Vivax Sporozoites Research Articles

Continuous Supply of Plasmodium vivax Sporozoites from Colonized Anopheles darlingi in the Peruvian Amazon.

In vitro culture of Plasmodium vivax liver stages underlies key understandings of the fundamental biology of this parasite, particularly the latent, hyponozoite stage, toward drug and vaccine development. Here, we report systematic production of Plasmodium vivax sporozoites in colonized Anopheles darlingi mosquitoes in the Peruvian Amazon. Human subject-derived P. vivax-infected blood was fed to Anopheles darlingi females using standard membrane feedings assays. Optimizing A. darlingi infection and sporozoite production included replacement of infected patient donor serum with naïve donor serum, comparing anticoagulants in processing blood samples, and addition of penicillin–streptomycin and ATP to infectious blood meals. Replacement of donor serum by naïve serum in the P. vivax donor blood increased oocysts in the mosquito midgut, and heparin, as anticoagulant, was associated with the highest sporozoite yields. Maintaining blood-fed mosquitoes on penicillin–streptomycin in sugar significantly extended mosquito survival which enabled greater sporozoite yield. In this study, we have shown that a robust P. vivax sporozoite production is feasible in a malaria-endemic setting where infected subjects and a stable A. darlingi colony are brought together, with optimized laboratory conditions.

Identifying Potential Plasmodium vivax Sporozoite Stage Vaccine Candidates: An Analysis of Genetic Diversity and Natural Selection.

Parasite antigen genetic diversity represents a great obstacle when designing a vaccine against malaria caused by Plasmodium vivax. Selecting vaccine candidate antigens has been focused on those fulfilling a role in invasion and which are conserved, thus avoiding specific-allele immune responses. Most antigens described to date belong to the blood stage, thereby blocking parasite development within red blood cells, whilst studying antigens from other stages has been quite restricted. Antigens from different parasite stages are required for developing a completely effective vaccine; thus, pre-erythrocyte stage antigens able to block the first line of infection becoming established should also be taken into account. However, few antigens from this stage have been studied to date. Several P. falciparum sporozoite antigens are involved in invasion. Since 77% of genes are orthologous amongst Plasmodium parasites, P. vivax sporozoite antigen orthologs to those of P. falciparum might be present in its genome. Although these genes might have high genetic diversity, conserved functionally-relevant regions (ideal for vaccine development) could be predicted by comparing genetic diversity patterns and evolutionary rates. This study was thus aimed at searching for putative P. vivax sporozoite genes so as to analyse their genetic diversity for determining their potential as vaccine candidates. Several DNA sequence polymorphism estimators were computed at each locus. The evolutionary force (drift, selection and recombination) drawing the genetic diversity pattern observed was also determined by using tests based on polymorphism frequency spectrum as well as the type of intra- and inter-species substitutions. Likewise, recombination was assessed both indirectly and directly. The results showed that sporozoite genes were more conserved than merozoite genes evaluated to date. Putative domains implied in cell traversal, gliding motility and hepatocyte interaction had a negative selection signal, being conserved amongst different species in the genus. PvP52, PvP36, PvSPATR, PvPLP1, PvMCP1, PvTLP, PvCelTOS, and PvMB2 antigens or functionally restricted regions within them would thus seem promising vaccine candidates and could be used when designing a pre-erythrocyte and/or multi-stage vaccine against P. vivax to avoid allele-specific immune responses that could reduce vaccine efficacy.

A novel immortalized hepatocyte-like cell line (imHC) supports in vitro liver stage development of the human malarial parasite Plasmodium vivax

BackgroundEradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay.MethodsA novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay.ResultsimHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening.ConclusionsimHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.

