Diagnostic performance and comparison of ultrasensitive and conventional rapid diagnostic test, thick blood smear and quantitative PCR for detection of low-density Plasmodium falciparum infections during a controlled human malaria infection study in Equatorial Guinea.

BACKGROUND: Progress towards malaria elimination has stagnated, partly because infections persisting at low parasite densities comprise a large reservoir contributing to ongoing malaria transmission and are difficult to detect. This study compared the performance of an ultrasensitive rapid diagnostic test (uRDT) designed to detect low density infections to a conventional RDT (cRDT), expert microscopy using Giemsa-stained thick blood smears (TBS), and quantitative polymerase chain reaction (qPCR) during a controlled human malaria infection (CHMI) study conducted in malaria exposed adults (NCT03590340). METHODS: Blood samples were collected from healthy Equatoguineans aged 18-35 years beginning on day 8 after CHMI with 3.2 × 103 cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ Challenge, strain NF54) administered by direct venous inoculation. qPCR (18s ribosomal DNA), uRDT (Alere™ Malaria Ag P.f.), cRDT [Carestart Malaria Pf/PAN (PfHRP2/pLDH)], and TBS were performed daily until the volunteer became TBS positive and treatment was administered. qPCR was the reference for the presence of Plasmodium falciparum parasites. RESULTS: 279 samples were collected from 24 participants; 123 were positive by qPCR. TBS detected 24/123 (19.5% sensitivity [95% CI 13.1-27.8%]), uRDT 21/123 (17.1% sensitivity [95% CI 11.1-25.1%]), cRDT 10/123 (8.1% sensitivity [95% CI 4.2-14.8%]); all were 100% specific and did not detect any positive samples not detected by qPCR. TBS and uRDT were more sensitive than cRDT (TBS vs. cRDT p = 0.015; uRDT vs. cRDT p = 0.053), detecting parasitaemias as low as 3.7 parasites/µL (p/µL) (TBS and uRDT) compared to 5.6 p/µL (cRDT) based on TBS density measurements. TBS, uRDT and cRDT did not detect any of the 70/123 samples positive by qPCR below 5.86 p/µL, the qPCR density corresponding to 3.7 p/µL by TBS. The median prepatent periods in days (ranges) were 14.5 (10-20), 18.0 (15-28), 18.0 (15-20) and 18.0 (16-24) for qPCR, TBS, uRDT and cRDT, respectively; qPCR detected parasitaemia significantly earlier (3.5 days) than the other tests. CONCLUSIONS: TBS and uRDT had similar sensitivities, both were more sensitive than cRDT, and neither matched qPCR for detecting low density parasitaemia. uRDT could be considered an alternative to TBS in selected applications, such as CHMI or field diagnosis, where qualitative, dichotomous results for malaria infection might be sufficient.

Multi-Dose Priming Regimens of PfSPZ Vaccine: Safety and Efficacy against Controlled Human Malaria Infection in Equatoguinean Adults.

Plasmodium falciparum sporozoites (PfSPZ) Vaccine is composed of radiation-attenuated, aseptic, purified cryopreserved PfSPZ. Multiple clinical trials empirically assessing two to six doses have shown multi-dose priming (-two to four doses the first week) to be optimal for protection in both 4- and 16-week regimens. In this randomized, double-blind, normal saline (NS), placebo-controlled trial, four groups (G) of 18- to 32-year-old Equatoguineans received multi-dose priming regimens with or without a delayed final dose at 4 or 16 weeks (9 × 105 PfSPZ/dose). The regimens were G1: days 1, 3, 5, 7, and 113; G2: days 1, 3, 5, and 7; G3: days 1, 3, 5, 7, and 29; and G4: days 1, 8, and 29). All doses were 9 × 105 PfSPZ. Tolerability, safety, immunogenicity, and vaccine efficacy (VE) against homologous-controlled human malaria infection (CHMI) 6-7 weeks after vaccination were assessed to down-select the best regimen. All four regimens were safe and well tolerated, with no significant differences in adverse events (AEs) between vaccinees (N = 84) and NS controls (N = 20) or between regimens. Out of 19 controls, 13 developed Pf parasitemia by quantitative polymerase chain reaction (qPCR) after CHMI. Only the vaccine regimen administered on study days 1, 8, and 29 gave significant protection (7/21 vaccinees versus 13/19 controls infected, VE 51.3%, P = 0.03, Barnard's test, two-tailed). There were no significant differences in antibodies against Pf circumporozoite protein (PfCSP), a major SPZ antigen, between protected and nonprotected vaccinees or controls pre-CHMI. The six controls not developing Pf parasitemia had significantly higher antibodies to blood stage antigens Pf exported protein 1 (PfEXP1) and Pf merozoite surface protein 1 (PfMSP1) than the controls who developed parasitemia, suggesting naturally acquired immunity against Pf-limited infections in controls. This study identified a safe, protective, 4-week, multi-dose prime vaccination regimen for assessment in future trials of PfSPZ Vaccine.

