Controlled human malaria infection with a clone of Plasmodium vivax with high-quality genome assembly.

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.

Malaria cross-sectional surveys identified asymptomatic infections of Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi in Surat Thani, a southern province of Thailand.

OBJECTIVES: Malaria cross-sectional surveys are rarely conducted in very low transmission settings. This study aimed to determine the prevalence and risk factors of Plasmodium infection in a near-elimination setting in southern Thailand. METHODS: Two cross-sectional surveys were conducted in areas of active transmission in the Surat Thani province of Thailand in January and May 2019. PCR was used to detect Plasmodium infection. RESULTS: The prevalence of Plasmodium blood infection was 0.45% and 0.61% in January and May 2019, respectively. The major parasite species was Plasmodium falciparum in January and Plasmodium vivax in May. Unexpectedly, Plasmodium knowlesi infections were also detected. Most infections, including those of Plasmodium knowlesi, were asymptomatic. Being male and staying outdoors at night-time were the only significant identified risk factors. Of people infected in January 28.0% were positive in May for the same parasite species, suggesting persistent asymptomatic infections. CONCLUSIONS: Despite the very low incidence rate in Surat Thani, most malaria infections were asymptomatic. Outdoor mosquito biting at night-time is likely an important mode of malaria transmission. Unexpectedly, asymptomatic Plasmodium knowlesi infection was found, confirming previous reports of such infection in mainland Southeast Asia.

Case Report: Case Series of Human Plasmodium knowlesi Infection on the Southern Border of Thailand.

Although human infections of Plasmodium knowlesi have been found throughout Southeast Asia, most cases originated from Malaysian Borneo. In Thailand, P. knowlesi malaria was considered extremely rare. However, during October 2017-September 2018, there was a surge in the number of reported P. knowlesi cases. Here, a series of six cases of P. knowlesi malaria found during this period in Songkhla and Narathiwat provinces of southern Thailand are presented. All cases were confirmed by polymerase chain reaction. The unprecedented case number in the affected area is a warning sign of an increasing P. knowlesi burden in the south of Thailand.

Distinct amino acid and lipid perturbations characterize acute versus chronic malaria.

Chronic malaria is a major public health problem and significant challenge for disease eradication efforts. Despite its importance, the biological factors underpinning chronic malaria are not fully understood. Recent studies have shown that host metabolic state can influence malaria pathogenesis and transmission, but its role in chronicity is not known. Here, with the goal of identifying distinct modifications in the metabolite profiles of acute versus chronic malaria, metabolomics was performed on plasma from Plasmodium-infected humans and nonhuman primates with a range of parasitemias and clinical signs. In rhesus macaques infected with Plasmodium coatneyi, significant alterations in amines, carnitines, and lipids were detected during a high parasitemic acute phase and many of these reverted to baseline levels once a low parasitemic chronic phase was established. Plasmodium gene expression, studied in parallel in the macaques, revealed transcriptional changes in amine, fatty acid, lipid and energy metabolism genes, as well as variant antigen genes. Furthermore, a common set of amines, carnitines, and lipids distinguished acute from chronic malaria in plasma from human Plasmodium falciparum cases. In summary, distinct host-parasite metabolic environments have been uncovered that characterize acute versus chronic malaria, providing insights into the underlying host-parasite biology of malaria disease progression.

Epidemiological profiles of recurrent malaria episodes in an endemic area along the Thailand-Myanmar border: a prospective cohort study.

BACKGROUND: In low malaria transmission areas, many people acquire multiple malaria infections within a single season. This study aimed to describe the pattern and epidemiological profile of malaria recurrence in a hypoendemic area of western Thailand and identify factors associated with having multiple malaria episodes. METHODS: An open cohort of 7000 residents in seven clusters along the Thai-Myanmar border was followed during a 6.5-year period (2011-mid 2017). Symptomatic and asymptomatic malaria infections were detected by passive case detection (PCD), weekly household visit, and mass blood surveys every 4-6 months. Malaria recurrence was defined as subsequent parasitaemic episodes occurred later than 7 days after receiving anti-malarial treatment. This study focused on analysis of recurrent episodes that occurred within 1 year after treatment. Numbers of malaria cases with single and multiple episodes were compared between clusters. Kaplan-Meier curve was performed to determine the intervals of recurrent episodes by Plasmodium species and age groups. The ordinal logistic model was used to determine factors associated with multiple malaria episodes, and to compare with single episodes, and those with no malaria infection. RESULTS: The cumulative incidence of malaria in the study area was 5.2% over the 6.5 years. Overall, 410 malaria patients were detected. Of these patients, 20% and 16% had multiple malaria episodes during the entire period and within 1 year after initial treatment, respectively. About 80% of repeated malaria episodes were caused by the same Plasmodium species as the primary infections. The median interval and interquartile range (IQR) between the first and second episode was 88 (43-175) days for all parasites, 56 (35-133) days for two Plasmodium falciparum episodes, and 90 (59-204) days for two Plasmodium vivax episodes. The interval between the episodes was increased with age. Factors significantly associated with multiple episodes of malaria infection included male sex, young age, Karen ethnicity, forest-related occupation, and having other malaria infected persons in the same house in the same period. CONCLUSIONS: People who have multiple malaria episodes may play an important role in maintaining malaria transmission in the area. Understanding epidemiological profiles of this group is important for planning strategies to achieve the elimination goal.