Relapse of a Plasmodium vivax Infection in an Iranian Patient: A Case Report

Introduction: Plasmodium vivax is the most prevalent in the malaria endemic areas of Iran. The number of Plasmodium vivax sporozoites that get to the liver remain latent, and after several months or years of inappropriate treatment period, they can reactivate and leads to the clinical manifestation of malaria. Case Presentation: A 49-year-old male was referred to a private clinical center in the capital city of Iran, Tehran, in October 2016 due to fever, chills, nausea, appetite, headache, and weakness. The patient had a history of clinical vivax malaria 1-year prior in Chabahar, in the province of Sistan and Baluchistan. The light microscope and nested PCR were applied for detecting the vivax malaria infection. In this patient, the relapse of P. vivax occurred after about 12 months, following the primary malaria infection. Conclusions: The report underlines that relapses should be considered in febrile individuals returning from malaria endemic areas of the country to malaria free areas. In addition, it highlights the occurrence of relapse in the patient with history using primaquine for a radical cure of liver latent parasites.

Vaccine Containing the Three Allelic Variants of the Plasmodium vivax Circumsporozoite Antigen Induces Protection in Mice after Challenge with a Transgenic Rodent Malaria Parasite.

Plasmodium vivax is the most common species that cause malaria outside of the African continent. The development of an efficacious vaccine would contribute greatly to control malaria. Recently, using bacterial and adenoviral recombinant proteins based on the P. vivax circumsporozoite protein (CSP), we demonstrated the possibility of eliciting strong antibody-mediated immune responses to each of the three allelic forms of P. vivax CSP (PvCSP). In the present study, recombinant proteins representing the PvCSP alleles (VK210, VK247, and P. vivax-like), as well as a hybrid polypeptide, named PvCSP-All epitopes, were generated. This hybrid containing the conserved C-terminal of the PvCSP and the three variant repeat domains in tandem were successfully produced in the yeast Pichia pastoris. After purification and biochemical characterization, they were used for the experimental immunization of C57BL/6 mice in a vaccine formulation containing the adjuvant Poly(I:C). Immunization with a recombinant protein expressing all three different allelic forms in fusion elicited high IgG antibody titers reacting with all three different allelic variants of PvCSP. The antibodies targeted both the C-terminal and repeat domains of PvCSP and recognized the native protein on the surface of P. vivax sporozoites. More importantly, mice that received the vaccine formulation were protected after challenge with chimeric Plasmodium berghei sporozoites expressing CSP repeats of P. vivax sporozoites (Pb/PvVK210). Our results suggest that it is possible to elicit protective immunity against one of the most common PvCSP alleles using soluble recombinant proteins expressed by P. pastoris. These recombinant proteins are promising candidates for clinical trials aiming to develop a multiallele vaccine against P. vivax malaria.

Biological, immunological and functional properties of two novel multi-variant chimeric recombinant proteins of CSP antigens for vaccine development against Plasmodium vivax infection

The circumsporozoite protein (CSP) of the malaria parasite Plasmodium vivax is a major pre-erythrocyte vaccine candidate. The protein has a central repeat region that belongs to one of repeat families (VK210, VK247, and the P. vivax-like). In the present study, computer modelling was employed to select chimeric proteins, comprising the conserved regions and different arrangements of the repeat elements (VK210 and VK247), whose structure is similar to that of the native counterparts. DNA encoding the selected chimeras (named CS127 and CS712) were synthetically constructed based on E. coli codons, then cloned and expressed. Mouse monoclonal antibodies (mAbs; anti-Pv-210-CDC and −Pv-247-CDC), recognized the chimeric antigens in ELISA, indicating correct conformation and accessibility of the B-cell epitopes. ELISA using IgG from plasma samples collected from 221 Iranian patients with acute P. vivax showed that only 49.32% of the samples reacted to both CS127 and CS712 proteins. The dominant subclass for the two chimeras was IgG1 (48% of the positive responders, OD492=0.777±0.420 for CS127; 48.41% of the positive responders, OD492=0.862±0.423 for CS712, with no statistically significant difference P>0.05; Wilcoxon signed ranks test). Binding assays showed that both chimeric proteins bound to immobilized heparan sulphate and HepG2 hepatocyte cells in a concentration-dependent manner, saturable at 80μg/mL. Additionally, anti-CS127 and −CS712 antibodies raised in mice recognized the native protein on the surface of P. vivax sporozoite with high intensity, confirming the presence of common epitopes between the recombinant forms and the native proteins. In summary, despite structural differences at the molecular level, the expression levels of both chimeras were satisfactory, and their conformational structure retained biological function, thus supporting their potential for use in the development of vivax-based vaccine.

