Detection of <em>Plasmodium</em> Sporozoites in <em>Anopheles</em> Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female ...

Mahidol University

Novel strategies to directly thwart malaria transmission are needed to maintain the gains achieved by current control measures. Transmission-blocking interventions (TBIs), namely vaccines and drugs targeting parasite or mosquito molecules required for vector-stage parasite development, have been recognized as promising approaches for preventing malaria transmission. However, the number of TBI targets is limited and their degree of conservation among the major vector-parasite systems causing human disease is unclear. Therefore, discovery and characterization of novel proteins involved in vector-stage parasite development of Plasmodium falciparum and Plasmodium vivax is paramount. We mined the recent Anopheles gambiae midgut lipid raft proteome for putative mosquito-derived TBI targets and characterized a secreted glycoconjugate of unknown function, AgSGU. We analyzed molecular variation in this protein among a range of anopheline mosquitoes, determined its transcriptomic and proteomic profiles, and conducted both standard and direct membrane feeding assays with P. falciparum (lab/field) and P. vivax (field) in An. gambiae and Anopheles dirus. We observed that α-AgSGU antibodies significantly reduced midgut infection intensity for both lab and field isolates of P. falciparum in An. gambiae and An. dirus. However, no transmission-reducing effects were noted when comparable concentrations of antibodies were included in P. vivax-infected blood meals. Although antibodies against AgSGU exhibit transmission-reducing activity, the high antibody titer required for achieving 80% reduction in oocyst intensity precludes its consideration as a malaria mosquito-based TBI candidate. However, our results suggest that P. falciparum and P. vivax ookinetes use a different repertoire of midgut surface glycoproteins for invasion and that α-AgSGU antibodies, as well as antibodies to other mosquito-midgut microvillar surface proteins, may prove useful as tools for interrogating Plasmodium-mosquito interactions. 

Differential roles of an Anopheline midgut GPI-anchored protein in mediating Plasmodium falciparum and Plasmodium vivax ookinete invasion.

The evaluation of Plasmodium vivax gametocyte infectiousness by the membrane feeding assay is herein described. While P. vivax cannot be cultured and different parasite isolates may infect mosquitoes at different rates, the protocol described in this chapter identifies critical parameters to be considered when performing the assay such as methods for the preparation of the mosquitoes, the size of the blood cup, and the blood volume used. In previous studies the data have shown that the membrane feeding assay is useful for studies of parasite biology, and the effects of transmission blocking drugs and vaccines. 

Membrane Feeding Assay to Determine the Infectiousness of Plasmodium vivax Gametocytes.

Plasmodium vivax is now the dominant Plasmodium species causing malaria in Thailand, yet little is known about naturally acquired immune responses to this parasite in this low-transmission region. The preerythrocytic stage of the P. vivax life cycle is considered an excellent target for a malaria vaccine, and in this study, we assessed the stability of the seropositivity and the magnitude of IgG responses to three different preerythrocytic P. vivax proteins in two groups of adults from a region of western Thailand where malaria is endemic. These individuals were enrolled in a yearlong cohort study, which comprised one group that remained P. vivax free (by quantitative PCR [qPCR] detection, n = 31) and another that experienced two or more blood-stage P. vivax infections during the year of follow up (n = 31). Despite overall low levels of seropositivity, IgG positivity and magnitude were long-lived over the 1-year period in the absence of qPCR-detectable blood-stage P. vivax infections. In contrast, in the adults with two or more P. vivax infections during the year, IgG positivity was maintained, but the magnitude of the response to P. vivax circumsporozoite protein 210 (CSP210) decreased over time. These findings demonstrate that long-term humoral immunity can develop in low-transmission regions. 

Acquisition and Longevity of Antibodies to Plasmodium vivax Preerythrocytic Antigens in Western Thailand.

The Thai-Myanmar border is a remaining hotspot for malaria transmission. Malaria transmission in this region continues year-round, with a major peak season in July-August, and a minor peak in October-November. Malaria elimination requires better knowledge of the mosquito community structure, dynamics and vectorial status to support effective vector control.

