadas. Los análisis crudos de regresión logística determinaron que había una asociación entre la ubicación del centro de salud (OR = 0,179 [IC de 95 % 0,064, 0,504]) y el tamaño del hogar (OR = 1,374 [IC de 95 % 1,056, 1,787]) con el cumplimiento. En el modelo ajustado se encontró que solo la ubicación del centro de salud (OR = 0,226 [IC de 95 % 0,056, 0,918]) estaba significativamente relacionada con el cumplimiento.Aunque el tamaño del hogar de los pacientes podría ser importante según el análisis crudo de regresión logística, en el análisis ajustado se determinó que la ubicación del centro de salud en que los pacientes reciben el tratamiento era el único factor importante asociado con el cumplimiento de seguimiento de los pacientes con malaria en tratamiento en Haití. Es posible que esta información sea útil para mejorar los resultados del tratamiento y contribuir al seguimiento de la resistencia a los antimaláricos en Haití.Identificar os fatores que afetam a adesão ao seguimento durante o tratamento da malária em pacientes com diagnóstico confirmado em dois centros de saúde no Haiti.Estudo observacional prospectivo de pacientes com malária em tratamento durante um período de seis semanas. Os retornos dos pacientes (consultas de seguimento) aos centros de saúde para consulta, conforme solicitado pelos médicos, foram registrados e usados para determinar a adesão. Os dados socioeconômicos foram obtidos a partir dos cadastros dos pacientes e por meio de entrevistas pós-tratamento. As práticas e procedimentos de gestão para reter pacientes no centro de saúde também foram analisados. Foram utilizadas estatísticas descritivas e correlação de Spearman para identificar fatores significativos, que foram então incluídos como variáveis em um modelo de regressão logística.Sessenta e oito por cento dos pacientes com malária concluíram o seguimento. A adesão foi superior no centro de saúde maior e mais estabelecido de Léogâne (67%) do que no de Cité Soleil (33%). Os perfis socioeconômicos dos pacientes nos dois centros de saúde divergiram em relação à escolaridade, diversidade religiosa, tamanho da família e porcentagem de indivíduos casados. As análises de regressão logística brutas identificaram a localização do centro de saúde (OR = 0,179 [IC 95% 0,064; 0,504]) e o número de residentes no domicílio (OR = 1,374 [IC 95% 1,056; 1,787]) como fatores associados à adesão. O modelo ajustado identificou apenas a localização do centro de saúde (OR = 0,226 [IC 95% 0,056; 0,918]) como fator significativamente associado à conformidade.Embora o número de residentes no domicílio dos pacientes possa ser importante de acordo com a análise de regressão logística bruta, na análise ajustada a localização do centro de saúde onde os pacientes receberam tratamento foi identificada como o único fator importante associado à adesão ao seguimento em pacientes com malária durante o tratamento no Haiti. Essas informações podem ser úteis para melhorar os desfechos do tratamento e contribuir para o monitoramento da resistência aos antimaláricos no Haiti.

Burkholderia pseudomallei, the etiological agent of melioidosis, has been hypothesized to be endemic throughout the Caribbean, including the impoverished nation of Haiti. However, because of the protean clinical manifestations, presence of asymptomatic infections, and limited medical diagnostic capacity, the identification of active melioidosis cases remains challenging. A seroepidemiological study was conducted using a novel enzyme-linked immunosorbent assay (ELISA) to detect antibodies toward B. pseudomallei in the native population. The performance of an indirect ELISA with purified lipopolysaccharide (LPS) from B. pseudomallei was evaluated using serum collected from rhesus macaques exposed to aerosolized B. pseudomallei. After optimization, serum collected from asymptomatic population members (n = 756) was screened for polyvalent (immunoglobulin M [IgM]/ immunoglobulin G [IgG]/ immunoglobulin A) and monoclonal (IgG or IgM) immunoglobulins against B. pseudomallei LPS. The population seroprevalence was 11.5% (95% confidence interval [CI]: 9.2, 13.8) for polyvalent immunoglobulins, 9.8% (95% CI: 7.7, 11.9) for IgG, and 1.7% (95% CI: 0.8, 2.6%) for IgM. The seroprevalence was not significantly different by gender (P = 0.16), but increased significantly (P < 0.001) with age, yielding an estimated annual seroconversion rate of 1.05% (95% CI: 0.81, 1.3). The detection of both recent (IgM+) and previous (IgG+) exposure to B. pseudomallei provides serological evidence that melioidosis is endemic in Haiti.

Introduction The governments of Haiti and the Dominican Republic have a binational agreement to work towards malaria elimination for the island of Hispaniola by the year 2020. Understanding malaria-related knowledge and behaviors can help inform elimination efforts. This study examined the association between social-behavioral factors, like bedtime and bed net ownership, with malaria seroconversion status among people in the Ouest and Sud-Est departments of Haiti. Methods In 2013, cross-sectional survey data ( n = 377) and blood samples were collected from a convenience sample of individuals within community, clinic and school settings. Logistic regression models were constructed to examine associations between social-behavioral factors and malaria exposure, adjusting for potential confounders. Results Compared to people going to bed between 6 and 8 pm, those going to bed between 8 and 10 pm were 2.67 (OR, 95% CI: 1.16–6.14) times as likely to have been exposed to malaria. Participants who reported going to bed after 10 pm were 5.96 times as likely to have had previous malaria exposure (OR, 95% CI: 2.26–15.7), compared to 6–8 pm. No significant associations were found between malaria exposure and either insecticide use nor bed net ownership. Discussion These findings are consistent with suspected feeding behaviors of Anopheles albimanus, which prefers feeding at night and outdoors. Study findings may improve overall understanding of malaria transmission in Haiti and potentially guide future studies conducted in this region.

Objectives To describe the epidemiology of malaria in pregnancy in Haiti. Methods Cross-sectional study among pregnant women in 6 departments of Haiti. After obtaining informed consent, whole blood samples and demographic surveys were collected to investigate malaria prevalence, anemia and socio-behavioral risk factors for infection, respectively. 311 pregnant women were screened for Plasmodium falciparum infection using a rapid diagnostic test (RDT), microscopy, and a novel, quantitative reverse-transcriptase polymerase chain reaction method (qRT-PCR). Results Overall, 1.2% (4/311) of pregnant women tested positive for malaria infection by both microscopy and RDT. However, using the qRT-PCR, 16.4% (51/311) of pregnant women were positive. The prevalence of malaria infection varied with geographic locations ranging between 0% to 46.4%. Additionally, 53% of pregnant women had some form of anemia; however no significant association was found between anemia and submicroscopic malaria infection. The socio-behavioral risk factors identified to be protective of malaria infection were marital status (P

Spondweni virus (SPONV) and Zika virus cause similar diseases in humans. We detected SPONV outside of Africa from a pool of Culex mosquitoes collected in Haiti in 2016. This finding raises questions about the role of SPONV as a human pathogen in Haiti and other Caribbean countries.