Proteogenomic analysis of the total and surface-exposed proteomes of Plasmodium vivax salivary gland sporozoites.

Plasmodium falciparum and Plasmodium vivax cause the majority of human malaria cases. Research efforts predominantly focus on P. falciparum because of the clinical severity of infection and associated mortality rates. However, P. vivax malaria affects more people in a wider global range. Furthermore, unlike P. falciparum, P. vivax can persist in the liver as dormant hypnozoites that can be activated weeks to years after primary infection, causing relapse of symptomatic blood stages. This feature makes P. vivax unique and difficult to eliminate with the standard tools of vector control and treatment of symptomatic blood stage infection with antimalarial drugs. Infection by Plasmodium is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver. The most advanced malaria vaccine for P. falciparum (RTS,S, a subunit vaccine containing of a portion of the major sporozoite surface protein) conferred limited protection in Phase III trials, falling short of WHO-established vaccine efficacy goals. However, blocking the sporozoite stage of infection in P. vivax, before the establishment of the chronic liver infection, might be an effective malaria vaccine strategy to reduce the occurrence of relapsing blood stages. It is also thought that a multivalent vaccine comprising multiple sporozoite surface antigens will provide better protection, but a comprehensive analysis of proteins in P. vivax sporozoites is not available. To inform sporozoite-based vaccine development, we employed mass spectrometry-based proteomics to identify nearly 2,000 proteins present in P. vivax salivary gland sporozoites. Analysis of protein post-translational modifications revealed extensive phosphorylation of glideosome proteins as well as regulators of transcription and translation. Additionally, the sporozoite surface proteins CSP and TRAP, which were recently discovered to be glycosylated in P. falciparum salivary gland sporozoites, were also observed to be similarly modified in P. vivax sporozoites. Quantitative comparison of the P. vivax and P. falciparum salivary gland sporozoite proteomes revealed a high degree of similarity in protein expression levels, including among invasion-related proteins. Nevertheless, orthologs with significantly different expression levels between the two species could be identified, as well as highly abundant, species-specific proteins with no known orthologs. Finally, we employed chemical labeling of live sporozoites to isolate and identify 36 proteins that are putatively surface-exposed on P. vivax salivary gland sporozoites. In addition to identifying conserved sporozoite surface proteins identified by similar analyses of other Plasmodium species, our analysis identified several as-yet uncharacterized proteins, including a putative 6-Cys protein with no known ortholog in P. falciparum.

Experimental evaluation of cryopreservative solutions to maintain in vitro and in vivo infectivity of P. berghei sporozoites.

The rodent malaria parasite Plasmodium berghei is an excellent model organism for laboratory-based experimental evaluation of anti-malarial therapeutics prior to studies with human malaria parasites. The rodent model is especially important for evaluation of pre-erythrocytic (PE) stage therapies, especially as current efforts to develop new PE vaccines and drugs is limited by access to P. falciparum and P. vivax sporozoites. Developing a more effective method for cryopreservation of sporozoites would help improve access to sporozoites for laboratories lacking suitable insectary facilities. In this study, P. berghei GFP-expressing sporozoites were purified from infected mosquitoes by manual dissection of salivary glands and different commercially-available, serum-free cryopreservative solutions were evaluated for efficient cryopreservation of the sporozoites. The cryopreservative solutions evaluated included CryoStor CS2, CryoSolutions DX5, CryoSolutions MC, Hestar 200, Voluven, Hetastarch, and Glycerolyte 57. The viability of fresh and post-thaw cryopreserved sporozoites was determined as a function of the relative sporozoite infectivity by infecting HC-04 cells in vitro, monitoring invasion and growth and development of liver stage parasites. Flow cytometer-based counting provided unbiased and fast quantitative assessment of parasite in vitro infection in infected HC-04 and in vivo infectivity was validated by injecting sporozoites IV into mice. CryoStor CS2 delivered the highest post-thaw recovery and infectivity of cryopreserved sporozoites. Sporozoites cryopreserved in CryoStor CS2 achieved 38% complete development of hepatic stages in HC-04 and 100% infectivity in mice. The cryopreservation method described here demonstrates a viable alternative for fresh Plasmodium sporozoites. The use of cryopreserved sporozoites should facilitate greater access to sporozoites for chemotherapeutic and vaccine research.

