Parasite Multiplication Rate Research Articles

PvDBPII elicits multiple antibody-mediated mechanisms that reduce growth in a Plasmodium vivax challenge trial

The receptor-binding domain, region II, of the Plasmodium vivax Duffy binding protein (PvDBPII) binds the Duffy antigen on the reticulocyte surface to mediate invasion. A heterologous vaccine challenge trial recently showed that a delayed dosing regimen with recombinant PvDBPII SalI variant formulated with adjuvant Matrix-MTM reduced the in vivo parasite multiplication rate (PMR) in immunized volunteers challenged with the Thai P. vivax isolate PvW1. Here, we describe extensive analysis of the polyfunctional antibody responses elicited by PvDBPII immunization and identify immune correlates for PMR reduction. A classification algorithm identified antibody features that significantly contribute to PMR reduction. These included antibody titre, receptor-binding inhibitory titre, dissociation constant of the PvDBPII-antibody interaction, complement C1q and Fc gamma receptor binding and specific IgG subclasses. These data suggest that multiple immune mechanisms elicited by PvDBPII immunization are likely to be associated with protection and the immune correlates identified could guide the development of an effective vaccine for P. vivax malaria. Importantly, all the polyfunctional antibody features that correlated with protection cross-reacted with both PvDBPII SalI and PvW1 variants, suggesting that immunization with PvDBPII should protect against diverse P. vivax isolates.

Intra-host Trypanosoma cruzi strain dynamics shape disease progression: the missing link in Chagas disease pathogenesis.

Chronic Chagasic cardiomyopathy develops years after infection in 20-40% of patients, but disease progression is poorly understood. Here, we assessed Trypanosoma cruzi parasite dynamics and pathogenesis over a 2.5-year period in naturally infected rhesus macaques. Individuals with better control of parasitemia were infected with a greater diversity of parasite strains compared to those with increasing parasitemia over time. Also, the in vivo parasite multiplication rate decreased with increasing parasite diversity, suggesting competition among strains or a stronger immune response in multiple infections. Significant differences in electrocardiographic (ECG) profiles were observed in Chagasic macaques compared to uninfected controls, suggesting early conduction defects, and changes in ECG patterns over time were observed only in macaques with increasing parasitemia and lower parasite diversity. Disease progression was also associated with plasma fibronectin degradation, which may serve as a biomarker. These data provide a novel framework for the understanding of Chagas disease pathogenesis, with parasite diversity shaping disease progression.IMPORTANCEChagas disease progression remains poorly understood, and patients at increased risk of developing severe cardiac disease cannot be distinguished from those who may remain asymptomatic. Monitoring of Trypanosoma cruzi strain dynamics and pathogenesis over 2-3 years in naturally infected macaques shows that increasing parasite diversity in hosts is detrimental to parasite multiplication and Chagasic cardiomyopathy disease progression. This provides a novel framework for the understanding of Chagas disease pathogenesis.

Vaccination with Plasmodium vivax Duffy-binding protein inhibits parasite growth during controlled human malaria infection.

There are no licensed vaccines against Plasmodium vivax. We conducted two phase 1/2a clinical trials to assess two vaccines targeting P. vivax Duffy-binding protein region II (PvDBPII). Recombinant viral vaccines using chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) vectors as well as a protein and adjuvant formulation (PvDBPII/Matrix-M) were tested in both a standard and a delayed dosing regimen. Volunteers underwent controlled human malaria infection (CHMI) after their last vaccination, alongside unvaccinated controls. Efficacy was assessed by comparisons of parasite multiplication rates in the blood. PvDBPII/Matrix-M, given in a delayed dosing regimen, elicited the highest antibody responses and reduced the mean parasite multiplication rate after CHMI by 51% (n = 6) compared with unvaccinated controls (n = 13), whereas no other vaccine or regimen affected parasite growth. Both viral-vectored and protein vaccines were well tolerated and elicited expected, short-lived adverse events. Together, these results support further clinical evaluation of the PvDBPII/Matrix-M P. vivax vaccine.