Incidence of Plasmodium falciparum malaria infection in 6-month to 45-year-olds on selected areas of Bioko Island, Equatorial Guinea.

BACKGROUND: Extensive malaria control measures have been implemented on Bioko Island, Equatorial Guinea over the past 16 years, reducing parasite prevalence and malaria-related morbidity and mortality, but without achieving elimination. Malaria vaccines offer hope for reducing the burden to zero. Three phase 1/2 studies have been conducted successfully on Bioko Island to evaluate the safety and efficacy of whole Plasmodium falciparum (Pf) sporozoite (SPZ) malaria vaccines. A large, pivotal trial of the safety and efficacy of the radiation-attenuated Sanaria® PfSPZ Vaccine against P. falciparum is planned for 2022. This study assessed the incidence of malaria at the phase 3 study site and characterized the influence of socio-demographic factors on the burden of malaria to guide trial design. METHODS: A cohort of 240 randomly selected individuals aged 6 months to 45 years from selected areas of North Bioko Province, Bioko Island, was followed for 24 weeks after clearance of parasitaemia. Assessment of clinical presentation consistent with malaria and thick blood smears were performed every 2 weeks. Incidence of first and multiple malaria infections per person-time of follow-up was estimated, compared between age groups, and examined for associated socio-demographic risk factors. RESULTS: There were 58 malaria infection episodes observed during the follow up period, including 47 first and 11 repeat infections. The incidence of malaria was 0.25 [95% CI (0.19, 0.32)] and of first malaria was 0.23 [95% CI (0.17, 0.30)] per person per 24 weeks (0.22 in 6-59-month-olds, 0.26 in 5-17-year-olds, 0.20 in 18-45-year-olds). Incidence of first malaria with symptoms was 0.13 [95% CI (0.09, 0.19)] per person per 24 weeks (0.16 in 6-59-month-olds, 0.10 in 5-17-year-olds, 0.11 in 18-45-year-olds). Multivariate assessment showed that study area, gender, malaria positivity at screening, and household socioeconomic status independently predicted the observed incidence of malaria. CONCLUSION: Despite intensive malaria control efforts on Bioko Island, local transmission remains and is spread evenly throughout age groups. These incidence rates indicate moderate malaria transmission which may be sufficient to support future larger trials of PfSPZ Vaccine. The long-term goal is to conduct mass vaccination programmes to halt transmission and eliminate P. falciparum malaria.

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.

Single-Dose Tafenoquine to Prevent Relapse of Plasmodium vivax Malaria.