A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus.

Theoretical Implications of a Pre-Erythrocytic Plasmodium vivax Vaccine for Preventing Relapses

Preventing malaria infection through vaccination requires preventing every sporozoite inoculated by mosquito bite: a major challenge for Plasmodium falciparum. Plasmodium vivax sporozoites consist of tachysporozoites causing primary infection and bradysporozoites leading to relapses. We hypothesise that a candidate P. vivax vaccine with low efficacy against primary infection may substantially reduce transmission by preventing relapses.

Distribution of malaria vectors and incidence of vivax malaria at Korean army installations near the demilitarized zone, Republic of Korea.

BackgroundAs a result of the reintroduction of malaria in the Republic of Korea (ROK) in 1993 and the threat to military and civilian populations, the Korea Military National Defense (MND) increased emphasis on vector control in 2012 at ROK Army (ROKA) installations located near the DMZ, while decreasing chemoprophylaxis, fearing potential drug resistance. Mosquito surveillance demonstrated a need for continuous monitoring of disease patterns among ROKA soldiers and vector malaria infection rates to ensure positive outcomes.MethodsAnopheles spp. were collected from May–October at three ROKA installations in three locations near the DMZ. Each of the areas included one installation <2 km and two installations 11–12 km from the DMZ in Paju and Yeoncheon counties, Gyeonggi Province. Anopheles spp. were identified by polymerase chain reaction (PCR) techniques and then assayed for the presence of vivax malaria sporozoites. The ROK MND reported vivax malaria patients monthly to Korea Centers for Disease Control and Prevention. Correlations for the incidence of Plasmodium vivax patients and infected Anopheles species were analysed using the Wilcoxon rank sum test, Pearson correlation test and liner regression analysis.ResultsA total of 4282 Anopheles spp. were collected. Anopheles kleini (69.5 %) was the most commonly collected, followed by Anopheles pullus (17.3 %), Anopheles belenrae (4.5 %), Anopheles sineroides (4.2 %), Anopheles sinensis sensu stricto (2.7 %), and Anopheles lesteri (1.9 %). Overall, 21 malaria patients were reported by the ROK MND. The monthly incidence of the malaria patients correlated with the monthly number of Plasmodium vivax sporozoite positive Anopheles spp. The monthly numbers of An.kleini demonstrated the highest correlations to the numbers of ROKA malaria patients throughout the mosquito season (P < 0.01). Anopheles spp. positive for P. vivax and malaria patients at ROKA installations located <2 km from the DMZ were higher than for ROKA installations located 11–12 km from DMZ.ConclusionThe number of Anopheles spp. positive for P. vivax sporozoites correlated with the monthly number of malaria cases and exposure of ROKA soldiers from May–October to P. vivax malaria infections. Malaria vector surveillance and vector control is warranted as part of an effective malaria management program at ROKA installations located near DMZ.

Circumsporozoite protein rates, blood-feeding pattern and frequency of knockdown resistance mutations in Anopheles spp. in two ecological zones of Mauritania.