Buffalo colostrum- A novel substitute of human serum for the cultivation of Plasmodium falciparum in vitro

In vitro cultivation of erythrocytic stages of Plasmodium falciparum requires supplementing the culture medium with human serum. The present study was carried out to explore an alternative to human serum. Different human serum samples were found to vary considerably in their ability to support the growth of erythrocytic stages of P. falciparum in vitro. These results strongly suggested the use of pooled human serum for comparing the growth of parasites in medium augmented with other supplements. Parasites could multiply for a few cycles only in RPMI (Roswell Park Memorial Institute) medium supplemented with serum obtained from pig, goat, sheep or buffalo. Continued cultivation could not be achieved using any one of these animal sera. Ability of bovine colostrum was investigated as an alternative to human serum. Buffalo colostrum, 10%(v/v) 'suitably prepared' supported the continuous growth and multiplication of P. falciparum . Morphologically both asexual and sexual stages appeared normal and healthy, but the multiplication rate of parasites grown in colostrum augmented medium was found to be lower than that in serum-supplemented medium. The one month of uninterrupted cultivation of P. falciparum registered 106 fold increase in parasite density compared to 1010 fold multiplication recorded in control culture with 10% serum supplement. Cow colostrum failed to support the growth and multiplication of parasites beyond 6 days in culture. The initial positive results with buffalo colostrum hold promise and should be explored further as a potential substitute for human serum for continuous in vitro propagation of erythrocytic stages of the malignant malaria parasite.

Mechanistic within-host models of the asexual Plasmodium falciparum infection: a review and analytical assessment

BackgroundMalaria blood-stage infection length and intensity are important drivers of disease and transmission; however, the underlying mechanisms of parasite growth and the host’s immune response during infection remain largely unknown. Over the last 30 years, several mechanistic mathematical models of malaria parasite within-host dynamics have been published and used in malaria transmission models.MethodsMechanistic within-host models of parasite dynamics were identified through a review of published literature. For a subset of these, model code was reproduced and descriptive statistics compared between the models using fitted data. Through simulation and model analysis, key features of the models were compared, including assumptions on growth, immune response components, variant switching mechanisms, and inter-individual variability.ResultsThe assessed within-host malaria models generally replicate infection dynamics in malaria-naïve individuals. However, there are substantial differences between the model dynamics after disease onset, and models do not always reproduce late infection parasitaemia data used for calibration of the within host infections. Models have attempted to capture the considerable variability in parasite dynamics between individuals by including stochastic parasite multiplication rates; variant switching dynamics leading to immune escape; variable effects of the host immune responses; or via probabilistic events. For models that capture realistic length of infections, model representations of innate immunity explain early peaks in infection density that cause clinical symptoms, and model representations of antibody immune responses control the length of infection. Models differed in their assumptions concerning variant switching dynamics, reflecting uncertainty in the underlying mechanisms of variant switching revealed by recent clinical data during early infection. Overall, given the scarce availability of the biological evidence there is limited support for complex models.ConclusionsThis study suggests that much of the inter-individual variability observed in clinical malaria infections has traditionally been attributed in models to random variability, rather than mechanistic disease dynamics. Thus, it is proposed that newly developed models should assume simple immune dynamics that minimally capture mechanistic understandings and avoid over-parameterization and large stochasticity which inaccurately represent unknown disease mechanisms.

Higher gametocyte production and mosquito infectivity in chronic compared to incident Plasmodium falciparum infections

Plasmodium falciparum gametocyte kinetics and infectivity may differ between chronic and incident infections. In the current study, we assess parasite kinetics and infectivity to mosquitoes among children (aged 5–10 years) from Burkina Faso with (a) incident infections following parasite clearance (n = 48) and (b) chronic asymptomatic infections (n = 60). In the incident infection cohort, 92% (44/48) of children develop symptoms within 35 days, compared to 23% (14/60) in the chronic cohort. All individuals with chronic infection carried gametocytes or developed them during follow-up, whereas only 35% (17/48) in the incident cohort produce gametocytes before becoming symptomatic and receiving treatment. Parasite multiplication rate (PMR) and the relative abundance of ap2-g and gexp-5 transcripts are positively associated with gametocyte production. Antibody responses are higher and PMR lower in chronic infections. The presence of symptoms and sexual stage immune responses are associated with reductions in gametocyte infectivity to mosquitoes. We observe that most incident infections require treatment before the density of mature gametocytes is sufficient to infect mosquitoes. In contrast, chronic, asymptomatic infections represent a significant source of mosquito infections. Our observations support the notion that malaria transmission reduction may be expedited by enhanced case management, involving both symptom-screening and infection detection.

