Insights into Plasmodium vivax Asymptomatic Malaria Infections and Direct Skin-Feeding Assays to Assess Onward Malaria Transmission in the Amazon.

Understanding the reservoir and infectivity of Plasmodium gametocytes to vector mosquitoes is crucial to align strategies aimed at malaria transmission elimination. Yet, experimental information is scarce regarding the infectivity of Plasmodium vivax for mosquitoes in diverse epidemiological settings where the proportion of asymptomatically infected individuals varies at a microgeographic scale. We measured the transmissibility of clinical and subclinical P. vivax malaria parasite carriers to the major mosquito vector in the Amazon Basin, Nyssorhynchus darlingi (formerly Anopheles). A total of 105 participants with natural P. vivax malaria infection were recruited from a cohort study in Loreto Department, Peruvian Amazon. Four of 18 asymptomatic individuals with P. vivax positivity by blood smear infected colony-grown Ny. darlingi (22%), with 2.6% (19 of 728) mosquitoes infected. In contrast, 77% (44/57) of symptomatic participants were infectious to mosquitoes with 51% (890 of 1,753) mosquitoes infected. Infection intensity was greater in symptomatic infections (mean, 17.8 oocysts/mosquito) compared with asymptomatic infections (mean, 0.28 oocysts/mosquito), attributed to parasitemia/gametocytemia level. Paired experiments (N = 27) using direct skin-feeding assays and direct membrane mosquito-feeding assays showed that infectivity to mosquitoes was similar for both methods. Longitudinal studies with longer follow-up of symptomatic and asymptomatic parasite infections are needed to determine the natural variations of disease transmissibility.

Malaria transmission structure in the Peruvian Amazon through antibody signatures to Plasmodium vivax.

BACKGROUND: The landscape of malaria transmission in the Peruvian Amazon is temporally and spatially heterogeneous, presenting different micro-geographies with particular epidemiologies. Most cases are asymptomatic and escape routine malaria surveillance based on light microscopy (LM). Following the implementation of control programs in this region, new approaches to stratify transmission and direct efforts at an individual and community level are needed. Antibody responses to serological exposure markers (SEM) to Plasmodium vivax have proven diagnostic performance to identify people exposed in the previous 9 months. METHODOLOGY: We measured antibody responses against 8 SEM to identify recently exposed people and determine the transmission dynamics of P. vivax in peri-urban (Iquitos) and riverine (Mazán) communities of Loreto, communities that have seen significant recent reductions in malaria transmission. Socio-demographic, geo-reference, LM and qPCR diagnosis data were collected from two cross-sectional surveys. Spatial and multilevel analyses were implemented to describe the distribution of seropositive cases and the risk factors associated with exposure to P. vivax. PRINCIPAL FINDINGS: Low local transmission was detected by qPCR in both Iquitos (5.3%) and Mazán (2.7%); however, seroprevalence indicated a higher level of (past) exposure to P. vivax in Mazán (56.5%) than Iquitos (38.2%). Age and being male were factors associated with high odds of being seropositive in both sites. Higher antibody levels were found in individuals >15 years old. The persistence of long-lived antibodies in these individuals could overestimate the detection of recent exposure. Antibody levels in younger populations (<15 years old) could be a better indicator of recent exposure to P. vivax. CONCLUSIONS: The large number of current and past infections detected by SEMs allows for detailed local epidemiological analyses, in contrast to data from qPCR prevalence surveys which did not produce statistically significant associations. Serological surveillance will be increasingly important in the Peruvian Amazon as malaria transmission is reduced by continued control and elimination efforts.

PvMSP8 as a Novel Plasmodium vivax Malaria Sero-Marker for the Peruvian Amazon.