BackgroundMosquitoes belonging to Anopheles gambiae species complex are the main malaria vector in Mauritania but data on their vector capacities, feeding habits and insecticide susceptibility are still scanty. The objectives of this study were to fill this gap.MethodsAdult Anopheles spp. mosquitoes were collected using pyrethrum spray catch method from two ecological zones of Mauritania: Nouakchott (Saharan zone) and Hodh Elgharbi region (Sahelian zone). Circumsporozoite proteins (CSP) for P. falciparum, P. vivax VK210 and P. vivax VK247 were detected by enzyme-linked immunosorbent assay (ELISA) from the female anopheline mosquitoes. To confirm CSP-ELISA results, polymerase chain reaction (PCR) was also performed. Blood meal identification was performed in all engorged females by partial sequencing of the mitochondrial cytochrome b gene. Molecular assessments of pyrethroid knockdown resistance (kdr) and insensitive acetylcholinesterase resistance (ace-1) were conducted.ResultsIn Nouakchott, the only species of Anopheles identified during the survey was Anopheles arabiensis (356 specimens). In Hodh Elgharbi, 1016 specimens of Anopheles were collected, including 578 (56.9 %) Anopheles rufipes, 410 (40.35 %) An. arabiensis, 20 (1.96 %) An. gambiae, 5 (0.5 %) An. pharoensis and 3 (0.3 %) An. funestus. Three of 186 female An. arabiensis collected in Nouakchott and tested by ELISA were found positive for Plasmodium vivax VK210, corresponding to a sporozoite rate of 1.6 %; however PCR confirmed infection by P. vivax sporozoite in only one of these. In Hodh Elgharbi, no mosquito was found positive for Plasmodium spp. infection. There was a statistically significant difference in the percentage of human blood-fed Anopheles spp. between Nouakchott (58.7 %, 47 of 80 blood-engorged An. arabiensis females) and Hodh Elgharbi (11.1 %, 2 of 18 blood-engorged mosquitoes). Analysis of the kdr polymorphisms showed 48.2 % (70/145) of East African kdr mutation (L1014S) in Nouakchott compared to 10 % (4/40) in Hodh Elgharbi region (P < 0.001). Nevertheless, West African kdr mutation (L1014F) was found only in An. gambiae populations (4/40, 10 %) from Hodh Elgharbi region. No ace-1 mutation was found in mosquito specimens from the two study zones.ConclusionsOverall, this study confirmed the autochthonous P. vivax malaria transmission in Nouakchott, involving An. arabiensis as the main vector. It also described for the first time the absence of ace-1 mutation, the co-occurrence of both West and East African kdr mutation in An. gambiae in Mauritania, and highlighted the regional variations in the prevalence and type of kdr mutations.

Antibody Profiling in Naïve and Semi-immune Individuals Experimentally Challenged with Plasmodium vivax Sporozoites.

BackgroundAcquisition of malaria immunity in low transmission areas usually occurs after relatively few exposures to the parasite. A recent Plasmodium vivax experimental challenge trial in malaria naïve and semi-immune volunteers from Colombia showed that all naïve individuals developed malaria symptoms, whereas semi-immune subjects were asymptomatic or displayed attenuated symptoms. Sera from these individuals were analyzed by protein microarray to identify antibodies associated with clinical protection.Methodology/Principal FindingsSerum samples from naïve (n = 7) and semi-immune (n = 9) volunteers exposed to P. vivax sporozoite-infected mosquito bites were probed against a custom protein microarray displaying 515 P. vivax antigens. The array revealed higher serological responses in semi-immune individuals before the challenge, although malaria naïve individuals also had pre-existing antibodies, which were higher in Colombians than US adults (control group). In both experimental groups the response to the P. vivax challenge peaked at day 45 and returned to near baseline at day 145. Additional analysis indicated that semi-immune volunteers without fever displayed a lower response to the challenge, but recognized new antigens afterwards.ConclusionClinical protection against experimental challenge in volunteers with previous P. vivax exposure was associated with elevated pre-existing antibodies, an attenuated serological response to the challenge and reactivity to new antigens.