Development and evaluation of a new Plasmodium falciparum 3D7 blood stage malaria cell bank for use in malaria volunteer infection studies

BackgroundNew anti-malarial therapeutics are required to counter the threat of increasing drug resistance. Malaria volunteer infection studies (VIS), particularly the induced blood stage malaria (IBSM) model, play a key role in accelerating anti-malarial drug development. Supply of the reference 3D7-V2 Plasmodium falciparum malaria cell bank (MCB) is limited. This study aimed to develop a new MCB, and compare the safety and infectivity of this MCB with the existing 3D7-V2 MCB, in a VIS. A second bank (3D7-V1) developed in 1995 was also evaluated.MethodsThe 3D7-V2 MCB was expanded in vitro using a bioreactor to produce a new MCB designated 3D7-MBE-008. This bank and 3D7-V1 were then evaluated using the IBSM model, where healthy participants were intravenously inoculated with blood-stage parasites. Participants were treated with artemether-lumefantrine when parasitaemia or clinical thresholds were reached. Safety, infectivity and parasite growth and clearance were evaluated.ResultsThe in vitro expansion of 3D7-V2 produced 200 vials of the 3D7-MBE-008 MCB, with a parasitaemia of 4.3%. This compares to 0.1% in the existing 3D7-V2 MCB, and < 0.01% in the 3D7-V1 MCB. All four participants (two per MCB) developed detectable P. falciparum infection after inoculation with approximately 2800 parasites. For the 3D7-MBE-008 MCB, the parasite multiplication rate of 48 h (PMR48) using non-linear mixed effects modelling was 34.6 (95% CI 18.5–64.6), similar to the parental 3D7-V2 line; parasitaemia in both participants exceeded 10,000/mL by day 8. Growth of the 3D7-V1 was slower (PMR48 of 11.5 [95% CI 8.5–15.6]), with parasitaemia exceeding 10,000 parasites/mL on days 10 and 8.5. Rapid parasite clearance followed artemether-lumefantrine treatment in all four participants, with clearance half-lives of 4.01 and 4.06 (weighted mean 4.04 [95% CI 3.61–4.57]) hours for 3D7-MBE-008 and 4.11 and 4.52 (weighted mean 4.31 [95% CI 4.16–4.47]) hours for 3D7-V1. A total of 59 adverse events occurred; most were of mild severity with three being severe in the 3D7-MBE-008 study.ConclusionThe safety, growth and clearance profiles of the expanded 3D7-MBE-008 MCB closely resemble that of its parent, indicating its suitability for future studies. Trial Registration: Australian New Zealand Clinical Trials registry numbers: P3487 (3D7-V1): ACTRN12619001085167. P3491 (3D7-MBE-008): ACTRN12619001079134

Role of human Pegivirus infections in whole Plasmodium falciparum sporozoite vaccination and controlled human malaria infection in African volunteers

BackgroundDiverse vaccination outcomes and protection levels among different populations pose a serious challenge to the development of an effective malaria vaccine. Co-infections are among many factors associated with immune dysfunction and sub-optimal vaccination outcomes. Chronic, asymptomatic viral infections can contribute to the modulation of vaccine efficacy through various mechanisms. Human Pegivirus-1 (HPgV-1) persists in immune cells thereby potentially modulating immune responses. We investigated whether Pegivirus infection influences vaccine-induced responses and protection in African volunteers undergoing whole P. falciparum sporozoites-based malaria vaccination and controlled human malaria infections (CHMI).MethodsHPgV-1 prevalence was quantified by RT-qPCR in plasma samples of 96 individuals before, post vaccination with PfSPZ Vaccine and after CHMI in cohorts from Tanzania and Equatorial Guinea. The impact of HPgV-1 infection was evaluated on (1) systemic cytokine and chemokine levels measured by Luminex, (2) PfCSP-specific antibody titers quantified by ELISA, (3) asexual blood-stage parasitemia pre-patent periods and parasite multiplication rates, (4) HPgV-1 RNA levels upon asexual blood-stage parasitemia induced by CHMI.ResultsThe prevalence of HPgV-1 was 29.2% (28/96) and sequence analysis of the 5′ UTR and E2 regions revealed the predominance of genotypes 1, 2 and 5. HPgV-1 infection was associated with elevated systemic levels of IL-2 and IL-17A. Comparable vaccine-induced anti-PfCSP antibody titers, asexual blood-stage multiplication rates and pre-patent periods were observed in HPgV-1 positive and negative individuals. However, a tendency for higher protection levels was detected in the HPgV-1 positive group (62.5%) compared to the negative one (51.6%) following CHMI. HPgV-1 viremia levels were not significantly altered after CHMI.ConclusionsHPgV-1 infection did not alter PfSPZ Vaccine elicited levels of PfCSP-specific antibody responses and parasite multiplication rates. Ongoing HPgV-1 infection appears to improve to some degree protection against CHMI in PfSPZ-vaccinated individuals. This is likely through modulation of immune system activation and systemic cytokines as higher levels of IL-2 and IL17A were observed in HPgV-1 infected individuals. CHMI is safe and well tolerated in HPgV-1 infected individuals. Identification of cell types and mechanisms of both silent and productive infection in individuals will help to unravel the biology of this widely present but largely under-researched virus.