International audience; Despite centuries of control efforts, the past three decades have witnessed a dramatic spread of many mosquito-borne diseases worldwide. The acceleration of urbanization, global warming, the intensification of intercontinental trade and travel, the co-evolution and adaptation between pathogens and mosquito vectors, and the development of insecticide resistance, have greatly contributed to the mosquito borne diseases worldwide. This chapter presents the current situation regarding the expansion of mosquito-borne diseases and theirs vectors worldwide, highlighting the factors that have contributed to these dramatic expansions. Furthermore, this chapter addresses the main difficulties encountered for vector control implementation using traditional approaches.

Madariaga virus (MADV), also known as South American eastern equine encephalitis virus, has been identified in animals and humans in South and Central America, but not previously in Hispaniola or the northern Caribbean. MADV was isolated from virus cultures of plasma from an 8-year-old child in a school cohort in the Gressier/Leogane region of Haiti, who was seen in April, 2015, with acute febrile illness (AFI). The virus was subsequently cultured from an additional seven AFI case patients from this same cohort in February, April, and May 2016. Symptoms most closely resembled those seen with confirmed dengue virus infection. Sequence data were available for four isolates: all were within the same clade, with phylogenetic and molecular clock data suggesting recent introduction of the virus into Haiti from Panama sometime in the period from October 2012-January 2015. Our data document the movement of MADV into Haiti, and raise questions about the potential for further spread in the Caribbean or North America., Author summary Madariaga virus (MADV) is the name given to what used to be called South American eastern equine encephalitis virus (EEEV), based on recent studies suggesting that MADV is distinct genetically from the EEEV circulating in North America. Until now, MADV has been found primarily in animals in South and Central America, with a limited number of human cases reported (most of whom had encephalitis). Our group has been responsible for a series of studies assessing the etiology of acute febrile illness (AFI) among children in a school cohort in Haiti. Unexpectedly, in April, 2015, we identified MADV on viral culture of plasma from a student with AFI in this cohort; an additional seven cases were identified on culture of samples from children with AFI in this same cohort in February, April, and May 2016. On sequence analysis, all strains were very similar genetically, and appear to have come from a strain introduced into Haiti from Panama sometime in the period from October 2012- January 2015. Symptoms of children were similar to those seen with dengue; none had encephalitis. Our data indicate that this virus, which has the potential for causing serious illness, has been recently introduced into Haiti, and raises the possibility that it might move into other parts of the Caribbean or North America.

Though plans to eliminate malaria from the island of Hispaniola have recently received much attention, arbovirus surveillance continues to be largely neglected in Haiti. To support surveillance efforts and encourage vector-control strategies, a cross-sectional study of dengue virus (DENV) and West Nile virus (WNV) transmission was conducted using standard seroepidemiological methods. Blood samples (N = 673) were collected from 278 males and 395 females from three locations in the Ouest and Sud-Est Departments of Haiti. Serum was separated and tested for the presence of anti-DENV and anti-WNV immunoglobulin G (IgG) antibodies using an indirect enzyme-linked immunosorbent assay (ELISA). Anti-DENV IgG antibodies were detected in 72.1% (95% confidence interval [CI] = 68.7, 75.5) of the sample population; with no significant differences in seroprevalence by study location, participant gender, or age group (P > 0.1, in all tests). Anti-WNV IgG antibodies were detected in only 1% (95% CI = 0.3, 1.8) of the sample population, all which originated from participants located in Gressier. The high prevalence of anti-DENV IgG antibodies among all age groups, including those in the youngest age group (2–5 years of age), suggests hyperendemic transmission of DENV in the Ouest and Sud-Est Departments of Haiti. In contrast, the relative absence of anti-WNV IgG antibodies, even among older population members, further supports the notion that WNV transmission in this population is largely absent. These findings highlight the large burden of disease from DENV and the need for enhanced arbovirus surveillance and implementation of vector control strategies throughout Haiti.

Human coronavirus (HCoV) NL63 is recognized as a common cause of upper respiratory infections and influenza-like illness. In screening children with acute undifferentiated febrile illness in a school cohort in rural Haiti, we identified HCoV-NL63 in blood samples from four children. Cases clustered over an 11-day period; children did not have respiratory symptoms, but two had gastrointestinal complaints. On phylogenetic analysis, the Haitian HCoV-NL63 strains cluster together in a highly supported monophyletic clade linked most closely with recently reported strains from Malaysia; two respiratory HCoV-NL63 strains identified in north Florida in the same general period form a separate clade, albeit again with close linkages with the Malaysian strains. Our data highlight the variety of presentations that may be seen with HCoV-NL63, and underscore the apparent ease with which CoV strains move among countries, with our data consistent with recurrent introduction of strains into the Caribbean (Haiti and Florida) from Asia.

Background Plasmodium vivax infections, while quite prevalent throughout South and Central America, are virtually non-existent in Haiti, where P. falciparum infections are detected in over 99% of malaria cases. Historically, few cases of P. vivax have been reported in Haiti; all of which were identified by microscopy and none were confirmed by molecular diagnostics. To further examine the transmission of P. vivax in Haiti, a cross-sectional seroepidemiological study was conducted. Methods Whole blood was collected from 814 community members and school children ranging in age between 2 and 80 years-of-age from four locations in the Ouest and Sud-Est Departments of Haiti. After separation of serum, samples were screened for antibodies toward P. vivax apical membrane antigen (AMA-1) and merozoite surface protein-119 (MSP-1) using an indirect enzyme-linked immunosorbent assay (ELISA). Results Of all participants screened, 4.42% (36/814) were seropositive for AMA-1, 4.55% (37/814) were seropositive for MSP-1, 7.99% (65/814) were seropositive to either antigen, and only 0.98% (7/814) were seropositive for both antigens. Seroconversion rates (SCR) for AMA-1, MSP-1, either AMA-1 or MSP-1, and for both AMA-1 and MSP-1 estimated from the cross-sectional seroprevalence indicated rates of P. vivax transmission of less than 1% per year. Conclusion Given the lack of historical evidence of P. vivax infections on the island of Hispaniola, the sparse serological evidence of antibodies toward P. vivax identified in the current study further support the notion that the transmission of P. vivax malaria might be extremely low or even completely absent in Haiti.

Burkholderia pseudomallei, the etiological agent of melioidosis, has been hypothesized to be endemic throughout the Caribbean, including the impoverished nation of Haiti. However, because of the protean clinical manifestations, presence of asymptomatic infections, and limited medical diagnostic capacity, the identification of active melioidosis cases remains challenging. A seroepidemiological study was conducted using a novel enzyme-linked immunosorbent assay (ELISA) to detect antibodies toward B. pseudomallei in the native population. The performance of an indirect ELISA with purified lipopolysaccharide (LPS) from B. pseudomallei was evaluated using serum collected from rhesus macaques exposed to aerosolized B. pseudomallei. After optimization, serum collected from asymptomatic population members (n = 756) was screened for polyvalent (immunoglobulin M [IgM]/ immunoglobulin G [IgG]/ immunoglobulin A) and monoclonal (IgG or IgM) immunoglobulins against B. pseudomallei LPS. The population seroprevalence was 11.5% (95% confidence interval [CI]: 9.2, 13.8) for polyvalent immunoglobulins, 9.8% (95% CI: 7.7, 11.9) for IgG, and 1.7% (95% CI: 0.8, 2.6%) for IgM. The seroprevalence was not significantly different by gender (P = 0.16), but increased significantly (P < 0.001) with age, yielding an estimated annual seroconversion rate of 1.05% (95% CI: 0.81, 1.3). The detection of both recent (IgM+) and previous (IgG+) exposure to B. pseudomallei provides serological evidence that melioidosis is endemic in Haiti.

