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Background: Plasmodium knowlesi, identified as the fifth human malaria parasite, has rapidly spread across various Southeast Asian countries, yet uncertainties persist regarding its human-mosquito-human transmission. Therefore, this study aims to explore the transmission potential of P. knowlesi from human blood to mosquitoes., Methods: A direct membrane-feeding assay was conducted by infecting laboratory-reared female Anopheles dirus mosquitoes with P. knowlesi-infected human blood from a single patient presenting with febrile malaria. Mosquitoes were dissected 7 days post-infection under a stereomicroscope to detect oocysts in the midgut, stained with mercurochrome. Salivary glands were examined 14 days post-infection for the presence of sporozoites. Malaria diagnosis employed microscopy by expert microscopists and nested PCR assays., Results: Upon dissecting 745 out of 1439 blood-fed An. dirus mosquitoes on day 7 post-infection, two oocysts were identified in the midguts of two mosquitoes (0.27%). An additional 694 mosquitoes were dissected for salivary glands on day 14 post-infection, with three mosquitoes (0.43%) exhibiting sporozoites. Further confirmation by nested-PCR assay verified these sporozoites as belonging to the P. knowlesi species., Conclusions: The findings underscore the potential transmission of P. knowlesi from human blood to mosquitoes. The significance of these findings necessitates further investigation, such as repeating similar experiments among natural vectors, to gain deeper insights into the transmission dynamics of P. knowlesi in Southeast Asia., (© 2024. The Author(s).)

Background: Plasmodium vivax is responsible for much of malaria outside Africa. Although most P. vivax infections in endemic areas are asymptomatic and have low parasite densities, they are considered a potentially important source of transmission. Several studies have demonstrated that asymptomatic P. vivax carriers can transmit the parasite to mosquitoes, but the efficiency has not been well quantified. The aim of this study is to determine the relationship between parasite density and mosquito infectivity, particularly at low parasitaemia., Methods: Membrane feeding assays were performed using serial dilutions of P. vivax-infected blood to define the relationship between parasitaemia and mosquito infectivity., Results: The infection rate (oocyst prevalence) and intensity (oocyst load) were positively correlated with the parasite density in the blood. There was a broad case-to-case variation in parasite infectivity. The geometric mean parasite density yielding a 10% mosquito infection rate was 33 (CI 95  9-120) parasites/µl or 4 (CI 95  1-17) gametocytes/µl. The geometric mean parasite density yielding a 50% mosquito infection rate was 146 (CI 95  36-586) parasites/µl or 13 (CI 95  3-49) gametocytes/µl., Conclusion: This study quantified the ability of P. vivax to infect Anopheles dirus at over a broad range of parasite densities. It provides important information about parasite infectivity at low parasitaemia common among asymptomatic P. vivax carriers., (© 2023. The Author(s).)

Background: Plasmodium vivax (P. vivax) is the dominant Plasmodium spp. causing the disease malaria in low-transmission regions outside of Africa. These regions often feature high proportions of asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. However, previous work has failed to address the presence and maintenance of such antibody responses to P. vivax particularly in low-transmission regions., Methods: We followed 34 patients in western Thailand after symptomatic P. vivax infections to monitor antibody kinetics over 9 months, during which no recurrent infections occurred. We assessed total IgG, IgG subclass and IgM levels to up to 52 P. vivax proteins every 2-4 weeks using a multiplexed Luminex® assay and identified protein-specific variation in antibody longevity. Mathematical modelling was used to generate the estimated half-life of antibodies, long-, and short-lived antibody-secreting cells., Results: Generally, an increase in antibody level was observed within 1-week post symptomatic infection, followed by an exponential decay of different rates. We observed mostly IgG1 dominance and IgG3 sub-dominance in this population. IgM responses followed similar kinetic patterns to IgG, with some proteins unexpectedly inducing long-lived IgM responses. We also monitored antibody responses against 27 IgG-immunogenic antigens in 30 asymptomatic individuals from a similar region. Our results demonstrate that most antigens induced robust and long-lived total IgG responses following asymptomatic infections in the absence of (detected) boosting infections., Conclusions: Our work provides new insights into the development and maintenance of naturally acquired immunity to P. vivax and will guide the potential use of serology to indicate immune status and/or identify populations at risk., (© 2022. The Author(s).)

Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, 2 healthy malaria-naive United Kingdom adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI. Parasitemia developed in both volunteers, and prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected RBCs. Following stringent safety screening, the parasite stabilate from one of these donors (PvW1) was thawed and used to inoculate 6 healthy malaria-naive United Kingdom adults by blood-stage CHMI, at 3 different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high-quality genome assembly by using a hybrid assembly method. We analyzed leading vaccine candidate antigens and multigene families, including the vivax interspersed repeat (VIR) genes, of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies.

Objective: 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., Methods: The qRT-PCR was used to detect the expression of the yellow-g gene in many organs of both male and female mosquitos. The yellow-g gene silencing was performed by dsRNA membrane feeding to An. dirus. These mosquitoes were later challenged by P. vivax-infected blood. The oocyst numbers were determined., Results: The yellow-g transcript was detected in several organs of both male and female An. dirus mosquitoes. Successful knockdown of yellow-g was achieved and resulted in reduced P. vivax infection in the mosquitoes. The decrease in yellow-g expression had no effect on the life span of the mosquitoes., Conclusions: These results support the yellow-g gene as having an important function in Plasmodium development in Anopheles mosquitoes., (© 2021 John Wiley & Sons Ltd.)

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 Plasmodium vivax are impeding these efforts. We hypothesized that a validated surveillance tool utilizing serological markers of recent exposure to P. vivax infection could help to identify areas of ongoing transmission. The objective of this exploratory study was to assess the ability of P. vivax serological exposure markers to detect residual transmission "hot-spots" in Western Thailand. Total IgG levels were measured against a panel of 23 candidate P. vivax 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 P. vivax infection in Thailand, including age, gender, and spatial location, as well as Plasmodium infection status itself. IgG levels to all proteins were significantly higher in the presence of a P. vivax infection ( n = 144) ( T -test, p < 0.0001). Overall seropositivity rates varied from 2.5% (PVX&#95;097625, merozoite surface protein 8) to 16.8% (PVX&#95;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, p < 0.05) and males (17/23 proteins, p < 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 P. vivax parasites in the last 9-months, based on their IgG antibody levels to a panel of eight previously validated P. vivax 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., Competing Interests: RL, MW, TT, and IM are inventors on patent PCT/US17/67926 on a system, method, apparatus, and diagnostic test for Plasmodium vivax. MH was employed by company CellFree Sciences Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chotirat, Nekkab, Kumpitak, Hietanen, White, Kiattibutr, Sa-angchai, Brewster, Schoffer, Takashima, Tsuboi, Harbers, Chitnis, Healer, Tham, Nguitragool, Mueller, Sattabongkot and Longley.)

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.

Background: 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., Methods: A cross-sectional survey of 4309 participants was conducted in three endemic areas in Kanchanaburi and Ratchaburi provinces of Thailand in 2012. The presence of P. falciparum and P. vivax parasites was determined using 18S rRNA qPCR. Gametocytes were also detected by pfs25 / pvs25 qRT-PCRs., Results: A total of 133 individuals were found infected with P. vivax (3.09%), 37 with P. falciparum (0.86%), and 11 with mixed P. vivax/ P. falciparum (0.26%). The clear majority of both P. vivax (91.7%) and P. falciparum (89.8%) infections were not accompanied by any febrile symptoms. Infections with either species were most common in adolescent and adult males. Recent travel to Myanmar was highly associated with P. falciparum (OR = 9.0, P = 0.001) but not P. vivax infections (P = 0.13). A large number of P. vivax (71.5%) and P. falciparum (72.0%) infections were gametocyte positive by pvs25/pfs25 qRT-PCR. Detection of gametocyte-specific pvs25 and pfs25 transcripts was strongly dependent on parasite density. pvs25 transcript numbers, a measure of gametocyte density, were also highly correlated with parasite density (r 2  = 0.82, P < 0.001)., Conclusions: Asymptomatic infections with Plasmodium spp. were common in western Thai communities in 2012. The high prevalence of gametocytes indicates that these infections may contribute substantially to the maintenance of local malaria transmission.