Low prevalence of Plasmodium and absence of malaria transmission in Conakry, Guinea: prospects for elimination.

BackgroundOver the past 15 years, mortality and morbidity due to malaria have been reduced substantially in sub-Saharan Africa and local elimination has been achieved in some settings. This study addresses the bio-ecology of larval and adult stages of malaria vectors, Plasmodium infection in Anopheles gambiae s.l. in the city of Conakry, Guinea, and discusses the prospect for malaria elimination.MethodsWater bodies were prospected to identify potential mosquito breeding sites for 6 days each in the dry season (January 2013) and in the rainy season (August 2013), using the dipping method. Adult mosquitoes were collected in 15 communities in the five districts of Conakry using exit traps and indoor spraying catches over a 1-year period (November 2012 to October 2013). Molecular approaches were employed for identification of Anopheles species, including An. coluzzii and An.gambiae s.s. Individual An.gambiae mosquitoes were tested for Plasmodium falciparum and P. vivax sporozoites using the VecTest™ malaria panel assay and an enzyme-linked immunosorbent assay. A systematic research of Ministry of Health statistical yearbooks was performed to determine malaria prevalence in children below the age of 5 years.ResultsCulex larval breeding sites were observed in large numbers throughout Conakry in both seasons. While Anopheles larval breeding sites were less frequent than Culex breeding sites, there was a high odds of finding An.gambiae mosquito larvae in agricultural sites during the rainy season. Over the 1-year study period, a total of 14,334 adult mosquitoes were collected; 14,135 Culex (98.6 %) and 161 (1.1 %) from the An.gambiae complex. One-hundred and twelve Anopheles mosquitoes, mainly collected from rice fields and gardens, were subjected to molecular analysis. Most of the mosquitoes were An. gambiae s.s. (n = 102; 91.1 %) while the remaining 10 (8.9 %) were An.melas. The molecular M form of An. gambiae s.s. was predominant (n = 89; 79.5 %). The proportions of kdr genotype in the An. gambiae s.s. M and S form were 65.2 and 81.8 % (n = 9), respectively. No sporozoite infection were detected in any of the mosquitoes tested. The prevalence of Plasmodium recorded in children aged below 5 years was relatively low and varied between 2.2 and 7.6 % from 2009 to 2012.ConclusionsThe low density of larval and adult stages of Anopheles mosquitoes, the absence of infected An.gambiae species and the low prevalence of Plasmodium in under 5-year-old children are important features that might facilitate malaria elimination in Conakry. The heterogeneity in species composition and resistance profiles call for vector control interventions that are tailored to the local bio-ecological setting.

Plasmodium vivax gametocyte infectivity in sub-microscopic infections.

BackgroundThe use of molecular techniques has put in the spotlight the existence of a large mass of malaria sub-microscopic infections among apparently healthy populations. These sub-microscopic infections are considered an important pool for maintained malaria transmission.MethodsIn order to assess the appearance of Plasmodium vivax gametocytes in circulation, gametocyte density and the parasite infectivity to Anopheles mosquitoes, a study was designed to compare three groups of volunteers either experimentally infected with P. vivax sporozoites (early infections; n = 16) or naturally infected patients (acute malaria, n = 16 and asymptomatic, n = 14). In order to determine gametocyte stage, a quantitative reverse transcriptase PCR (RT-qPCR) assay targeting two sexual stage-specific molecular markers was used. Parasite infectivity was assessed by membrane feeding assays (MFA).ResultsIn early infections P. vivax gametocytes could be detected starting at day 7 without giving rise to infected mosquitoes during 13 days of follow-up. Asymptomatic carriers, with presumably long-lasting infections, presented the highest proportion of mature gametocytes and were as infective as acute patients.ConclusionsThis study shows the potential role of P. vivax asymptomatic carriers in malaria transmission should be considered when new policies are envisioned to redirect malaria control strategies towards targeting asymptomatic infections as a tool for malaria elimination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1104-1) contains supplementary material, which is available to authorized users.