As part of on-going arboviral surveillance activity in a semi-rural region in Haiti, Chikungunya virus (CHIKV)-positive mosquito pools were identified in 2014 (the peak of the Caribbean Asian-clade epidemic), and again in 2016 by RT-PCR. In 2014, CHIKV was only identified in Aedes aegypti (11 positive pools/124 screened). In contrast, in sampling in 2016, CHIKV was not identified in Ae. aegypti, but, rather, in (a) a female Aedes albopictus pool, and (b) a female Culex quinquefasciatus pool. Genomic sequence analyses indicated that the CHIKV viruses in the 2016 mosquito pools were from the East-Central-South African (ECSA) lineage, rather than the Asian lineage. In phylogenetic studies, these ECSA lineage strains form a new ECSA subgroup (subgroup IIa) together with Brazilian ECSA lineage strains from an isolated human outbreak in 2014, and a mosquito pool in 2016. Additional analyses date the most recent common ancestor of the ECSA IIa subgroup around May 2007, and the 2016 Haitian CHIKV genomes around December 2015. Known CHIKV mutations associated with improved Ae. albopictus vector competence were not identified. Isolation of this newly identified lineage from Ae. albopictus is of concern, as this vector has a broader geographic range than Ae. aegypti, especially in temperate areas of North America, and stresses the importance for continued vector surveillance.

Currently, there are only limited data available on rates of major diagnostic categories of illnesses among Haitian children. We have established a cohort of 1,245 students attending schools run by the Christianville Foundation in the Gressier/Leogane region of Haiti, for whom our group provides primary medical care. Among 1,357 clinic visits during the 2012-2013 academic year, the main disease categories (with rates per 1,000 child years of observation) included acute respiratory infection (ARI) (385.6 cases/1,000 child years of observation), gastrointestinal complaints (277.8 cases/1,000 child years), febrile illness (235.0 cases/1,000 child years), and skin infections (151.7 cases/1,000 child years). The most common diarrheal pathogen was enteroaggregative Escherichia coli (present in 17% of children with diarrhea); Vibrio cholerae O1 and norovirus were the next most common. Our data highlight the importance of better defining etiologies for ARI and febrile illnesses and continuing problems of diarrheal illness in this region, including mild cases of cholera, which would not have been diagnosed without laboratory screening.

Mayaro virus has been associated with small outbreaks in northern South America. We isolated this virus from a child with acute febrile illness in rural Haiti, confirming its role as a cause of mosquitoborne illness in the Caribbean region. The clinical presentation can mimic that of chikungunya, dengue, and Zika virus infections.

We report the detection and isolation of enterovirus D68 from the blood of a 6-year-old child in rural Haiti, who presented with high fever and clinical signs suggestive of pneumonia. On phylogenetic analysis, this Haitian isolate was virtually identical to an enterovirus D68 strain circulating in New York during the same time period.

Community engagement has become an increasingly important focus of global health programs. Arbovirus emergence in the Americas (Zika and chikungunya virues), and global goals for malaria and lymphatic filariasis elimination, mean that community-based mosquito control has taken on a new salience. But how should mosquito control initiatives be designed and implemented in ways that best engage local people? What are the challenges and trade-offs of different strategies, not only for effectiveness but also for scale-up? In this paper, we describe the social and political dynamics of a pilot study in a small town in northern Haiti. With the aim of developing a culturally-competent approach to larval source management (LSM), our pilot project combined larval surveillance with environmental management, social engagement, community education, and larvicide application. Orientated around a network of ‘Mosquito Police’ (Polisye Kont Moustik, in Haitian Creole), our approach integrated elements of formative research, social learning, and community participation. Here, we reflect on the challenges we encountered in the field, from larval mapping, staff management, education and behavior change, engagement with formal and informal leaders, and community-based environmental cleanup. We discuss how these programmatic efforts were influenced and shaped by a complex range of social, cultural, political, and economic realities, and conclude by discussing the implications of our community-based approach for the elimination of lymphatic filariasis and malaria, and other vector-borne diseases, in Haiti.

Ten chikungunya virus isolates from human plasma collected in Haiti from May to August 2014, in the midst of a chikungunya fever outbreak, were fully sequenced. The resulting genomic sequences are nearly identical, and phylogenetic analyses indicate they belong to the Asian lineage of the virus.

Malaria remains a significant public health issue in Haiti, with chloroquine (CQ) used almost exclusively for the treatment of uncomplicated infections. Recently, single dose primaquine (PQ) was added to the Haitian national malaria treatment policy, despite a lack of information on the prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency within the population. G6PD deficient individuals who take PQ are at risk of developing drug induced hemolysis (DIH). In this first study to examine G6PD deficiency rates in Haiti, 22.8% (range 14.9%-24.7%) of participants were found to be G6PD deficient (class I, II, or III) with 2.0% (16/800) of participants having severe deficiency (class I and II). Differences in deficiency were observed by gender, with males having a much higher prevalence of severe deficiency (4.3% vs. 0.4%) compared to females. Male participants were 1.6 times more likely to be classified as deficient and 10.6 times more likely to be classified as severely deficient compared to females, as expected. Finally, 10.6% (85/800) of the participants were considered to be at risk for DIH. Males also had much higher rates than females (19.3% vs. 4.6%) with 4.9 times greater likelihood (p value 0.000) of having an activity level that could lead to DIH. These findings provide useful information to policymakers and clinicians who are responsible for the implementation of PQ to control and manage malaria in Haiti.

BACKGROUND: The identification and correction of malnutrition in children remains a critical public health issue in many developing countries. In this study, the rate of anemia and standardized indicators of physical growth were investigated in schoolchildren from Haiti. METHODS: Hemoglobin concentrations, height, and weight were collected from schoolchildren (n = 615) and evaluated by World Health Organization (WHO) standardized classifications for anemia and child growth. Data on glucose-6-dehydrogenase (G6PD) deficiency and current or previous malaria infection available for all students; a subset of anemia and non-anemic males (n = 43) were tested for serum ferritin. School A (n = 504) had a school-based supplemental nutrition program, while School B (n = 111) did not. RESULTS: Anemia was identified in 48.3% of students (95% CI: 44.3, 52.3), and not associated with BMI Z scores (P = 0.28) or serum ferritin concentrations (P = 0.17). The average Z scores for body mass index (BMI) of students at School A was within the normal range, while Z scores for BMI at School B were below average. Compared to school A, students at School B had significantly higher (P

Throughout many developing and tropical countries around the world, malaria remains a significant threat to human health. One barrier to malaria elimination is the ability to safely administer primaquine chemotherapy for the radical cure of malaria infections in populations with a high prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency. In the current study, a field trial of the world's first quantitative, point-of-care assay for measuring G6PD activity was conducted in Haiti. The performance of the CareStart Biosensor Analyzer was compared with the gold standard spectrophotometric assay and genotyping of the G6PD allele in schoolchildren (N = 343) from the Ouest Department of Haiti. In this population, 19.5% of participants (67/343) had some form of G6PD deficiency (< 60% residual activity) and 9.9% (34/343) had moderate-to-severe G6PD deficiency (< 30% residual activity). Overall, 18.95% of participants had the presence of the A-allele (65/343) with 7.87% (27/343) considered at high risk for drug-induced hemolysis (hemizygous males and homozygous females). Compared with the spectrophotometric assay, the sensitivity and specificity to determine participants with < 60% residual activity were 53.7% and 94.6%, respectively; for participants with 30% residual activity, the sensitivity and specificity were 5.9% and 99.7%, respectively. The biosensor overestimated the activity in deficient individuals and underestimated it in participants with normal G6PD activity, indicating the potential for a systematic measurement error. Thus, we suggest that the current version of the biosensor lacks adequate sensitivity and should be improved prior to its use as a point-of-care diagnostic for G6PD deficiency.