Natural human Plasmodium infections in major Anopheles mosquitoes in western Thailand.

Thailand is aiming to eliminate malaria by the year 2024. Plasmodium vivax has now become the dominant species causing malaria within the country, and a high proportion of infections are asymptomatic. A better understanding of antibody dynamics to P. vivax antigens in a low-transmission setting, where acquired immune responses are poorly characterized, will be pivotal for developing new strategies for elimination, such as improved surveillance methods and vaccines. The objective of this study was to characterize total IgG antibody levels to 11 key P. vivax proteins in a village of western Thailand.

Asymptomatic Plasmodium vivax infections induce robust IgG responses to multiple blood-stage proteins in a low-transmission region of western Thailand.

Low-density asymptomatic infections of Plasmodium spp. are common in low endemicity areas worldwide, but outside Africa, their contribution to malaria transmission is poorly understood. Community-based studies with highly sensitive molecular diagnostics are needed to quantify the asymptomatic reservoir of Plasmodium falciparum and P. vivax infections in Thai communities.

Very high carriage of gametocytes in asymptomatic low-density Plasmodium falciparum and P. vivax infections in western Thailand.

There was recently an outbreak of malaria in Ubon Ratchathani Province, northeastern Thailand. In the absence of information on malaria vector transmission dynamics, this study aimed to identify the anopheline vectors and their role in malaria transmission. Adult female Anopheles mosquitoes were collected monthly by human-landing catch in Na Chaluai District of Ubon Ratchathani Province during January 2014-December 2015. Field-captured mosquitoes were identified to species using morphology-based keys and molecular assays (allele-specific polymerase chain reaction, AS-PCR), and analysed for the presence of Plasmodium falciparum and Plasmodium vivax using an enzyme-linked immunosorbent assay (ELISA) for the detection of circumsporozoite proteins (CSP). A total of 1,229 Anopheles females belonging to 13 species were collected. Four anopheline taxa were most abundant: Members of the Anopheles barbirostris complex, comprising 38% of the specimens, species of the Anopheles hyrcanus group (18%), Anopheles nivipes (17%) and Anopheles philippinensis (12%). The other nine species comprised 15% of the collections. Plasmodium infections were detected in two of 668 pooled samples of heads/thoraces, Anopheles dirus (1/29) and An. philippinensis (1/97). The An. dirus pool had a mixed infection of P. vivax-210 and P. vivax-247, whereas the An. philippinensis pool was positive only for the latter protein variant. Both positive ELISA samples were confirmed by nested PCR. This study is the first to incriminate An. dirus and An. philippinensis as natural malaria vectors in the area where the outbreak occurred. This information can assist in designing and implementing a more effective malaria control programme in the province.

Natural Plasmodium vivax infections in Anopheles mosquitoes in a malaria endemic area of northeastern Thailand.

Malaria is transmitted by Plasmodium parasites through the bite of female Anopheles mosquitoes. One of the most important mosquito vectors in the Greater Mekong Subregion is Anopheles dirus. This study reports RNA sequencing (RNA-Seq) transcriptome analysis of An. dirus at 18 hours and 7 days after a P. vivax-infected blood meal, which represent infection at the ookinete and oocyst parasite developmental stages, respectively. Following infection, 582 An. dirus transcripts were modulated. The 2,408 P. vivax transcripts could be classified into ookinete-specific, two-stage, and oocyst-specific groups. Results were validated by quantitative reverse transcription polymerase chain reaction. Gene ontology analysis of the vector and parasite revealed several biological pathways for both, providing a better understanding of Anopheles-Plasmodium interactions at the ookinete and oocyst stages.

Transcriptome analysis of Anopheles dirus and Plasmodium vivax at ookinete and oocyst stages.

