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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, we fed Anopheles dirus with blood samples from 94 individuals who had natural P. vivax infection with parasitemias spanning four orders of magnitude. We found that the mosquito infection rate is positively correlated with blood parasitemia and that infection begins to rise when parasitemia is >10 parasites/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.

Abstract. 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 ×700 using a natural light source, whereas expert laboratory microscopy consisted of a 20-minute read (number of parasites per 500 leukocytes) at ×1,000 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. falciparumpositive slides and 93.9% of the P. vivax-positive slides had a parasitemia of less than 500/L. 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.

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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.autophagy | LC3-associated phagocytosis | IFN-γ | malaria S everal hundred million cases of human malaria are reported annually, and nearly 600,000 people die from the disease each year (1). Of the five species that infect humans, Plasmodium vivax is not only the most geographically widespread, but also the most prevalent malarial parasite in areas outside Africa. As such, it has caused massive morbidity in these regions of the world. Although malaria caused by P. vivax was previously regarded as benign compared with that caused by Plasmodium falciparum, the recent alarming increase in both the severity and the drug resistance of P. vivax infection has raised concern (2).The widespread distribution of P. vivax has been attributed to the parasite's ability to remain dormant in the liver for years before reactivation (3). The molecular mechanism responsible for P. vivax dormancy is unknown, and knowledge of Plasmodiumhepatocyte interactions remains very limited. Nonetheless, because the number of liver-stage parasites is in the range of 100, whereas in the blood stage as many as 10 13 organisms may be found (4), intervention at the liver stage would seem to offer a better strategy for parasite elimination. A prerequisite to this route of malaria control and the development of novel therapies is a better understanding of liver-stage Plasmodium and its interactions with host hepatocytes. IFN-γ was previously shown to exhibit antimalarial activity against the liver stag...

BackgroundThe 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.MethodsClinical 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.ResultsThe 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.ConclusionsBased on the analysis of different culture variables, an improved and feasible protocol for continuous culture of P. vivax was developed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0815-z) contains supplementary material, which is available to authorized users.

Controlled human malaria infection (CHMI) provides a highly informative means to investigate hostpathogen 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, two healthy malaria-naïve UK 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 red blood cells. Following stringent safety screening, the parasite stabilate from one of these donors ("PvW1") was thawed and used to inoculate six healthy malaria-naïve UK adults by blood-stage CHMI, at three 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 analysed 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.

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.