Brush border membrane vesicles from whole Aedes aegypti larvae (AaBBMVw) are confirmed to be valid preparations for membrane transport studies. The Abdul-Rauf and Ellar method was used to isolate AaBBMVw that were frozen, stored for several months, transported to a distant site, thawed and used to study Na + -coupled, 3 H-labeled, phenylalanine (Phe) uptake. The affinity for all components of the uptake was very high with half maximal values in the sub-micromolar range. By contrast a K 0.5 Phe of 0.2 mM and a K 0.5 Na of 26 mM were calculated from Phe-induced electrical currents in Xenopus oocytes that were heterologously expressing the Anopheles gambiae symporter (co-transporter), AgNAT8, in a buffer with 98 mM Na + . What accounts for the >1000-fold discrepancy in affinity for substrates between the BBMV and oocyte experiments? Is it because Ae. aegypti were used to isolate BBMVw whereas An. gambiae were used to transfect oocytes? More likely, it is because BBMVw were exposed to [Na + ] in the micromolar range with the transporter(s) being surrounded by native lipids. By contrast, the oocyte measurements were made at [Na + ] 100,000 times higher with AgNAT8 surrounded by foreign frog lipids. The results show that AaBBMVw are osmotically sealed; the time-course has a Na + -induced overshoot, the pH optimum is ∼7 and the K 0.5 values for Phe and Na + are very low. The transport is virtually unchanged when Na + is replaced by K + or Li + but decreased by Rb + . This approach to resolving discrepancies between electrical data on solute transporters such as AgNAT8 that are over-expressed in oocytes and flux data on corresponding transporters that are highly expressed in native membrane vesicles, may serve as a model for similar studies that add membrane biochemistry to molecular biology in efforts to identify targets for the development of new methods to control disease-vector mosquitoes.

Haiti is one of two remaining malaria-endemic countries in the Caribbean. To decrease malaria transmission in Haiti, primaquine was recently added to the malaria treatment public health policy. One limitation of primaquine is that, at certain doses, primaquine can cause hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDd). In this study, we genotyped two mutations (A376G and G202A), which confer the most common G6PDd variant in West African populations, G6PDd A−. We estimated the frequency of G6PDd A− in a sample of febrile patients enrolled in an on-going malaria study who represent a potential target population for a primaquine mass drug administration. We found that 33 of 168 individuals carried the G6PDd A− allele (includes A− hemizygous males, A− homozygous or heterozygous females) and could experience toxicity if treated with primaquine. These data inform discussions on safe and effective primaquine dosing and future malaria elimination strategies for Haiti.

SUMMARYGlycolysis, the citric acid cycle and other metabolic pathways of living organisms generate potentially toxic acids within all cells. One ubiquitous mechanism for ridding cells of the acids is to expel H+ in exchange for extracellular Na+, mediated by electroneutral transporters called Na+/H+ exchangers (NHEs) that are driven by Na+ concentration gradients. The exchange must be important because the human genome contains 10 NHEs along with two Na+/H+antiporters (NHAs). By contrast, the genomes of two principal disease vector mosquitoes, Anopheles gambiae and Aedes aegypti, contain only three NHEs along with the two NHAs. This shortfall may be explained by the presence of seven nutrient amino acid transporters (NATs) in the mosquito genomes. NATs transport Na+ stoichiometrically linked to an amino acid into the cells by a process called symport or co-transport. Three of the mosquito NATs and two caterpillar NATs have previously been investigated after heterologous expression in Xenopus laevis oocytes and were found to be voltage driven (electrophoretic). Moreover, the NATs are present in the same membrane as the H+ V-ATPase, which generates membrane potentials as high as 120 mV. We review evidence that the H+V-ATPase moves H+ out of the cells and the resulting membrane potential (Vm) drives Na+ linked to an amino acid into the cells via a NAT. The H+ efflux by the V-ATPase and Na+ influx by the NAT comprise the same ion exchange as that mediated by an NHE; so the V and NAT working together constitute an NHE that we call NHEVNAT. As the H+ V-ATPase is widely distributed in mosquito epithelial cells and there are seven NATs in the mosquito genomes, there are potentially seven NHEVNATs that could replace the missing NHEs. We review published evidence in support of this hypothesis and speculate about broader functions of NHEVNATs.

SUMMARYWe have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes(Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned,nor have the encoded proteins been localized or characterized.The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia.A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.

Hispaniola, comprising Haiti and the Dominican Republic, has been identified as a candidate for malaria elimination. However, incomplete surveillance data in Haiti hamper efforts to assess the impact of ongoing malaria control interventions. Characteristics of the genetic diversity of Plasmodium falciparum populations can be used to assess parasite transmission, which is information vital to evaluating malaria elimination efforts. Here we characterize the genetic diversity of P. falciparum samples collected from patients at seven sites in Haiti using 12 microsatellite markers previously employed in population genetic analyses of global P. falciparum populations. We measured multiplicity of infections, level of genetic diversity, degree of population geographic substructure, and linkage disequilibrium (defined as non-random association of alleles from different loci). For low transmission populations like Haiti, we expect to see few multiple infections, low levels of genetic diversity, high degree of population structure, and high linkage disequilibrium. In Haiti, we found low levels of multiple infections (12.9%), moderate to high levels of genetic diversity (mean number of alleles per locus = 4.9, heterozygosity = 0.61), low levels of population structure (highest pairwise Fst = 0.09 and no clustering in principal components analysis), and moderate linkage disequilibrium (ISA = 0.05, P