Infectivity of symptomatic and asymptomatic Plasmodium vivax infections to a Southeast Asian vector, Anopheles dirus.

Plasmodium vivax is now the predominant species causing malarial infection and disease in most non-African areas, but little is known about its transmission efficiency from human to mosquitoes. Because the majority of Plasmodium infections in endemic areas are low density and asymptomatic, it is important to evaluate how well these infections transmit. Using membrane feeding apparatus, Anopheles dirus were fed with blood samples from 94 individuals who had natural P. vivax infections with parasitemias spanning four orders of magnitude. We found that the mosquito infection rate was positively correlated with blood parasitemia and that infection began to rise when parasitemia was >10parasites/µl. Below this threshold, mosquito infection is rare and associated with very few oocysts. These findings provide useful information for assessing the human reservoir of transmission and for establishing diagnostic sensitivity required to identify individuals who are most infective to mosquitoes.

LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage.

IFN-γ is a major regulator of immune functions and has been shown to induce liver-stage Plasmodium elimination both in vitro and in vivo. The molecular mechanism responsible for the restriction of liver-stage Plasmodium downstream of IFN-γ remains uncertain, however. Autophagy, a newly described immune defense mechanism, was recently identified as a downstream pathway activated in response to IFN-γ in the control of intracellular infections. We thus hypothesized that the killing of liver-stage malarial parasites by IFN-γ involves autophagy induction. Our results show that whereas IFN-γ treatment of human hepatocytes activates autophagy, the IFN-γ-mediated restriction of liver-stage Plasmodium vivax depends only on the downstream autophagy-related proteins Beclin 1, PI3K, and ATG5, but not on the upstream autophagy-initiating protein ULK1. In addition, IFN-γ enhanced the recruitment of LC3 onto the parasitophorous vacuole membrane (PVM) and increased the colocalization of lysosomal vesicles with P. vivax compartments. Taken together, these data indicate that IFN-γ mediates the control of liver-stage P. vivax by inducing a noncanonical autophagy pathway resembling that of LC3-associated phagocytosis, in which direct decoration of the PVM with LC3 promotes the fusion of P. vivax compartments with lysosomes and subsequent killing of the pathogen. Understanding the hepatocyte response to IFN-γ during Plasmodium infection and the roles of autophagy-related proteins may provide an urgently needed alternative strategy for the elimination of this human malaria.

Improvement of culture conditions for long-term in vitro culture of Plasmodium vivax.

BACKGROUND: The study of the biology, transmission and pathogenesis of Plasmodium vivax is hindered due to the lack of a robustly propagating, continuous culture of this parasite. The current culture system for P. vivax parasites still suffered from consistency and difficulties in long-term maintenance of parasites in culture and for providing sufficient biological materials for studying parasite biology. Therefore, further improvement of culture conditions for P. vivax is needed. METHODS: Clinical samples were collected from patients diagnosed with P. vivax in western Thailand. Leukocyte-depleted P. vivax infected blood samples were cultured in a modified McCoy's 5A medium at 5% haematocrit under hypoxic condition (5% O2, 5% CO2, and 90% N2). Reticulocytes purified from adult peripheral blood were added daily to maintain 4% reticulocytes. Parasites were detected by microscopic examination of Giemsa-stained smears and molecular methods. RESULTS: The effects of culture variables were first analysed in order to improve the culture conditions for P. vivax. Through analysis of the sources of host reticulocytes and nutrients of culture medium, the culture conditions better supporting in vitro growth and maturation of the parasites were identified. Using this system, three of 30 isolates could be maintained in vitro for over 26 months albeit parasite density is low. CONCLUSIONS: Based on the analysis of different culture variables, an improved and feasible protocol for continuous culture of P. vivax was developed.

A rapid sensitive, flow cytometry-based method for the detection of Plasmodium vivax-infected blood cells.

BACKGROUND: Plasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation. These features complicate detection and quantification by flow cytometry (FC) using standard nucleic acid-based staining methods. A simple antibody-based FC method was developed for rapid parasite detection along with simultaneous detection of other parasite and erythrocyte markers. METHODS: Clinical samples were collected from patients diagnosed with P. vivax at a district Malaria Clinic in Kanchanaburi, Thailand. One µL of infected blood was washed, fixed, stained with a Plasmodium pan-specific anti-PfBiP antibody conjugated with Alexa Fluor 660, and analysed by FC. Additional primary conjugated antibodies for stage-specific markers of P. vivax for late trophozoite-early schizonts (MSP1-Alexa Fluor 660), late-stage schizonts (DBP-Alexa Fluor 555), and sexual stages (Pvs16) were used to differentiate intra-erythrocytic developmental stages. RESULTS: The percentages of P. vivax-infected cells determined by the FC method and manually by microscopic examination of Giemsa-stained thick blood smears were positively correlated by Spearman's rank correlation coefficient (R2=0.93843) from 0.001 to 1.00% P. vivax-infected reticulocytes. CONCLUSIONS: The FC-based method is a simple, robust, and efficient method for detecting P. vivax-infected reticulocytes.

Comparison of field and expert laboratory microscopy for active surveillance for asymptomatic Plasmodium falciparum and Plasmodium vivax in western Thailand.

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.