BACKGROUND: Treatment of Plasmodium vivax malaria requires the clearing of asexual parasites, but relapse can be prevented only if dormant hypnozoites are cleared from the liver (a treatment termed "radical cure"). Tafenoquine is a single-dose 8-aminoquinoline that has recently been registered for the radical cure of P. vivax. METHODS: This multicenter, double-blind, double-dummy, parallel group, randomized, placebo-controlled trial was conducted in Ethiopia, Peru, Brazil, Cambodia, Thailand, and the Philippines. We enrolled 522 patients with microscopically confirmed P. vivax infection (>100 to <100,000 parasites per microliter) and normal glucose-6-phosphate dehydrogenase (G6PD) activity (with normal activity defined as ≥70% of the median value determined at each trial site among 36 healthy male volunteers who were otherwise not involved in the trial). All patients received a 3-day course of chloroquine (total dose of 1500 mg). In addition, patients were assigned to receive a single 300-mg dose of tafenoquine on day 1 or 2 (260 patients), placebo (133 patients), or a 15-mg dose of primaquine once daily for 14 days (129 patients). The primary outcome was the Kaplan-Meier estimated percentage of patients who were free from recurrence at 6 months, defined as P. vivax clearance without recurrent parasitemia. RESULTS: In the intention-to-treat population, the percentage of patients who were free from recurrence at 6 months was 62.4% in the tafenoquine group (95% confidence interval [CI], 54.9 to 69.0), 27.7% in the placebo group (95% CI, 19.6 to 36.6), and 69.6% in the primaquine group (95% CI, 60.2 to 77.1). The hazard ratio for the risk of recurrence was 0.30 (95% CI, 0.22 to 0.40) with tafenoquine as compared with placebo (P<0.001) and 0.26 (95% CI, 0.18 to 0.39) with primaquine as compared with placebo (P<0.001). Tafenoquine was associated with asymptomatic declines in hemoglobin levels, which resolved without intervention. CONCLUSIONS: Single-dose tafenoquine resulted in a significantly lower risk of P. vivax recurrence than placebo in patients with phenotypically normal G6PD activity. (Funded by GlaxoSmithKline and Medicines for Malaria Venture; DETECTIVE ClinicalTrials.gov number, NCT01376167 .).

Tafenoquine versus Primaquine to Prevent Relapse of Plasmodium vivax Malaria.

BACKGROUND: Tafenoquine, a single-dose therapy for Plasmodium vivax malaria, has been associated with relapse prevention through the clearance of P. vivax parasitemia and hypnozoites, termed "radical cure." METHODS: We performed a phase 3, prospective, double-blind, double-dummy, randomized, controlled trial to compare tafenoquine with primaquine in terms of safety and efficacy. The trial was conducted at seven hospitals or clinics in Peru, Brazil, Colombia, Vietnam, and Thailand and involved patients with normal glucose-6-phosphate dehydrogenase (G6PD) enzyme activity and female patients with moderate G6PD enzyme deficiency; all patients had confirmed P. vivax parasitemia. The patients were randomly assigned, in a 2:1 ratio, to receive a single 300-mg dose of tafenoquine or 15 mg of primaquine once daily for 14 days (administered under supervision); all patients received a 3-day course of chloroquine and were followed for 180 days. The primary safety outcome was a protocol-defined decrease in the hemoglobin level (>3.0 g per deciliter or ≥30% from baseline or to a level of <6.0 g per deciliter). Freedom from recurrence of P. vivax parasitemia at 6 months was the primary efficacy outcome in a planned patient-level meta-analysis of the current trial and another phase 3 trial of tafenoquine and primaquine (per-protocol populations), and an odds ratio for recurrence of 1.45 (tafenoquine vs. primaquine) was used as a noninferiority margin. RESULTS: A protocol-defined decrease in the hemoglobin level occurred in 4 of 166 patients (2.4%; 95% confidence interval [CI], 0.9 to 6.0) in the tafenoquine group and in 1 of 85 patients (1.2%; 95% CI, 0.2 to 6.4) in the primaquine group, for a between-group difference of 1.2 percentage points (95% CI, -4.2 to 5.0). In the patient-level meta-analysis, the percentage of patients who were free from recurrence at 6 months was 67.0% (95% CI, 61.0 to 72.3) among the 426 patients in the tafenoquine group and 72.8% (95% CI, 65.6 to 78.8) among the 214 patients in the primaquine group. The efficacy of tafenoquine was not shown to be noninferior to that of primaquine (odds ratio for recurrence, 1.81; 95% CI, 0.82 to 3.96). CONCLUSIONS: Among patients with normal G6PD enzyme activity, the decline in hemoglobin level with tafenoquine did not differ significantly from that with primaquine. Tafenoquine showed efficacy for the radical cure of P. vivax malaria, although tafenoquine was not shown to be noninferior to primaquine. (Funded by GlaxoSmithKline and Medicines for Malaria Venture; GATHER ClinicalTrials.gov number, NCT02216123 .).

Antimalarial activity of single-dose DSM265, a novel plasmodium dihydroorotate dehydrogenase inhibitor, in patients with uncomplicated Plasmodium falciparum or Plasmodium vivax malaria infection: a proof-of-concept, open-label, phase 2a study.