Haiti and the Dominican Republic, which share the island of Hispaniola, are the last locations in the Caribbean where malaria still persists. Malaria is an important public health concern in Haiti with 17,094 reported cases in 2014. Further, on January 12, 2010, a record earthquake devastated densely populated areas in Haiti including many healthcare and laboratory facilities. Weakened infrastructure provided fertile reservoirs for uncontrolled transmission of infectious pathogens. This situation results in unique challenges for malaria epidemiology and elimination efforts. To help Haiti achieve its malaria elimination goals by year 2020, the Laboratoire National de Santé Publique and Henry Ford Health System, in close collaboration with the Direction d’Épidémiologie, de Laboratoire et de Recherches and the Programme National de Contrôle de la Malaria, hosted a scientific meeting on “Elimination Strategies for Malaria in Haiti” on January 29-30, 2015 at the National Laboratory in Port-au-Prince, Haiti. The meeting brought together laboratory personnel, researchers, clinicians, academics, public health professionals, and other stakeholders to discuss main stakes and perspectives on malaria elimination. Several themes and recommendations emerged during discussions at this meeting. First, more information and research on malaria transmission in Haiti are needed including information from active surveillance of cases and vectors. Second, many healthcare personnel need additional training and critical resources on how to properly identify malaria cases so as to improve accurate and timely case reporting. Third, it is necessary to continue studies genotyping strains of Plasmodium falciparum in different sites with active transmission to evaluate for drug resistance and impacts on health. Fourth, elimination strategies outlined in this report will continue to incorporate use of primaquine in addition to chloroquine and active surveillance of cases. Elimination of malaria in Haiti will require collaborative multidisciplinary approaches, sound strategic planning, and strong ownership of strategies by the Haiti Ministère de la Santé Publique et de la Population.

Antimalarial drugs are a key tool in malaria elimination programs. With the emergence of artemisinin resistance in southeast Asia, an effort to identify molecular markers for surveillance of resistant malaria parasites is underway. Non-synonymous mutations in the kelch propeller domain (K13-propeller) in Plasmodium falciparum have been associated with artemisinin resistance in samples from southeast Asia, but additional studies are needed to characterize this locus in other P. falciparum populations with different levels of artemisinin use. Here, we sequenced the K13-propeller locus in 82 samples from Haiti, where limited government oversight of non-governmental organizations may have resulted in low-level use of artemisinin-based combination therapies. We detected a single-nucleotide polymorphism (SNP) at nucleotide 1,359 in a single isolate. Our results contribute to our understanding of the global genomic diversity of the K13-propeller locus in P. falciparum populations.

Chloroquine (CQ) has been used for malaria treatment in Haiti for several decades, but reports of CQ resistance are scarce. The efficacy of CQ in patients with uncomplicated Plasmodium falciparum undergoing treatment in Haiti was evaluated. Malaria patients were enrolled, treated with CQ, and monitored over a 42-day period. The treatment outcomes were evaluated on day 28 by microscopy. The P. falciparum slide-confirmed rate was 9.5% (121 of 1,277). Malaria infection was seasonal, with peak observations between October and January; 88% (107 of 121) of patients consented to participate. Sixty patients successfully completed the 42-day follow-up, whereas 47 patients withdrew consent or were lost to follow-up. The mean parasite density declined rapidly within the first few days after treatment. Seven patients did not clear their malaria infections and were clinically asymptomatic; therefore, they were considered late parasitological failures. About 90% (95% confidence interval = 84.20-97.90) of patients had no detectable parasitemia by day 28 and remained malaria-free to day 42. Testing for recrudescence, reinfection, and CQ serum levels was not done in the seven patients, and therefore, their CQ resistance status is unresolved. CQ resistance surveillance by patient follow-up, in vitro drug sensitivity studies, and molecular markers is urgently needed in Haiti.

Xanthurenic acid (XA), produced as a byproduct during the biosynthesis of insect eye pigment (ommochromes), is a strong inducer of Plasmodium gametogenesis at very low concentrations. In previous studies, it was shown that XA is present in Anopheles stephensi (Diptera: Culicidae) mosquito salivary glands and that during blood feeding the mosquitoes ingested their own saliva into the midgut. Considering these two facts together, it is therefore likely that XA is discharged with saliva during blood feeding and is swallowed into the midgut where it exerts its effect on Plasmodium gametocytes. However, the quantities of XA in the salivary glands and midgut are unknown. In this study, we used high performance liquid chromatography with electrochemical detection to detect and quantify XA in the salivary glands and midgut. Based on the results of this study, we found 0.28+/-0.05 ng of XA in the salivary glands of the mosquitoes, accounting for 10% of the total XA content in the mosquito whole body. The amounts of XA in the salivary glands reduced to 0.13+/-0.06 ng after mosquitoes ingested a blood meal. Approximately 0.05+/-0.01 ng of XA was detected in the midgut of nonblood fed An. stephensi mosquitoes. By adding synthetic tryptophan as a source of XA into larval rearing water (2 mM) or in sugar meals (10 mM), we evaluated whether XA levels in the mosquito (salivary glands, midgut, and whole body) were boosted and the subsequent effect on infectivity of Plasmodium berghei in the treated mosquito groups. A female specific increase in XA content was observed in the whole body and in the midgut of mosquito groups where tryptophan was added either in the larval water or sugar meals. However, XA in the salivary glands was not affected by tryptophan addition to larval water, and surprisingly it reduced when tryptophan was added to sugar meals. The P. berghei oocyst loads in the mosquito midguts were lower in mosquitoes fed tryptophan treated sugar meals than in mosquitoes reared on tryptophan treated larval water. Our results suggest that mosquito nutrition may have a significant impact on whole body and midgut XA levels in mosquitoes. We discuss the observed parasite infectivity results in relation to XA's relationship with malaria parasite development in mosquitoes.

The influence of indoor microclimate on survival of female Anopheles gambiae sensu stricto Giles (Diptera: culicidae) mosquitoes fed on different nutrition sources was evaluated in a semifield experimental hut exposed to ambient climate in western Kenya. Cages of mosquitoes (n approximately 50 per cage) were placed in nine positions within the hut combining three different sides and three different heights. At each height and side, mosquitoes were offered either human blood (once every 2 d), glucose (6% wt:vol) or a combination of the two diets over three experiments so that each cage position received one diet source. The effect of diet on survival was significant with mean survival times of 14 d for mosquitoes fed blood alone, 29 d for sugar alone and 33 d for blood plus sugar. Sugar availability decreased the odds of mortality approximately 85% compared with the blood group. Micro heterogeneities of temperature but not relative humidity also influenced survival although to a much lesser extent. The side but not height within the hut at which mosquitoes were placed, influenced survival but could not be explained by either temperature or relative humidity differences. The potential influence of seemingly minor heterogeneities of indoor microclimate upon vector longevity and vectorial capacity may merit further investigation. Also, the availability of sugar was shown to be a potentially crucial determinant of vectorial capacity. Compared with blood alone, the availability of sugar served to increase survival potential of vectors beyond ages at which they are old enough to transmit malaria.

Sickle cell disease is a growing global health concern because infants born with the disorder in developing countries are now surviving longer with little access to diagnostic and management options. In Haiti, the current state of sickle cell disease/trait in the population is unclear. To inform future screening efforts in Haiti, we assayed sickle hemoglobin mutations using traditional hemoglobin solubility tests (HST) and add-on techniques, which incorporated spectrophotometry and insoluble hemoglobin separation. We also generated genotype data as a metric for HST performance. We found 19 of 202 individuals screened with HST were positive for sickle hemoglobin, five of whom did not carry the HbS allele. We show that spectrophotometry and insoluble hemoglobin separation add-on techniques could resolve false positives associated with the traditional HST approach, with some limitations. We also discuss the incorporation of insoluble hemoglobin separation observation with HST in suboptimal screening settings like Haiti.