The measurement of recent malaria exposure can support malaria control efforts. This study evaluated serological responses to an in-house Plasmodium vivax Merozoite Surface Protein 8 (PvMSP8) expressed in a Baculovirus system as sero-marker of recent exposure to P. vivax (Pv) in the Peruvian Amazon. In a first evaluation, IgGs against PvMSP8 and PvMSP10 proteins were measured by Luminex in a cohort of 422 Amazonian individuals with known history of Pv exposure (monthly data of infection status by qPCR and/or microscopy over five months). Both serological responses were able to discriminate between exposed and non-exposed individuals in a good manner, with slightly higher performance of anti-PvMSP10 IgGs (area under the curve AUC = 0.78 [95% CI = 0.72-0.83]) than anti-PvMSP8 IgGs (AUC = 0.72 [95% CI = 0.67-0.78]) (p = 0.01). In a second evaluation, the analysis by ELISA of 1251 plasma samples, collected during a population-based cross-sectional survey, confirmed the good performance of anti-PvMSP8 IgGs for discriminating between individuals with Pv infection at the time of survey and/or with antecedent of Pv in the past month (AUC = 0.79 [95% CI = 0.74-0.83]). Anti-PvMSP8 IgG antibodies can be considered as a good biomarker of recent Pv exposure in low-moderate transmission settings of the Peruvian Amazon.

Heterogeneity in response to serological exposure markers of recent Plasmodium vivax infections in contrasting epidemiological contexts.

BACKGROUND: Antibody responses as serological markers of Plasmodium vivax infection have been shown to correlate with exposure, but little is known about the other factors that affect antibody responses in naturally infected people from endemic settings. To address this question, we studied IgG responses to novel serological exposure markers (SEMs) of P. vivax in three settings with different transmission intensity. METHODOLOGY: We validated a panel of 34 SEMs in a Peruvian cohort with up to three years' longitudinal follow-up using a multiplex platform and compared results to data from cohorts in Thailand and Brazil. Linear regression models were used to characterize the association between antibody responses and age, the number of detected blood-stage infections during follow-up, and time since previous infection. Receiver Operating Characteristic (ROC) analysis was used to test the performance of SEMs to identify P. vivax infections in the previous 9 months. PRINCIPAL FINDINGS: Antibody titers were associated with age, the number of blood-stage infections, and time since previous P. vivax infection in all three study sites. The association between antibody titers and time since previous P. vivax infection was stronger in the low transmission settings of Thailand and Brazil compared to the higher transmission setting in Peru. Of the SEMs tested, antibody responses to RBP2b had the highest performance for classifying recent exposure in all sites, with area under the ROC curve (AUC) = 0.83 in Thailand, AUC = 0.79 in Brazil, and AUC = 0.68 in Peru. CONCLUSIONS: In low transmission settings, P. vivax SEMs can accurately identify individuals with recent blood-stage infections. In higher transmission settings, the accuracy of this approach diminishes substantially. We recommend using P. vivax SEMs in low transmission settings pursuing malaria elimination, but they are likely to be less effective in high transmission settings focused on malaria control.

Temporal and Microspatial Heterogeneity in Transmission Dynamics of Coendemic Plasmodium vivax and Plasmodium falciparum in Two Rural Cohort Populations in the Peruvian Amazon.

BACKGROUND: Malaria is highly heterogeneous: its changing malaria microepidemiology needs to be addressed to support malaria elimination efforts at the regional level. METHODS: A 3-year, population-based cohort study in 2 settings in the Peruvian Amazon (Lupuna, Cahuide) followed participants by passive and active case detection from January 2013 to December 2015. Incidence and prevalence rates were estimated using microscopy and polymerase chain reaction (PCR). RESULTS: Lupuna registered 1828 infections (1708 Plasmodium vivax, 120 Plasmodium falciparum; incidence was 80.7 infections/100 person-years (95% confidence interval [CI] , 77.1-84.5). Cahuide detected 1046 infections (1024 P vivax, 20 P falciparum, 2 mixed); incidence was 40.2 infections/100 person-years (95% CI, 37.9-42.7). Recurrent P vivax infections predominated onwards from 2013. According to PCR data, submicroscopic predominated over microscopic infections, especially in periods of low transmission. The integration of parasitological, entomological, and environmental observations evidenced an intense and seasonal transmission resilient to standard control measures in Lupuna and a persistent residual transmission after severe outbreaks were intensively handled in Cahuide. CONCLUSIONS: In 2 exemplars of complex local malaria transmission, standard control strategies failed to eliminate submicroscopic and hypnozoite reservoirs, enabling persistent transmission.