Malaria transmission dynamics surrounding the first nationwide long-lasting insecticidal net distribution in Papua New Guinea.

BackgroundThe major malaria vectors of Papua New Guinea exhibit heterogeneities in distribution, biting behaviour and malaria infection levels. Long-lasting, insecticide-treated nets (LLINs), distributed as part of the National Malaria Control Programme, are the primary intervention targeting malaria transmission. This study evaluated the impact of LLINs on anopheline density, species composition, feeding behaviour, and malaria transmission.MethodsMosquitoes were collected by human landing catch in 11 villages from East Sepik Province and Madang Province. Mosquitoes were collected for 3 years (1 year before distribution and 2 years after), and assayed to determine mosquito species and Plasmodium spp. infection prevalence. The influence of weather conditions and the presence of people and animals on biting density was determined. Determinants of biting density and sporozoite prevalence were analysed by generalized estimating equations (GEE).ResultsMosquito biting rates and entomological inoculation rates decreased significantly after the distribution. Plasmodium falciparum and P. vivax sporozoite prevalence decreased in year 2, but increased in year 3, suggesting the likelihood of resurgence in transmission if low biting rates are not maintained. An earlier shift in the median biting time of Anopheles punctulatus and An. farauti s.s. was observed. However, this was not accompanied by an increase in the proportion of infective bites occurring before 2200 hours. A change in species composition was observed, which resulted in dominance of An. punctulatus in Dreikikir region, but a decrease in An. punctulatus in the Madang region. When controlling for village and study year, An. farauti s.s., An. koliensis and An. punctulatus were equally likely to carry P. vivax sporozoites. However, An. punctulatus was significantly more likely than An. farauti s.s. (OR 0.14; p = 0.007) or An. koliensis (OR 0.27; p < 0.001) to carry P. falciparum sporozoites.ConclusionsLLINs had a significant impact on malaria transmission, despite exophagic and crepuscular feeding behaviours of dominant vectors. Changes in species composition and feeding behaviour were observed, but their epidemiological significance will depend on their durability over time.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1067-7) contains supplementary material, which is available to authorized users.

A simple and efficient method for cryopreservation and recovery of viable Plasmodium vivax and P. falciparum sporozoites

Plasmodium falciparum and Plasmodium vivax sporozoites are the crucial stages of malaria parasites that initiate infection in humans. However, studies to develop new vaccines and drugs targeting these infective stages remain insufficient due to limited availability of sporozoites for research. This is a consequence of relatively few facilities that are established to produce sporozoites of human malaria parasites, sporozoites remaining viable for only a few days, and infected mosquitoes being a biohazard, making them difficult to transport. Cryopreservation of sporozoites offers the potential to alleviate these limitations and enhance sporozoite availability. These experiments were performed to evaluate methods for cryopreservation of P. vivax and P. falciparum sporozoites. Sporozoites, isolated in sterile buffer from infected mosquitoes by manual dissection of salivary glands, were cryopreserved using several types of commercially available serum-free cryoprotective solutions. The efficiency of cryopreservation was validated by a standard in vitro gliding motility assay as a measure of sporozoite activity. Viability of infective sporozoites was defined as percent gliding of sporozoites attached to the coverslip. Significant differences were observed among the cryopreservation media and protocols evaluated, with CryoStor CS2 giving the best results for both P. falciparum and P. vivax, whereas Hestar 200 worked efficiently only for P. vivax sporozoites. Further improvement in recovery of viable sporozoites would be anticipated using automated controlled-rate freezing equipment. Our results demonstrate that cryopreservation provides an alternative for experimental studies that currently rely on fresh P. falciparum and P. vivax sporozoites.

The suitability of laboratory-bred Anopheles cracens for the production of Plasmodium vivax sporozoites.