Administering primaquine (PQ) to treat malaria patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency can pose a serious risk of drug-induced hemolysis (DIH). New easy to use point-of-care rapid diagnostic tests are being developed as an alternative to labor-intensive spectrophotometric methods, but they require field testing before they can be used at scale. This study screened 456 participants in Gressier, Haiti using the Access Bio CareStart qualitative G6PD rapid detection test compared with the laboratory-based Trinity Biotech quantitative spectrophotometric assay. Findings suggest that the CareStart test was 90% sensitive for detecting individuals with severe deficiency and 84.8% sensitive for detecting individuals with moderate and severe deficiency compared with the Trinity Biotech assay. A high negative predictive value of 98.2% indicates excellent performance in determining those patients able to take PQ safely. The CareStart G6PD test holds much value for screening malaria patients to determine eligibility for PQ therapy.

Background In Haiti where chloroquine (CQ) is widely used for malaria treatment, reports of resistance are scarce. However, recent identification of CQ resistance genotypes in one site is suggestive of an emerging problem. Additional studies are needed to evaluate genetic mutations associated with CQ resistance, especially in the Plasmodium falciparum multi-drug resistance- 1 gene (pfmdr 1) while expanding the already available information on P. falciparum CQ transporter gene (pfcrt) in Haiti. Methods Blood samples were collected on Whatman filter cards (FTA) from eight clinics spread across Haiti. Following the confirmation of P. falciparum in the samples, PCR protocols were used to amplify regions of pfmdr 1and pfcrt codons of interest, (86, 184, 1034, 1042, and 1246) and (72-76), respectively. Sequencing and site-specific restriction enzyme digestions were used to analyse these DNA fragments for the presence of single nucleotide polymorphisms (SNPs) known to confer resistance to anti-malarial drugs. Results P. falciparum infection was confirmed in160 samples by amplifying a segment of the P. falciparum 18S small subunit ribosomal RNA gene (pfssurrn a). The sequence of pfmdr1 in 54 of these samples was determined between codons 86,184 codons 1034, 1042 and 1246. No sequence differences from that of the NF54 clone 3D7 were found among the 54 samples except at codon 184, where a non-silent mutation was found in all samples predicted to alter the amino acid sequence replacing tyrosine with phenylalanine (Y184F). This altered sequence was also confirmed by restriction enzyme digestion. The sequence of pfmdr 1 at codons 86, 184, 1034 and 1042 encoded the NF SN haplotype. The sequence of pfcrt codons 72-76 from 79 samples was determined and found to encode CVMNK, consistent with a CQ sensitive genotype. Conclusion The presence of the Y184F mutation in pfmdr1 of P. falciparum parasites in Haiti may have implications for resistance to antimalarial drugs. The absence of mutation in pfcrt at codon 76 among 79 isolates tested suggests that sensitivity to CQ in Haiti remains common. Wide-spread screening of the pfmdr1 and pfcrt especially among patients experiencing treatment failure may be a useful tool in early detection of the emergence of antimalarial drug resistance in Haiti.

Background Zika virus (ZIKV), first isolated in Uganda in 1947, is currently spreading rapidly through South America and the Caribbean. In Brazil, infection has been linked with microcephaly and other serious complications, leading to declaration of a public health emergency of international concern; however, there currently are only limited data on the virus (and its possible sources and manifestations) in the Caribbean. Methods From May, 2014-February, 2015, in conjunction with studies of chikungunya (CHIKV) and dengue (DENV) virus infections, blood samples were collected from children in the Gressier/Leogane region of Haiti who presented to a school clinic with undifferentiated febrile illness. Samples were initially screened by RT-PCR for CHIKV and DENV, with samples negative in these assays further screened by viral culture. Findings Of 177 samples screened, three were positive for ZIKV, confirmed by viral sequencing; DENV-1 was also identified in culture from one of the three positive case patients. Patients were from two different schools and 3 different towns, with all three cases occurring within a single week, consistent with the occurrence of an outbreak in the region. Phylogenetic analysis of known full genome viral sequences demonstrated a close relationship with ZIKV from Brazil; additional analysis of the NS5 gene, for which more sequences are currently available, showed the Haitian strains clustering within a monophyletic clade distinct from Brazilian, Puerto Rican and Guatemalan sequences, with all part of a larger clade including isolates from Easter Island. Phylogeography also clarified that at least three major African sub-lineages exist, and confirmed that the South American epidemic is most likely to have originated from an initial ZIKV introduction from French Polynesia into Easter Island, and then to the remainder of the Americas. Conclusions ZIKV epidemics in South America, as well as in Africa, show complex dissemination patterns. The virus appears to have been circulating in Haiti prior to the first reported cases in Brazil. Factors contributing to transmission and the possible linkage of this early Haitian outbreak with microcephaly remain to be determined., Author Summary Zika virus is currently spreading rapidly through the Americas, including the Caribbean, where it has emerged as a major public health problem due to the linkage with birth defects, including microcephaly. We report the isolation of Zika virus from 3 children in rural Haiti in December, 2014, as part of a study of acute undifferentiated febrile illness that was being conducted by our research group; from one of these children, we also isolated dengue virus serotype 1. On analysis of nucleotide sequence data from these and Zika strains from other locales, the South American/Haitian sequences cluster within the Asian clade and clearly branch out from a sequence circulating in Easter Island, which originated, in turn, from French Polynesia. On further analysis of one specific gene sequence for which more data were available, there appeared to be slight separation of Haitian strains and the strains from Brazil, Suriname, Puerto Rico and Guatemala, with molecular clock analysis suggesting that Zika virus was present in Haiti as early as mid-2013. These findings raise questions about the origin of Zika virus in the Caribbean, and subsequent patterns of circulation of the virus within the Americas.

Background Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. Methods DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. Results Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch’s T-test p-value = 0.53 and permutations p-value = 0.59). Conclusion This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on alternative malaria treatment options in Haiti.

The effect of age and dietary factors of Anopheles gambiae (Diptera: Culicidae) on the infectivity of natural Plasmodium falciparum parasites was studied. Mosquitoes of various ages (1-3, 4-7 and 8-11 day old) and those fed blood (either single or double meals) and sugar meals were experimentally co-infected with P. falciparum gametocytes obtained from different naturally infected human volunteers. On day 7, midguts were examined for oocyst infection to determine whether mosquito age or diets have significant effects on parasite infectivity. The age of the mosquitoes did not significantly influence the oocyst infection rates (chi2 = 48.32, df = 40, P = 0.172) or oocyst load (# of oocysts/midgut) (P = 0.14) observed. Oocyst load between groups was not significantly different. Similarly, the type of diet (either blood or sugar) did not influence oocyst infection rates (chi2 = 16.52, df = 19, P = 0.622). However, an increase in oocyst infection rates resulted after previous feeding on double blood meals (35%) compared to single blood meals (25%), with comparable oocyst load. These observations are in agreement with those reported in previous studies suggesting that increased mosquito nutritional reserves resulting from increased dietary resources is favorable for malaria infectivity. This field-based study indicates that vector competence of An. gambiae to natural P. falciparum parasites does not vary with age and that nutritional resources acquired prior to an infectious blood meal plays a crucial role in mosquito-parasite relationships.