Microsatellite analysis reveals connectivity among geographically distant transmission zones of Plasmodium vivax in the Peruvian Amazon: A critical barrier to regional malaria elimination.

Despite efforts made over decades by the Peruvian government to eliminate malaria, Plasmodium vivax remains a challenge for public health decision-makers in the country. The uneven distribution of its incidence, plus its complex pattern of dispersion, has made ineffective control measures based on global information that lack the necessary detail to understand transmission fully. In this sense, population genetic tools can complement current surveillance. This study describes the genetic diversity and population structure from September 2012 to March 2015 in three geographically distant settlements, Cahuide (CAH), Lupuna (LUP) and Santa Emilia (STE), located in the Peruvian Amazon. A total 777 P. vivax mono-infections, out of 3264, were genotyped. Among study areas, LUP showed 19.7% of polyclonal infections, and its genetic diversity (Hexp) was 0.544. Temporal analysis showed a significant increment of polyclonal infections and Hexp, and the introduction and persistence of a new parasite population since March 2013. In STE, 40.1% of infections were polyclonal, with Hexp = 0.596. The presence of four genetic clusters without signals of clonal expansion and infections with lower parasite densities compared against the other two areas were also found. At least four parasite populations were present in CAH in 2012, where, after June 2014, malaria cases decreased from 213 to 61, concomitant with a decrease in polyclonal infections (from 0.286 to 0.18), and expectedly variable Hexp. Strong signals of gene flow were present in the study areas and wide geographic distribution of highly diverse parasite populations were found. This study suggests that movement of malaria parasites by human reservoirs connects geographically distant malaria transmission areas in the Peruvian Amazon. The maintenance of high levels of parasite genetic diversity through human mobility is a critical barrier to malaria elimination in this region.

Micro-heterogeneity of malaria transmission in the Peruvian Amazon: a baseline assessment underlying a population-based cohort study.

BACKGROUND: Understanding the dynamics of malaria transmission in diverse endemic settings is key for designing and implementing locally adapted and sustainable control and elimination strategies. A parasitological and epidemiological survey was conducted in September-October 2012, as a baseline underlying a 3-year population-based longitudinal cohort study. The aim was to characterize malaria transmission patterns in two contrasting ecological rural sites in the Peruvian Amazon, Lupuna (LUP), a riverine environment, and Cahuide (CAH), associated with road-linked deforestation. METHODS: After a full population census, 1941 individuals 3 years and older (829 in LUP, 1112 in CAH) were interviewed, clinically examined and had a blood sample taken for the detection of malaria parasites by microscopy and PCR. Species-specific parasite prevalence was estimated overall and by site. Multivariate logistic regression models assessed risk factors for parasite infection by PCR, while SaTScan detected spatial clusters of PCR-positive individuals within each site. In addition, data from routine malaria surveillance in the period 2009-2012 were obtained. RESULTS: Parasite prevalence by PCR was higher in CAH than in LUP for Plasmodium vivax (6.2% vs. 3.9%) and for Plasmodium falciparum (2.6% vs. 1.2%). Among PCR-confirmed infections, asymptomatic (Asy) parasite carriers were always more common than symptomatic (Sy) infections for P. vivax (Asy/Sy ratio: 2/1 in LUP and 3.7/1 in CAH) and for P. falciparum (Asy/Sy ratio: 1.3/1 in LUP and 4/1 in CAH). Sub-patent (Spat) infections also predominated over patent (Pat) infections for both species: P. vivax (Spat/Pat ratio: 2.8/1 in LUP and 3.7/1 in CAH) and P. falciparum malaria (Spat/Pat ratio: 1.9/1 in LUP and 26/0 in CAH). For CAH, age, gender and living in a household without electricity were significantly associated with P. vivax infection, while only age and living in a household with electricity was associated with P. falciparum infection. For LUP, only household overcrowding was associated with P. falciparum infection. The spatial analysis only identified well-defined clusters of P. vivax and P. falciparum infected individuals in CAH. Reported malaria incidence indicated that malaria transmission has long occurred in LUP with primarily seasonal patterns, and confirmed a malaria outbreak in CAH since May 2012. CONCLUSIONS: This parasitological and epidemiological baseline assessment demonstrates that malaria transmission and parasite prevalence is heterogeneous in the Peruvian Amazon, and influenced by local socio-demographics and ecological contexts. Riverine and road construction/deforestation contexts must be taken into account in order to carry out effective anti-malaria control and elimination efforts.