BACKGROUND: DSM265 is a novel, long-duration inhibitor of plasmodium dihydroorotate dehydrogenase (DHODH) with excellent selectivity over human DHODH and activity against blood and liver stages of Plasmodium falciparum. This study aimed to assess the efficacy of DSM265 in patients with P falciparum or Plasmodium vivax malaria infection. METHODS: This proof-of-concept, open-label, phase 2a study was conducted at the Asociación Civil Selva Amazónica in Iquitos, Peru. Patients aged 18-70 years, weighing 45-90 kg, who had clinical malaria (P falciparum or P vivax monoinfection) and fever within the previous 24 h were eligible. Exclusion criteria were clinical or laboratory signs of severe malaria, inability to take oral medicine, and use of other antimalarial treatment in the preceding 14 days. Patients were divided into cohorts of those with P falciparum (cohort a) or P vivax (cohort b) infection. Two initial cohorts received single oral doses of 400 mg DSM265. Patients were followed up for efficacy for 28 days and safety for 35 days. Further cohorts received escalated or de-escalated doses of DSM265, after safety and efficacy assessment of the initial dose. The primary endpoints were the proportion of patients achieving PCR-adjusted adequate clinical and parasitological response (ACPR) by day 14 for patients infected with P falciparum and the proportion of patients achieving a crude cure by day 14 for those infected with P vivax. Cohort success, the criteria for dose escalation, was defined as ACPR (P falciparum) or crude cure (P vivax) in at least 80% of patients in the cohort. The primary analysis was done in the intention-to-treat population (ITT) and the per-protocol population, and safety analyses were done in all patients who received the study drug. This study is registered at ClinicalTrials.gov (NCT02123290). FINDINGS: Between Jan 12, 2015, and Dec 2, 2015, 45 Peruvian patients (24 with P falciparum [cohort a] and 21 with P vivax [cohort b] infection) were sequentially enrolled. For patients with P falciparum malaria in the per-protocol population, all 11 (100%) in the 400 mg group and eight (80%) of ten in the 250 mg group achieved ACPR on day 14. In the ITT analysis, 11 (85%) of 13 in the 400 mg group and eight (73%) of 11 in the 250 mg group achieved ACPR at day 14. For the patients with P vivax malaria, the primary endpoint was not met. In the per-protocol analysis, none of four patients who had 400 mg, three (50%) of six who had 600 mg, and one (25%) of four who had 800 mg DSM265 achieved crude cure at day 14. In the ITT analysis, none of five in the 400 mg group, three (33%) of nine in the 600 mg group, and one (14%) of seven in the 800 mg group achieved crude cure at day 14. During the 28-day extended observation of P falciparum patients, a resistance-associated mutation in the gene encoding the DSM265 target DHODH was observed in two of four recurring patients. DSM265 was well tolerated. The most common adverse events were pyrexia (20 [44%] of 45) and headache (18 [40%] of 45), which are both common symptoms of malaria, and no patients had any treatment-related serious adverse events or adverse events leading to study discontinuation. INTERPRETATION: After a single dose of DSM265, P falciparum parasitaemia was rapidly cleared, whereas against P vivax, DSM265 showed less effective clearance kinetics. Its long duration of action provides the potential to prevent recurrence of P falciparum after treatment with a single dose, which should be further assessed in future combination studies. FUNDING: The Global Health Innovative Technology Fund, the Bill & Melinda Gates Foundation, the National Institutes of Health (R01 AI103058), the Wellcome Trust, and the UK Department of International Development.

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.

Accelerating to Zero: Strategies to Eliminate Malaria in the Peruvian Amazon.

AbstractIn February 2014, the Malaria Elimination Working Group, in partnership with the Peruvian Ministry of Health (MoH), hosted its first international conference on malaria elimination in Iquitos, Peru. The 2-day meeting gathered 85 malaria experts, including 18 international panelists, 23 stakeholders from different malaria-endemic regions of Peru, and 11 MoH authorities. The main outcome was consensus that implementing a malaria elimination project in the Amazon region is achievable, but would require: 1) a comprehensive strategic plan, 2) the altering of current programmatic guidelines from control toward elimination by including symptomatic as well as asymptomatic individuals for antimalarial therapy and transmission-blocking interventions, and 3) the prioritization of community-based active case detection with proper rapid diagnostic tests to interrupt transmission. Elimination efforts must involve key stakeholders and experts at every level of government and include integrated research activities to evaluate, implement, and tailor sustainable interventions appropriate to the region.