BackgroundA stenogamous colony of Anopheles cracens (A. dirus B) established 20 years ago in a Thai insectary proved susceptible to Plasmodium vivax. However, routine sporozoite production by feeding on field-collected blood samples has not been described. The setting-up of an A. cracens colony in an insectary on the Thai-Myanmar border and the process of using P. vivax field samples for the production of infectious sporozoites are described.MethodsThe colony was started in 2012 from egg batches that were sent from the Department of Parasitology, Faculty of Medicine, University of Chiang Mai, to the Shoklo Malaria Research Unit (SMRU), on wet filter paper in sealed Petri dishes. From May 2013 to December 2014, P. vivax-infected blood samples collected from patients seeking care at SMRU clinics were used for membrane feeding assays and sporozoite production.ResultsMosquitoes were fed on blood samples from 55 patients, and for 38 (69 %) this led to the production sporozoites. The average number of sporozoites obtained per mosquito was 26,112 (range 328–79,310). Gametocytaemia was not correlated with mosquito infectiousness (p = 0.82), or with the number of the sporozoites produced (Spearman’s ρ = −0.016, p = 0.905). Infectiousness did not vary with the date of collection or the age of the patient. Mosquito survival was not correlated with sporozoite load (Spearman’s ρ = 0.179, p = 0.282).ConclusionConsistent and routine P. vivax sporozoites production confirms that A. cracens is highly susceptible to P. vivax infection. Laboratory-bred colonies of this vector are suitable for experimental transmission protocols and thus constitute a valuable resource.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0830-0) contains supplementary material, which is available to authorized users.

Plasmodium vivax liver stage development and hypnozoite persistence in human liver-chimeric mice.

Plasmodium vivax malaria is characterized by periodic relapses of symptomatic blood stage parasite infections likely initiated by activation of dormant liver stage parasites-hypnozoites. The lack of tractable P. vivax animal models constitutes an obstacle in examining P. vivax liver stage infection and drug efficacy. To overcome this obstacle, we have used human liver-chimeric (huHep) FRG KO mice as a model for P. vivax infection. FRG KO huHep mice support P. vivax sporozoite infection, liver stage development, and hypnozoite formation. We show complete P. vivax liver stage development, including maturation into infectious exo-erythrocytic merozoites as well as the formation and persistence of hypnozoites. Prophylaxis or treatment with the antimalarial primaquine can prevent and eliminate liver stage infection, respectively. Thus, P. vivax-infected FRG KO huHep mice are a model to investigate liver stage development and dormancy and may facilitate the discovery of drugs targeting relapsing malaria.

Modeling the dynamics of Plasmodium vivax infection and hypnozoite reactivation in vivo.

The dynamics of Plasmodium vivax infection is characterized by reactivation of hypnozoites at varying time intervals. The relative contribution of new P. vivax infection and reactivation of dormant liver stage hypnozoites to initiation of blood stage infection is unclear. In this study, we investigate the contribution of new inoculations of P. vivax sporozoites to primary infection versus reactivation of hypnozoites by modeling the dynamics of P. vivax infection in Thailand in patients receiving treatment for either blood stage infection alone (chloroquine), or the blood and liver stages of infection (chloroquine + primaquine). In addition, we also analysed rates of infection in a study in Papua New Guinea (PNG) where patients were treated with either artesunate, or artesunate + primaquine. Our results show that up to 96% of the P. vivax infection is due to hypnozoite reactivation in individuals living in endemic areas in Thailand. Similar analysis revealed the around 70% of infections in the PNG cohort were due to hypnozoite reactivation. We show how the age of the cohort, primaquine drug failure, and seasonality may affect estimates of the ratio of primary P. vivax infection to hypnozoite reactivation. Modeling of P. vivax primary infection and hypnozoite reactivation provides important insights into infection dynamics, and suggests that 90–96% of blood stage infections arise from hypnozoite reactivation. Major differences in infection kinetics between Thailand and PNG suggest the likelihood of drug failure in PNG.