Predominance of asymptomatic and sub-microscopic infections characterizes the Plasmodium gametocyte reservoir in the Peruvian Amazon.

Malaria transmission requires that Anopheles mosquitoes ingest Plasmodium gametocyte stages circulating in the human bloodstream. In the context of malaria elimination, understanding the epidemiology of gametocytes relative to all Plasmodium infections and the contribution of asymptomatic and sub-microscopic parasite carriers to the gametocyte reservoir is necessary, especially in low endemic settings with predominance of P.vivax. A 13-month longitudinal study was conducted in two communities (n = 1935 individuals) of Loreto Department, Peru, with five active screenings for Plasmodium infections and gametocyte stages by quantitative real-time PCR (qPCR) and reverse transcription (RT)-qPCR, respectively. Parasite prevalence by qPCR was 7.2% for P.vivax (n = 520/7235; range by survey 6.0%-8.1%) and 3.2% for P.falciparum (n = 235/7235; range by survey 0.4%-7.7%). Sub-microscopic infections accounted for 73.5% of P.vivax (range by survey 60%-89%) and almost the totality of P.falciparum cases. Gametocytes were found in 28.4% P.vivax infections (range by survey 18.7%-34.1%), with a peak of 61.5% in one community at the start of the transmission season. About 59.8% of all P.vivax gametocyte carriers were asymptomatic and 31.9% were sub-microscopic. Age patterns for gametocyte prevalence paralleled asexual stage infections and peaked among >15-25 year old individuals. Asexual parasite density was found to be the strongest predictor for P.vivax gametocyte presence in longitudinal multivariate analysis (odds ratio 2.33 [95% confidence interval 1.96, 2.78]; P<0.001). Despite significant differences in seasonality patterns and P.vivax prevalence found at the local scale, sub-microscopic and asymptomatic infections predominate and contribute significantly to the gametocyte reservoir in different communities of the Peruvian Amazon. Control and elimination campaigns need sensitive tools to detect all infections that escape routine malaria surveillance, which may contribute to maintain transmission in the region.

[Long-lasting insecticide - treated bednet ownership, retention and usage one year after their distribution in Loreto, Peru]. / Posesión, retención y uso de mosquiteros tratados con insecticidas de larga duración luego de un año de su distribución en Loreto, Perú