Estimation of parasite age and synchrony status in Plasmodium falciparum infections

Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any one time there is a limited spread of parasites at a particular time (hours) post-invasion. Patients presenting with older parasites, and with asynchronous infections, have been reported to have higher risks of fatal outcomes, associated with higher parasite biomass and multiplication rates respectively. However, practical tools to assess synchrony and estimate parasite age post-invasion in patient samples are lacking. We have developed a novel method based on three genes differentially expressed over the parasite intra-erythrocytic lifecycle, and applied it to samples from patients with uncomplicated malaria attending two health clinics in Ghana. We found that most patients presented with synchronous infections, and with parasites within 12 h of erythrocyte invasion. Finally we investigated if clinical features such as fever and parasite density could act as predictors of parasite age and synchrony. The new method is a simple and practicable approach to study parasite dynamics in naturally-infected patients, and is a significant improvement on the subjective microscopical methods for parasite staging in vivo, aiding patient management.

Comparative analysis of asexual and sexual stage Plasmodium falciparum development in different red blood cell types

BackgroundRed blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development.MethodsApproximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy.ResultsMerozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p > 0.05).ConclusionsParasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.

Ethanolic extract of Croton blanchetianus Ball induces mitochondrial defects in Leishmania amazonensis promastigotes.

Leishmaniasis is a neglected disease caused by Leishmania. Chemotherapy remains the mainstay for leishmaniasis control; however, available drugs fail to provide a parasitological cure, and are associated with high toxicity. Natural products are promising leads for the development of novel chemotherapeutics against leishmaniasis. This work investigated the leishmanicidal properties of ethanolic extract of Croton blanchetianus (EECb) on Leishmania infantum and Leishmania amazonensis, and found that EECb, rich in terpenic compounds, was active against promastigote and amastigote forms of both Leishmania species. Leishmania infantum promastigotes and amastigotes presented IC50 values of 208.6 and 8.8 μg/mL, respectively, whereas Leishmania amazonensis promastigotes and amastigotes presented IC50 values of 73.6 and 3.1 μg/mL, respectively. Promastigotes exposed to EECb (100 µg/mL) had their body cellular volume reduced and altered to a round shape, and the flagellum was duplicated, suggesting that EECb may interfere with the process of cytokinesis, which could be the cause of the decline in the parasite multiplication rate. Regarding possible EECb targets, a marked depolarization of the mitochondrial membrane potential was observed. No cytotoxic effects of EECb were observed in murine macrophages at concentrations below 60 µg/mL, and the CC50 obtained was 83.8 µg/mL. Thus, the present results indicated that EECb had effective and selective effects against Leishmania infantum and Leishmania amazonensis, and that these effects appeared to be mediated by mitochondrial dysfunction.