Brush border membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs) contain an H(+) V-ATPase (V), a Na(+)/H(+) antiporter, NHA1 (A) and a Na(+)-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles. AgNAT8 was cloned from Anopheles gambiae, localized in BBMs and characterized in Xenopus laevis oocytes. AgNHA1 was cloned and localized in BBMs but characterization in oocytes was compromised by an endogenous cation conductance. AeBBMVWs complement Xenopus oocytes for characterizing membrane proteins, can be energized by voltage from the V-ATPase and are in their natural lipid environment. BBMVs from caterpillars were used in radio-labeled solute uptake experiments but approximately 10,000 mosquito larvae are needed to equal 10 caterpillars. By contrast, functional AeBBMVWs can be prepared from 10,000 whole larvae in 4h. Na(+)-coupled (3H)phenylalanine uptake mediated by AeNAT8 in AeBBMVs can be compared to the Phe-induced inward Na(+) currents mediated by AgNAT8 in oocytes. Western blots and light micrographs of samples taken during AeBBMVW isolation are labeled with antibodies against all of the VAN components. The use of AeBBMVWs to study coupling between electrogenic V-ATPases and the electrophoretic transporters is discussed.

Two principal strategies are used to energize membranes in living organisms, a Na+ strategy and a voltage strategy. In the Na+ strategy a primary Na+/K+ ATPase imposes both Na+ and K+ concentration gradients across cell membranes with Na+ high outside and K+ high inside the cells. The Na+ gradient, Δ[Na+] is used to drive diverse secondary transporters. For example, in many animal cells Δ[Na+] drives Na+ inwardly coupled to H+ outwardly, mediated by Na+/H+ exchangers (NHEs). They provide the principal means by which metabolically produced acids are ejected from mammalian cells [70]. In the voltage strategy the electron transport system of prokaryotes or H+ V-ATPases of eukaryotes, impose a voltage gradient, ΔΨ, across biological membranes with the outside positive. The ΔΨ drives secondary (Na+ or K+)/nH+ antiport that is mediated by Na+/H+ antiporters (NHAs). The stoichiometry of NHEs is 1Na+ to 1H+ so they are independent of the membrane potential and are said to be electroneutral. The stoichiometry of NHAs is 1Na+ or K+ to more than 1H+ so they are driven both by the ion gradients and the membrane potential and are said to be electrophoretic. NHAs operate in the opposite direction from NHEs, moving nH+ inwardly and Na+ or K+ outwardly. ΔΨ also drives Na+- or K+-coupled nutrient amino acid uptake that is mediated by electrophoretic (Na+ or K+) amino acid symporters (NATs) [11]. In eukaryotic cells the primary sources of voltage gradients across plasma membranes have classically been considered to be K+, Na+, or other ionic diffusion potentials. Thus, K+ diffusion potentials dominate the resting potential and Na+ diffusion potentials dominate the action potential in squid axon and many other nerves. Only recently are ΔΨs generated by H+ V-ATPases becoming recognized as the energy source for electrophoretic transporters in animal cells [35, 65, 90]. The H+ V-ATPases translocate H+ outwardly across the cell membrane leaving their partner anion (gegenion) behind. Thus, they charge the capacitance of the membrane resulting in a transmembrane voltage, with the outside positive. The translocated H+s exchange with more numerous Na+s or K+s in the outside bulk solution, transforming the H+ electrochemical gradient to a Na+ or K+ electrochemical gradient which in turn drives Na+- or K+-coupled amino acid symport via a NAT into the cells. Membrane energization by H+ V-ATPases is accomplished by a five-phase system consisting of (1) the bulk solution inside the cells, (2) the inside solution/membrane interface, (3) the membrane, (4) the outside solution/membrane interface, and (5) the outside bulk solution [36, 49, 50].

In the fifth paragraph of the results section the last two sentences contain incorrect data. The correct text should read: In addition, 302 children were selected from one school in Mwea division. After getting consent from their guardians and parents, only 285 pupils from the school were asked to donate a finger prick blood sample for malaria parasite examination. The median age was 7.0 years old (Range: 4½ – 10 years). There were more boys (53%) than girls (47%) examined in the survey. No malaria parasites were found.

SUMMARYThe nutrient amino acid transporter (NAT) subfamily is the largest subdivision of the sodium neurotransmitter symporter family (SNF; also known as SLC6; HUGO). There are seven members of the NAT population in the African malaria mosquito Anopheles gambiae, two of which, AgNAT6 and AgNAT8,preferably transport indole- and phenyl-branched substrates, respectively. The relative expression and distribution of these aromatic NATs were examined with transporter-specific antibodies in Xenopus oocytes and mosquito larval alimentary canal, representing heterologous and tissue expression systems, respectively. NAT-specific aromatic-substrate-induced currents strongly corresponded with specific accumulation of both transporters in the plasma membrane of oocytes. Immunolabeling revealed elevated expressions of both transporters in specific regions of the larval alimentary canal,including salivary glands, cardia, gastric caeca, posterior midgut and Malpighian tubules. Differences in relative expression densities and spatial distribution of the transporters were prominent in virtually all of these regions, suggesting unique profiles of the aromatic amino acid absorption. For the first time reversal of the location of a transporter between apical and basal membranes was identified in posterior and anterior epithelial domains corresponding with secretory and absorptive epithelial functions,respectively. Both aromatic NATs formed putative homodimers in the larval gut whereas functional monomers were over-expressed heterologously in Xenopus oocytes. The results unequivocally suggest functional synergy between substrate-specific AgNAT6 and AgNAT8 in intracellular absorption of aromatic amino acids. More broadly, they suggest that the specific selectivity, regional expression and polarized membrane docking of NATs represent key adaptive traits shaping functional patterns of essential amino acid absorption in the metazoan alimentary canal and other tissues.