Genome-Scale Protein Microarray Comparison of Human Antibody Responses in Plasmodium vivax Relapse and Reinfection.

Large scale antibody responses in Plasmodium vivax malaria remains unexplored in the endemic setting. Protein microarray analysis of asexual-stage P. vivax was used to identify antigens recognized in sera from residents of hypoendemic Peruvian Amazon. Over 24 months, of 106 participants, 91 had two symptomatic P. vivax malaria episodes, 11 had three episodes, 3 had four episodes, and 1 had five episodes. Plasmodium vivax relapse was distinguished from reinfection by a merozoite surface protein-3α restriction fragment length polymorphism polymerase chain reaction (MSP3α PCR-RFLP) assay. Notably, P. vivax reinfection subjects did not have higher reactivity to the entire set of recognized P. vivax blood-stage antigens than relapse subjects, regardless of the number of malaria episodes. The most highly recognized P. vivax proteins were MSP 4, 7, 8, and 10 (PVX_003775, PVX_082650, PVX_097625, and PVX_114145); sexual-stage antigen s16 (PVX_000930); early transcribed membrane protein (PVX_090230); tryptophan-rich antigen (Pv-fam-a) (PVX_092995); apical merozoite antigen 1 (PVX_092275); and proteins of unknown function (PVX_081830, PVX_117680, PVX_118705, PVX_121935, PVX_097730, PVX_110935, PVX_115450, and PVX_082475). Genes encoding reactive proteins exhibited a significant enrichment of non-synonymous nucleotide variation, an observation suggesting immune selection. These data identify candidates for seroepidemiological tools to support malaria elimination efforts in P. vivax-endemic regions.

Infection of laboratory-colonized Anopheles darlingi mosquitoes by Plasmodium vivax.

Anopheles darlingi Root is the most important malaria vector in the Amazonia region of South America. However, continuous propagation of An. darlingi in the laboratory has been elusive, limiting entomological, genetic/genomic, and vector-pathogen interaction studies of this mosquito species. Here, we report the establishment of an An. darlingi colony derived from wild-caught mosquitoes obtained in the northeastern Peruvian Amazon region of Iquitos in the Loreto Department. We show that the numbers of eggs, larvae, pupae, and adults continue to rise at least to the F6 generation. Comparison of feeding Plasmodium vivax ex vivo of F4 and F5 to F1 generation mosquitoes showed the comparable presence of oocysts and sporozoites, with numbers that corresponded to blood-stage asexual parasitemia and gametocytemia, confirming P. vivax vectorial capacity in the colonized mosquitoes. These results provide new avenues for research on An. darlingi biology and study of An. darlingi-Plasmodium interactions.

Microgeographical differences of Plasmodium vivax relapse and re-infection in the Peruvian Amazon.

To determine the magnitude of Plasmodium vivax relapsing malaria in rural Amazonia, we carried out a study in four sites in northeastern Peru. Polymerase chain reaction-restriction fragment length polymorphism of PvMSP-3α and tandem repeat (TR) markers were compared for their ability to distinguish relapse versus reinfection. Of 1,507 subjects with P. vivax malaria, 354 developed > 1 episode during the study; 97 of 354 (27.5%) were defined as relapse using Pvmsp-3α alone. The addition of TR polymorphism analysis significantly reduced the number of definitively defined relapses to 26 of 354 (7.4%) (P < 0.05). Multivariate logistic regression modeling showed that the probability of having > 1 infection was associated with the following: subjects in Mazan (odds ratio [OR] = 2.56; 95% confidence interval [CI] 1.87, 3.51), 15-44 years of age (OR = 1.49; 95% CI 1.03, 2.15), traveling for job purposes (OR = 1.45; 95%CI 1.03, 2.06), and travel within past month (OR = 1.46; 95% CI 1.0, 2.14). The high discriminatory capacity of the molecular tools shown here is useful for understanding the micro-geography of malaria transmission.

Human host-derived cytokines associated with Plasmodium vivax transmission from acute malaria patients to Anopheles darlingi mosquitoes in the Peruvian Amazon.