Fetal Hemoglobin Promotes P. Falciparum Growth in Sickle Cell Disease Erythrocytes

Introduction: We have demonstrated that sickle hemoglobin (HbS) polymerization in low oxygen (O2) is the main driver of sickle cell trait (AS) resistance to P.falciparum malaria. This suggests that homozygous sickle cell disease (SS) individuals should have even greater resistance to malaria. Instead SS individuals infected by P. falciparum often have increased malaria morbidity and mortality compared to individuals with normal hemoglobin (AA) or AS. The reasons for this paradox are poorly understood. We propose that fetal hemoglobin (HbF) inhibits polymerization thereby allowing parasite proliferation. To test this hypothesis the following experiments were performed. Methods: AA and SS erythrocytes were collected within two weeks of the assay date. P. falciparum 3D7 parasites were used for growth assays. Growth assays were performed at 1, 3, 5, 7.5, 10 and 16% O2 using synchronized schizonts obtained by magnet purification via the MACS system. Staging was performed via light microscopy analysis of May-Grünwald-Giemsa-stained cytospins. Parasite Multiplication Rate (PMR) was determined via flow cytometry using parasitemia at 64 and 16 hours post infection (hpi) as previously described. Results: In contrast to AS erythrocytes, SS RBCs contain varying amounts of HbF that differ by cell and by individual. In hypoxic SS RBCs with low (3.8%) HbF, P. falciparum parasites remain immature (early trophozoites) in 1, 3, and 5% O2 (Fig. 1) at 36 hpi, when they should have matured into schizonts. However, in SS RBCs with high (22.6%) HbF, 12, 16 and 27% of parasites matured into schizonts in 1, 3 and 5% O2 respectively compared to 0, 0, and 1% in the SS RBCs with 3.8% HbF. Preliminary data also demonstrate that the parasite multiplication rate (PMR), a surrogate of proliferation, in SS RBCs improves with increasing O2 and HbF. Using SS/Hereditary Persistence of Fetal Hemoglobin RBCs with 42% HbF, proliferation in 5% O2 or higher is exponential with a PMR of 2.6 or greater (Fig. 2). In RBCs isolated from SS patients treated with hydroxyurea with HbF of 26.4 or 21%, PMR is 2 or more in 7.5% O2 or greater. Conclusion: These data demonstrate that HbF promotes P. falciparum growth in SS erythrocytes. In addition, the data initiates a resolution of the malaria paradox. HbS in both hypoxic AS and SS surely inhibits P. falciparum growth due to HbS polymerization at low O2. But high levels of anti-sickling HbF observed in erythrocytes from many individuals with SS reverses this inhibition despite the increased content of HbS in SS individuals. Even small increases in P. falciparum proliferation within the broadly compromised SS host may enhance the malaria associated morbidity and mortality seen in this population. These data further suggest the importance of anti-malarial prophylaxis in patients with SS especially those treated with hydroxyurea or anti-sickling agents. Disclosures No relevant conflicts of interest to declare.

Modelling pathogen load dynamics to elucidate mechanistic determinants of host-Plasmodium falciparum interactions.

SummaryDuring infection, increasing pathogen load stimulates both protective and harmful aspects of the host response. The dynamics of this interaction are hard to quantify in humans, but doing so could improve understanding of mechanisms of disease and protection. We sought to model the contributions of parasite multiplication rate and host response to observed parasite load in individual subjects with Plasmodium falciparum malaria, using only data obtained at the time of clinical presentation, and then to identify their mechanistic correlates. We predicted higher parasite multiplication rates and lower host responsiveness in severe malaria cases, with severe anemia being more insidious than cerebral malaria. We predicted that parasite growth-inhibition was associated with platelet consumption, lower expression of CXCL10 and type-1 interferon-associated genes, but increased cathepsin G and matrix metallopeptidase 9 expression. We found that cathepsin G and matrix metallopeptidase 9 directly inhibit parasite invasion into erythrocytes. Parasite multiplication rate was associated with host iron availability and higher complement factor H levels, lower expression of gametocyte-associated genes but higher expression of translation-associated genes in the parasite. Our findings demonstrate the potential of using explicit modelling of pathogen load dynamics to deepen understanding of host-pathogen interactions and identify mechanistic correlates of protection.

Temperature Dependence of Plasmodium falciparum Erythrocytic Stage Development.