Background: 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., Methods: Plasma samples from 546 volunteers enrolled in a cross-sectional survey conducted in 2012 in Kanchanaburi Province were utilized. Total IgG levels to 11 different proteins known or predicted to be involved in reticulocyte binding or invasion (ARP, GAMA, P41, P12, PVX&#95;081550, and five members of the PvRBP family), as well as the leading pre-erythrocytic vaccine candidate (CSP) were measured using a multiplexed bead-based assay. Associations between IgG levels and infection status, age, and spatial location were explored., Results: Individuals from a low-transmission region of western Thailand reacted to all 11 P. vivax recombinant proteins. Significantly greater IgG levels were observed in the presence of a current P. vivax infection, despite all infected individuals being asymptomatic. IgG levels were also higher in adults (18 years and older) than in children. For most of the proteins, higher IgG levels were observed in individuals living closer to the Myanmar border and further away from local health services., Conclusions: Robust IgG responses were observed to most proteins and IgG levels correlated with surrogates of exposure, suggesting these antigens may serve as potential biomarkers of exposure, immunity, or both.

Background: 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., Methods: Adult Anopheles mosquitoes were collected using CDC light traps and cow bait in 7 villages along the Thai-Myanmar border in January 2011 - March 2013. Mosquitoes were determined to species by morphological characters. Plasmodium-positivity was determined by circumsporozoite protein ELISA., Results: The 2986 Anopheles mosquitoes collected were assigned to 26 species, with Anopheles minimus sensu lato (s.l.) (40.32%), An. maculatus s.l. (21.43%), An. annularis s.l. (14.43%), An. kochi (5.39%), An. tessellatus (5.26%), and An. barbirostris s.l. (3.52%) being the top six most abundant species. Plasmodium-infected mosquitoes were found in 22 positive samples from 2906 pooled samples of abdomens and heads/thoraxes. Four mosquito species were found infected with Plasmodium: An. minimus s.l., An. maculatus s.l., An. annularis s.l. and An. barbirostris s.l. The infectivity rates of these mosquitoes were 0.76, 0.37, 0.72, and 1.74%, respectively. Consistent with a change in malaria epidemiology to the predominance of P. vivax in this area, 20 of the 22 infected mosquito samples were P. vivax-positive. The four potential vector species all displayed apparent seasonality in relative abundance. While An. minimus s.l. was collected through the entire year, its abundance peaked in the season immediately after the wet season. In comparison, An. maculatus s.l. numbers showed a major peak during the wet season. The two potential vector species, An. annularis s.l. and An. barbirostris s.l., both showed peak abundance during the transition from wet to dry season. Moreover, An. minimus s.l. was more abundant in indoor collections, whereas An. annularis s.l. and An. barbirostris s.l. were more abundant in outdoor collections, suggesting their potential role in outdoor malaria transmission., Conclusions: This survey confirmed the major vector status of An. minimus s.l. and An. maculatus s.l. and identified An. annularis s.l. and An. barbirostris s.l. as additional vectors with potential importance in malaria transmission after the wet season.

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., (Copyright © 2016 Longley et al.)

We evaluated the efficacy of the OptiMAL assay in a cross-sectional malaria survey in western Thailand from April to August 2001. Expert microscopy of Giemsa-stained thick and thin blood films was used as the gold standard. Positive control lines were evident in 99% (1,128 of 1,137) of the assays tested. However, 34% (384 of 1,128) of assays produced an aberrant result (a positive P. falciparum-specific line and a negative panmalarial line). False-positive panmalarial and Plasmodium falciparum-specific lines occurred in 25.9% (270 of 1,042) and 60.3% (628 of 1,042) of microscopy-negative samples, respectively. Due to the preponderance of false-positive test results, it was necessary to develop subjective criteria for test positivity based on line intensity. For determination of assay performance during this study, we therefore considered all test lines that were scored as intermediate or strong as positive and lines that were faint as negative. Using these criteria, we determined that the sensitivity of the OptiMAL assay for P. falciparum was 25% with > 500 parasites/microl and 10.5% with > 100 parasites/microl, while for P. vivax, the sensitivity at the same parasite rates was 100% and 41.7%, respectively. Further studies are required to determine whether the problems we identified are limited to this particular lot of OptiMAL assays.