OBJECTIVES: To assess long-lasting insecticide - treated bednet (LLITN) ownership, retention and usage one year after their distribution in Loreto, Peru. MATERIALS AND METHODS: From July to September 2007, 26,185 LLITNs Olyset Net ® were delivered in 194 targeted communities in the Peruvian Amazon region, covering 45,331 people. Two cross-sectional community-based surveys in December 2007 and July 2008 were undertaken in a sample of 60 targeted communities (30.9% out of the total). A semi-structured questionnaire was used to collect necessary data to calculate LLITN ownership, retention and usage indicators. RESULTS: High LLITN household ownership was showed in both surveys (99.9% and 98.7%, respectively). LLITN/ person ratio decreased from 0.58 at the end of the LLITN delivery to 0.51 in the second survey, estimating LLITN retention of 87% after 1 year of intervention. In the first survey, 99.0% of all children <5 years and 96,0% of pregnant women slept under a LLITN the previous night, while in the second survey, 77.7% of children <5 years and 66.3% of pregnant women slept under a LLITN the previous night. Big mesh size of LLITN had low people's acceptability, reaching only 39.0% in the second survey. CONCLUSIONS: Although universal LLITN household ownership remained high, their LLITN usage levels have decreased during one-year follow-up period.

Posesión, retención y uso de mosquiteros tratados con insecticidas de larga duración luego de un año de su distribución en Loreto, Perú / Long-lasting insecticide - treated bednet ownership, retention and usage one year after their distribution in Loreto, Peru

Objetivos. Evaluar la posesión, retención y uso de mosquiteros tratados con insecticida de larga duración (MTILD) luego de un año de su distribución en Loreto, Perú. Materiales y métodos. De julio a septiembre de 2007 se distribuyeron 26 185 MTILD Olyset Net® en 194 comunidades objetivo de Loreto, que protegieron a 45 331 personas. Posteriormente, se realizaron visitas de monitoreo (primera en diciembre de 2007 y segunda en julio de 2008) a los hogares de una muestra de 60 comunidades objetivo (30,9 por ciento del total), colectándose mediante un cuestionario semiestructurado los datos necesarios para el cálculo de indicadores de posesión, retención y uso de los MTILD. Resultados. En ambas visitas, la posesión de MTILD en hogares fue elevada (99,9 por ciento y 98,7 por ciento, respectivamente). La razón MTILD/persona disminuyó de 0,58 al momento de la distribución a 0,51 en la segunda visita, estimándose una retención al año de MTILD de 87 por ciento. En la primera visita, 99,0 por ciento de los niños <5 años y 96,0 por ciento de las embarazadas durmieron bajo un MTILD la noche anterior, mientras que en la segunda visita, 77,7 por ciento de los niños<5 años y 66,3 por ciento de las embarazadas durmieron bajo un MTILD la noche anterior. El tamaño de los agujeros de la trama de los MTILD tuvo una baja aceptabilidad por parte del a población durante la segunda visita (39,0 por ciento). Conclusiones. Si bien la posesión de MTILD en los hogares se ha mantenido elevada, su uso por la población beneficiada ha disminuido durante el año de seguimiento.

Objectives. To assess long-lasting insecticide - treated bednet (LLITN) ownership, retention and usage one year after their distribution in Loreto, Peru. Materials and methods. From July to September 2007, 26,185 LLITNs Olyset Net ® were delivered in 194 targeted communities in the Peruvian Amazon region, covering 45,331 people. Two cross-sectional community-based surveys in December 2007 and July 2008 were undertaken in a sample of 60 targeted communities (30.9 percent out of the total). A semi-structured questionnaire was used to collect necessary data to calculate LLITN ownership, retention and usage indicators. Results. High LLITN household ownership was showed in both surveys (99.9 percent and 98.7 percent, respectively). LLITN/ person ratio decreased from 0.58 at the end of the LLITN delivery to 0.51 in the second survey, estimating LLITN retention of 87 percent after 1 year of intervention. In the first survey, 99.0 percent of all children <5 years and 96,0 percent of pregnant women slept under a LLITN the previous night, while in the second survey, 77.7 percent of children <5 years and 66.3 percent of pregnant women slept under a LLITN the previous night. Big mesh size of LLITN had low people´s acceptability, reaching only 39.0 percent in the second survey. Conclusions. Although universal LLITN household ownership remained high, their LLITN usage levels have decreased during one-year follow-up period.