Infection of mosquitoes by humans is not always successful in the setting of patent gametocytemia. This study tested the hypothesis that pro- or anti-inflammatory cytokines are associated with transmission of Plasmodium vivax to Anopheles darlingi mosquitoes in experimental infection. Blood from adults with acute, non-severe P. vivax malaria was fed to laboratory-reared F1 An. darlingi mosquitoes. A panel of cytokines at the time of mosquito infection was assessed in patient sera and levels compared among subjects who did and did not infect mosquitoes. Overall, blood from 43 of 99 (43%) subjects led to mosquito infection as shown by oocyst counts. Levels of IL-10, IL-6, TNF-α, and IFN-γ were significantly elevated in vivax infection and normalized 3 weeks later. The anti-inflammatory cytokine IL-10 was significantly higher in nontransmitters compared with top transmitters but was not in TNF-α and IFN-γ. The IL-10 elevation during acute malaria was associated with P. vivax transmission blocking.

Whole genome sequencing analysis of Plasmodium vivax using whole genome capture.

BACKGROUND: Malaria caused by Plasmodium vivax is an experimentally neglected severe disease with a substantial burden on human health. Because of technical limitations, little is known about the biology of this important human pathogen. Whole genome analysis methods on patient-derived material are thus likely to have a substantial impact on our understanding of P. vivax pathogenesis and epidemiology. For example, it will allow study of the evolution and population biology of the parasite, allow parasite transmission patterns to be characterized, and may facilitate the identification of new drug resistance genes. Because parasitemias are typically low and the parasite cannot be readily cultured, on-site leukocyte depletion of blood samples is typically needed to remove human DNA that may be 1000X more abundant than parasite DNA. These features have precluded the analysis of archived blood samples and require the presence of laboratories in close proximity to the collection of field samples for optimal pre-cryopreservation sample preparation. RESULTS: Here we show that in-solution hybridization capture can be used to extract P. vivax DNA from human contaminating DNA in the laboratory without the need for on-site leukocyte filtration. Using a whole genome capture method, we were able to enrich P. vivax DNA from bulk genomic DNA from less than 0.5% to a median of 55% (range 20%-80%). This level of enrichment allows for efficient analysis of the samples by whole genome sequencing and does not introduce any gross biases into the data. With this method, we obtained greater than 5X coverage across 93% of the P. vivax genome for four P. vivax strains from Iquitos, Peru, which is similar to our results using leukocyte filtration (greater than 5X coverage across 96% ). CONCLUSION: The whole genome capture technique will enable more efficient whole genome analysis of P. vivax from a larger geographic region and from valuable archived sample collections.

High degree of Plasmodium vivax diversity in the Peruvian Amazon demonstrated by tandem repeat polymorphism analysis.

Molecular tools to distinguish strains of Plasmodium vivax are important for studying the epidemiology of malaria transmission. Two sets of markers-tandem repeat (TR) polymorphisms and MSP3α-were used to study Plasmodium vivax in patients in the Peruvian Amazon region of Iquitos. Of 110 patients, 90 distinct haplotypes were distinguished using 9 TR markers. An MSP3α polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using HhaI and AluI revealed 8 and 9 profiles, respectively, and 36 profiles when analyzed in combination. Combining TR and PCR-RFLP markers, 101 distinct molecular profiles were distinguished among these 110 patients. Nine TR markers arrayed along a 100 kB stretch of a P. vivax chromosome containing the gene for circumsporozoite protein showed non-linear linkage disequilibrium (I(SA) = 0.03, P = 0.001). These findings demonstrate the potential use of TR markers for molecular epidemiology studies.

Socio-demographics and the development of malaria elimination strategies in the low transmission setting.

This analysis presents a comprehensive description of malaria burden and risk factors in Peruvian Amazon villages where malaria transmission is hypoendemic. More than 9000 subjects were studied in contrasting village settings within the Department of Loreto, Peru, where most malaria occurs in the country. Plasmodium vivax is responsible for more than 75% of malaria cases; severe disease from any form of malaria is uncommon and death rare. The association between lifetime malaria episodes and individual and household covariates was studied using polychotomous logistic regression analysis, assessing effects on odds of some vs. no lifetime malaria episodes. Malaria morbidity during lifetime was strongly associated with age, logging, farming, travel history, and living with a logger or agriculturist. Select groups of adults, particularly loggers and agriculturists acquire multiple malaria infections in transmission settings outside of the main domicile, and may be mobile human reservoirs by which malaria parasites move within and between micro-regions within malaria endemic settings. For example, such individuals might well be reservoirs of transmission by introducing or reintroducing malaria into their home villages and their own households, depending on vector ecology and the local village setting. Therefore, socio-demographic studies can identify people with the epidemiological characteristic of transmission risk, and these individuals would be prime targets against which to deploy transmission blocking strategies along with insecticide treated bednets and chemoprophylaxis.