Microscopy of Giemsa-stained thick and thin films by a skilled microscopist has remained the standard laboratory method for the diagnosis of malaria. However, diagnosis of malaria with this method is problematic since interpretation of results requires considerable expertise, particularly at low parasite levels. We compared the efficacy of "field" and "expert laboratory" microscopy for active surveillance of Plasmodium falciparum and P. vivax in western Thailand. Field microscopy consisted of an approximately five-minute read (50-100 fields) of a thick film at x700 using a natural light source, whereas expert laboratory microscopy consisted of a 20-minute read (number of parasites per 500 leukocytes) at x1,000 using a high-quality, well-maintained microscope with an artificial light source. All discordant and 20% of concordant results were cross-checked blindly. A total of 3,004 blood films collected between May and November 2000 were included in the study, of which 156 (5.2%) were positive for P. falciparum, 177 (5.9%) for P. vivax, and 4 (0.1%) for both P. falciparum and P. vivax by expert microscopy. A total of 84.4% (135 of 160) of the P. falciparum-positive slides and 93.9% of the P. vivax-positive slides had a parasitemia of less than 500/microL. Field microscopy was specific (99.3%) but not sensitive (10.0%) for the diagnosis of P. falciparum malaria, with a positive predictive value (PPV) of 43.2% and a negative predictive value (NPV) of 95.1%. The corresponding specificity and sensitivity for the diagnosis of P. vivax malaria were 99.2% and 7.1%, respectively, with a PPV of 38.7% and an NPV of 93.9%. Field microscopy, as defined in this study, is not an effective method for active malaria surveillance in western Thailand, where prevalence and parasitemia rates are low.

Rapid antigen assays provide an effective tool for the detection of malaria in symptomatic patients. However, the efficacy of these devices for detecting asymptomatic malaria, where parasite levels are normally significantly lower than in symptomatic patients, is less well established. We evaluated the efficacy of a new combined Plasmodium falciparum-Plasmodim vivax immunochromatographic test (ICT Malaria Pf/Pv) in a cross-sectional malaria survey of the village of Ban Kong Mong Tha, Kanchanaburi Provice, Thailand, from August to December 2000. A total of 1,976 bleeds were made from 559 individuals over the course of the study. Blinded microscopy of thick and thin blood films was used as the gold standard; all discordant and 10% of concordant results were cross-checked. Of 1,976 ICT Malaria Pf/Pv dipsticks tested, 98.3% (n = 1,943) performed as expected, as evidenced by the appearance of the control line. The ICT Malaria Pf/Pv test was both sensitive (100.0%) and specific (99.7 %) for the diagnosis of falciparum malaria with parasitemias of > or = 500 trophozoites/microL; however, only 15.9% (13/82) of infected individuals had parasitemia rates this high. When P. falciparum parasitemia rates were < 500/microL, the sensitivity of the diagnosis was only 23.3%, with a positive predictive value (PPV) and a negative predictive value (NPV) of 76.2 and 97.2%, respectively. The ICT Malaria Pf/Pv test was specific, but not sensitive, for the diagnosis of vivax malaria with parasite rates of > or = 500 trophozoites/microl, with sensitivity, specificity, PPV, and NPV of 66.7%, 99.9%, 66.7%, and 99.9%, respectively. At parasite rates of < 500/microL, corresponding values were 0.0%, 99.9%, 0%, and 95.1%. Because of the relatively high cost of these assays, low parasite rates found in the majority of asymptomatic individuals, and low sensitivity of this assay with rates of < 500/microl, use of this assay as a tool for active case detection is of limited value in western Thailand.