Anopheles gambiae larvae (Diptera: Culicidae) live in freshwater with low Na(+) concentrations yet they use Na(+) for alkalinization of the alimentary canal, for electrophoretic amino acid uptake and for nerve function. The metabolic pathway by which larvae accomplish these functions has anionic and cationic components that interact and allow the larva to conserve Na(+) while excreting H(+) and HCO(3)(-). The anionic pathway consists of a metabolic CO(2) diffusion process, carbonic anhydrase and Cl(-)/HCO(3)(-) exchangers; it provides weak HCO(3)(-) and weaker CO(3)(2-) anions to the lumen. The cationic pathway consists of H(+) V-ATPases and Na(+)/H(+) antiporters (NHAs), Na(+)/K(+) P-ATPases and Na(+)/H(+) exchangers (NHEs) along with several (Na(+) or K(+)):amino acid(+/-) symporters, a.k.a. nutrient amino acid transporters (NATs). This paper considers the cationic pathway, which provides the strong Na(+) or K(+) cations that alkalinize the lumen in anterior midgut then removes them and restores a lower pH in posterior midgut. A key member of the cationic pathway is a Na(+)/H(+) antiporter, which was cloned recently from Anopheles gambiae larvae, localized strategically in plasma membranes of the alimentary canal and named AgNHA1 based upon its phylogeny. A phylogenetic comparison of all cloned NHAs and NHEs revealed that AgNHA1 is the first metazoan NHA to be cloned and localized and that it is in the same clade as electrophoretic prokaryotic NHAs that are driven by the electrogenic H(+) F-ATPase. Like prokaryotic NHAs, AgNHA1 is thought to be electrophoretic and to be driven by the electrogenic H(+) V-ATPase. Both AgNHA1 and alkalophilic bacterial NHAs face highly alkaline environments; to alkalinize the larva mosquito midgut lumen, AgNHA1, like the bacterial NHAs, would have to move nH(+) inwardly and Na(+) outwardly. Perhaps the alkaline environment that led to the evolution of electrophoretic prokaryotic NHAs also led to the evolution of an electrophoretic AgNHA1 in mosquito larvae. In support of this hypothesis, antibodies to both AgNHA1 and H(+) V-ATPase label the same membranes in An. gambiae larvae. The localization of H(+) V-ATPase together with (Na(+) or K(+)):amino acid(+/-) symporter, AgNAT8, on the same apical membrane in posterior midgut cells constitutes the functional equivalent of an NHE that lowers the pH in the posterior midgut lumen. All NATs characterized to date are Na(+) or K(+) symporters so the deduction is likely to have wide application. The deduced colocalization of H(+) V-ATPase, AgNHA1 and AgNAT8, on this membrane forms a pathway for local cycling of H(+) and Na(+) in posterior midgut. The local H(+) cycle would prevent unchecked acidification of the lumen while the local Na(+) cycle would regulate pH and support Na(+):amino acid(+/-) symport. Meanwhile, a long-range Na(+) cycle first transfers Na(+) from the blood to gastric caeca and anterior midgut lumen where it initiates alkalinization and then returns Na(+) from the rectal lumen to the blood, where it prevents loss of Na(+) during H(+) and HCO(3)(-) excretion. The localization of H(+) V-ATPase and Na(+)/K(+)-ATPase in An. gambiae larvae parallels that reported for Aedes aegypti larvae. The deduced colocalization of the two ATPases along with NHA and NAT in the alimentary canal constitutes a cationic pathway for Na(+)-conserving midgut alkalinization and de-alkalinization which has never been reported before.

Background The origin of highly competent malaria vectors has been linked to productive larval habitats in the field, but there isn't solid quantitative or qualitative data to support it. To test this, the effect of larval habitat soil substrates on larval development time, pupation rates and vector competence of Anopheles gambiae to Plasmodium falciparum were examined. Methods Soils were collected from active larval habitats with sandy and clay substrates from field sites and their total organic matter estimated. An. gambiae larvae were reared on these soil substrates and the larval development time and pupation rates monitored. The emerging adult mosquitoes were then artificially fed blood with infectious P. falciparum gametocytes from human volunteers and their midguts examined for oocyst infection after seven days. The wing sizes of the mosquitoes were also measured. The effect of autoclaving the soil substrates was also evaluated. Results The total organic matter was significantly different between clay and sandy soils after autoclaving (P = 0.022). A generalized liner model (GLM) analysis identified habitat type (clay soil, sandy soil, or lake water) and autoclaving (that reduces presence of microbes) as significant factors affecting larval development time and oocyst infection intensities in adults. Autoclaving the soils resulted in the production of significantly smaller sized mosquitoes (P = 0.008). Autoclaving clay soils resulted in a significant reduction in Plasmodium falciparum oocyst intensities (P = 0.041) in clay soils (unautoclaved clay soils (4.28 ± 0.18 oocysts/midgut; autoclaved clay soils = 1.17 ± 0.55 oocysts/midgut) although no difference (P = 0.480) in infection rates was observed between clay soils (10.4%), sandy soils (5.3%) or lake water (7.9%). Conclusion This study suggests an important nutritional role for organic matter and microbial fauna on mosquito fitness and vector competence. It shows that the quality of natural aquatic habitats of mosquito larvae may influence malaria parasite transmission potential by An. gambiae. This information can be important in targeting larval habitats for malaria control.

We studied the effects of high temperature, 30 and 32 versus 27 C on early Plasmodium falciparumdevelopment in Anopheles gambiae experimentally infected with gametocytes from 30 volunteers with mean density of 264.1 gametocytes/ml blood (range: 16-1,536/ml). From several batches of mosquitoes, fed by membrane feeding, midguts of individual mosquitoes were dissected at 24 hr for ookinete enumeration and at 7 days to quantify oocysts. There were temperature-related differences in mean ookinete intensity per mosquito midgut, with 9.71 6 1.6 at 27 C, 9.85 6 2.32 at 30 C, and 3.89 6 0.81 at 32 C. The prevalence of oocyst infection decreased with an increase in temperatures from 15.9 to 8.5 to 6.4% at 27, 30, and 32 C, respectively. The average oocyst intensities for the infected mosquitoes increased with temperatures from 2.9 at 27 C to 3.5 at 30 C, and to 3.3 at 32 C. However, the success of infections was reduced at 30 and 32 C, and resulted in greater losses during consecutive inter-stage parasite development. The most significant impact of high temperatures occurred at the transition between macrogametocytes and ookinetes, whereas the transition between ookinetes and oocysts apparently was not affected. In contrast to other reports, exposure of mosquitoes infected with natural parasites to high temperatures did not eliminate preoocyst stages, as has been observed from laboratory studies using the NF-54 strain of P. falciparum. This observation of parasite resistance to high temperatures is consistent with the natural situation in tropical environments where perennial malaria transmission occurs during hot dry seasons. Species of Anopheles mosquitoes transmit malaria parasites after supporting a sporogonic development from preoocyst stag

The survival of female Anopheles gambiae s.s. mosquitoes inside two village house types (grass-thatched and iron-roofed) was studied in relation to diet and ambient indoor microclimatic conditions. Two batches of 20–30, 1-day-old laboratory-bred mosquitoes were maintained inside cages in the grass-thatched (n = 2) and iron-roofed (n = 2) houses and fed daily, one group on 10% glucose and the other on human blood. Throughout the experiments, indoor temperature and relative humidity of the houses were recorded, and mortality of mosquitoes monitored daily until all had died. The experiments were replicated thrice. There was no significant variation in the overall mean temperature (P = 0.93) or relative humidity profiles (P = 0.099) between the two house types, although the iron-roofed houses recorded higher temperature peaks. A Kaplan-Meier survival analysis showed that the mean survival times of mosquitoes were 8 and 10 days in the two grass-thatched huts and 7 and 10 days in the two iron-roof houses for mosquitoes feeding on blood and sugar meals, respectively. The mean survival times of mosquitoes maintained inside similar house types differed only due to diet. In the proportionality of hazards model (Cox regression), the dietary regimes significantly influenced the probability of survival (P = 0.0001), with mosquitoes surviving longer on sugar meals than on blood. Microclimatic factors inside houses also significantly influenced mosquito survival. Although higher peak temperatures were recorded in corrugated iron-roofed houses, the survival of the mosquitoes resting in them did not differ significantly from that in grass-thatched houses. However, the impact of these temperatures on the development of malaria parasites inside the vector needs to be investigated.