Standard and direct membrane-feeding assays (SMFA and DMFA) are fundamental assays to evaluate efficacy of transmission-blocking intervention (TBI) candidates against Plasmodium falciparum and vivax. To compare different candidates precisely, it is crucial to understand the error range of measured activity, usually expressed as percent inhibition in either oocyst intensity (% transmission reducing activity, %TRA), or in prevalence of infected mosquitoes (% transmission blocking activity, %TBA). To this end, mathematical models have been proposed for P. falciparum SMFA (PfSMFA), but such study for DMFA is limited. In this study, we analyzed P. vivax DMFA (PvDMFA) data from 22,236 mosquitoes tested from 96 independent assays. While the two assays are quite different, a zero-inflated negative binomial (ZINB) model could reasonably explain the PvDMFA results, as it has for PfSMFA. Our simulation studies based on the ZINB model revealed it is better to report %TRA values with a proper error range, rather than observed %TBA both in SMFA and DMFA. Furthermore, the simulations help in designing a better assay and aid in estimating an error range of a %TRA value when the uncertainty is not reported. This study strongly supports future TBI development by providing a rational method to compare different candidates.

Evaluation and modeling of direct membrane-feeding assay with Plasmodium vivax to support development of transmission blocking vaccines.

Our previous transcriptome analysis of Anopheles dirus revealed upregulation of the An. dirus yellow-g gene upon ingestion of Plasmodium vivax-infected blood. This gene belongs to the yellow gene family, but its role regarding P. vivax infection is not known and remains to be validated. The aim of this study was to investigate the role of the An. dirus yellow-g gene in P. vivax infection.

Anopheles dirus yellow-g mediates Plasmodium vivax infection.

Thailand is aiming for malaria elimination by the year 2030. However, the high proportion of asymptomatic infections and the presence of the hidden hypnozoite stage of  are impeding these efforts. We hypothesized that a validated surveillance tool utilizing serological markers of recent exposure to  infection could help to identify areas of ongoing transmission. The objective of this exploratory study was to assess the ability of  serological exposure markers to detect residual transmission "hot-spots" in Western Thailand. Total IgG levels were measured against a panel of 23 candidate  serological exposure markers using a multiplexed bead-based assay. A total of 4,255 plasma samples from a cross-sectional survey conducted in 2012 of endemic areas in the Kanchanaburi and Ratchaburi provinces were assayed. We compared IgG levels with multiple epidemiological factors that are associated with an increased risk of  infection in Thailand, including age, gender, and spatial location, as well as  infection status itself. IgG levels to all proteins were significantly higher in the presence of a  infection ( = 144) (-test,  < 0.0001). Overall seropositivity rates varied from 2.5% (PVX_097625, merozoite surface protein 8) to 16.8% (PVX_082670, merozoite surface protein 7), with 43% of individuals seropositive to at least 1 protein. Higher IgG levels were associated with older age (>18 years,  < 0.05) and males (17/23 proteins,  < 0.05), supporting the paradigm that men have a higher risk of infection than females in this setting. We used a Random Forests algorithm to predict which individuals had exposure to  parasites in the last 9-months, based on their IgG antibody levels to a panel of eight previously validated  proteins. Spatial clustering was observed at the village and regional level, with a moderate correlation between PCR prevalence and sero-prevalence as predicted by the algorithm. Our data provides proof-of-concept for application of such surrogate markers as evidence of recent exposure in low transmission areas. These data can be used to better identify geographical areas with asymptomatic infection burdens that can be targeted in elimination campaigns.

Chalermpon Kumpitak

Mahidol Vivax Research Unit,
Faculty of Tropical Medicine,
Mahidol Vivax Research Unit, Faculty of Tropical Medicine

Mahidol University

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Chalermpon Kumpitak

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Mahidol Vivax Research Unit,Faculty of Tropical Medicine,Mahidol Vivax Research Unit, Faculty of Tropical Medicine

Mahidol University

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Mahidol Vivax Research Unit,
Faculty of Tropical Medicine,
Mahidol Vivax Research Unit, Faculty of Tropical Medicine