Lack of molecular correlates of Plasmodium vivax ookinete development.

Previous studies of Plasmodium vivax transmission to Anopheles spp. mosquitoes have not been able to predict mosquito infectivity on the basis of microscopic or molecular quantification of parasites (total parasites in the sample or total number of gametocytes) in infected blood. Two methods for production of P. vivax ookinete cultures in vitro, with yields of 10(6) macrogametocytes, 10(4) zygotes, and 10(3) ookinetes, respectively, per 10 mL of P. vivax-infected patient blood with approximately 0.01% parasitemia, were used to study P. vivax sexual stage development. The quantity of gametocytes, determined by counting Giemsa-stained blood smears, and quantity and type of gametocyte as determined by quantitative reverse transcriptase-polymerase chain reaction for Pvalpha tubulin II and macrogametocyte-specific pvg377 did not predict ookinete yield. Factors that affect the efficiency of in vitro P. vivax ookinete transformation remain poorly understood.

Polymerase chain reaction detection of Plasmodium vivax and Plasmodium falciparum DNA from stored serum samples: implications for retrospective diagnosis of malaria.

Polymerase chain reaction (PCR) detection of Plasmodium DNA is highly sensitive in diagnosing malaria. The specimen of choice for this assay has been whole blood samples from malaria patients. To retrospectively determine malaria infection rates in populations or cohorts for whom stored serum samples are available, we determined the ability of a nested PCR assay to detect Plasmodium DNA in stored serum samples. The PCR result was positive in 20 of 23 serum samples from patients with microscopy-confirmed malaria and negative in 8 of 8 healthy controls, resulting in a sensitivity of 87% and specificity of 100%. In all positive samples, species were correctly identified by PCR except for one case where a mixed infection was detected. The PCR is able to detect Plasmodium DNA in serum samples frozen up to 2.5 years and has the potential for the retrospective identification of malaria parasitemia in patient cohorts to determine potential interactions of malaria and other diseases such as human immunodeficiency virus/acquired immunodeficiency syndrome.

Experimental infection of the neotropical malaria vector Anopheles darlingi by human patient-derived Plasmodium vivax in the Peruvian Amazon.

Malaria transmission from humans to mosquitoes is modulated by human host immune factors. Understanding mechanisms by which the human host response may impair parasite infectivity for mosquitoes has direct implications for the development of transmission-blocking vaccines. We hypothesized that despite a low transmission intensity of malaria in the Peruvian Amazon region of Iquitos, transmission-blocking immunity against Plasmodium vivax might be common, given an unexpectedly high proportion of asymptomatic parasitemic individuals in this region. To test this hypothesis, the ability of symptomatic P. vivax malaria patients to experimentally infect wild-caught outbred Anopheles darlingi mosquitoes was tested using the indirect membrane feeding technique. Only half (52/102) of P. vivax parasitemic patients successfully infected mosquitoes. Transmitters were more likely to have gametocytes (OR 6.35, P = 0.003), high parasitemia (OR 3.79, P = 0.024), and, in terms of basic clinical parameters, a slower pulse rate (mean +/- SD: 82.3 +/- 12.3 versus 88.7 +/- 13.5, P = 0.016) than non-transmitters. Log(10) gametocytemia and log(10) real-time reverse transcriptase Pvs25 PCR quantifying gametocytes were significantly and positively correlated with oocyst counts (correlation coefficient 0.505, R2 = 0.26, P = 0.001). These experiments are the first to establish a system of determining transmission patterns in experimental infection of outbred natural neotropical malaria vectors in the Amazon region. Patients with P. vivax inefficiently infect outbred An. darlingi mosquitoes, raising the possibility that some degree of naturally occurring transmission-blocking immunity is present on a population basis in the Peruvian Amazon, an area of low intensity of malaria transmission.