.Plasmodium falciparum infection causes febrile illness and severe disease with multiple organ failure and death when treatment is delayed. Antipyretic treatment is standard, and inducing hypothermia has been proposed to protect the brain in cerebral malaria. Here, we investigated the temperature dependence of asexual-stage parasite development and parasite multiplication in vitro. Plasmodium falciparum laboratory strain TM267 was incubated for 2 hours (short exposure) or 48 hours (continuous exposure) at different temperatures (32°C, 34°C, 35°C, 38°C, 39°C, and 40°C). The starting parasite developmental stage (ring, trophozoite, or schizont) varied between experiments. The parasite multiplication rate (PMR) was reduced under both hyper- and hypothermic conditions; after continuous exposure, the mean PMR ± SD was 9.1 ± 1.2 at 37°C compared with 2.4 ± 1.8 at 32°C, 2.3 ± 0.4 at 34°C, and 0.4 ± 0.1 at 40°C (P < 0.01). Changes in PMR were not significant after 2-hour exposure at temperatures ranging from 32°C to 40°C. Morphological changes in parasite cytoplasm and nucleus could be observed after long exposure to low or high temperature. After 48-hour incubation, rosette formation (≥ 2 uninfected red blood cells bound to infected red blood cells) was decreased at 34°C or 39°C compared with that at 37°C. In conclusion, both hyper- and hypothermia reduce PMR and delay erythrocytic stage development of P. falciparum, subsequently reducing rosette formation.

Effect of Blastocystis sp. in dengue patients-Increase in the treatment cost and exacerbation of symptoms.

Increasing incidences of dengue have become a global health threat with major clinical manifestation including high fever and gastrointestinal symptoms. These symptoms were also expressed among Blastocystis sp. infected individuals, a parasite commonly seen in human stools. This parasite has been previously reported to replicate faster upon exposure to high temperature. The present study is a hospitalized-based cross-sectional study involved the collection of faecal sample from dengue patients. Stool examination was done by in vitro cultivation to isolate Blastocystis sp. Growth pattern of all the positive isolates were analyzed to identify the multiplication rate of Blastocystis sp. isolated from dengue patients. Distribution of Blastocystis sp. among dengue patients was 23.6%. Dengue patients who were positive for Blastocystis sp. infection denoted a significantly higher fever rate reaching 38.73°C (p<0.05) compared to the non-Blastocystis sp. infected patients (38.44°C). It was also found that Blastocystis sp. infected patients complained of frequenting the toilet more than five times a day (p<0.05) compared to those who were non-Blastocystis sp. infected. At the same time, the duration of hospitalization was significantly longer (p<0.05) for Blastocystis sp. infected dengue patients compared to the non-Blastocystis sp. infected patients. Besides, Blastocystis sp. isolated from dengue patients (in vivo thermal stress) showed a higher growth rate compared to the non-dengue isolated which was exposed to high temperature (in vitro thermal stress). Our findings suggest that presence of Blastocystis sp. during dengue infection could trigger the increase of temperature which could be due to highly elevated pro inflammatory cytokines by both parasitic and virus infection. This could justify why the temperature in Blastocystis sp. infected dengue patients is higher compared to the non-Blastocystis sp. infected patients. Higher temperature could have triggered a greater parasite multiplication rate that contributed to the aggravation of the gastrointestinal symptoms.

Growth Rate of Plasmodium falciparum: Analysis of Parasite Growth Data From Malaria Volunteer Infection Studies.

BackgroundGrowth rate of malaria parasites in the blood of infected subjects is an important measure of efficacy of drugs and vaccines.MethodsWe used log-linear and sine-wave models to estimate the parasite growth rate of the 3D7 strain of Plasmodium falciparum using data from 177 subjects from 14 induced blood stage malaria (IBSM) studies conducted at QIMR Berghofer. We estimated parasite multiplication rate per 48 hour (PMR48), PMR per life-cycle (PMRLC), and parasite life-cycle duration. We compared these parameters to those from studies conducted elsewhere with infections induced by IBSM (n=66), sporozoites via mosquito bite (n=336) or injection (n=51).ResultsThe parasite growth rate of 3D7 in QIMR Berghofer studies was 0.75/day (95% CI: 0.73–0.77/day), PMR48 was 31.9 (95% CI: 28.7–35.4), PMRLC was 16.4 (95% CI: 15.1–17.8) and parasite life-cycle was 38.8 hour (95% CI: 38.3–39.2 hour). These parameters were similar to estimates from IBSM studies elsewhere (0.71/day, 95% CI: 0.67–0.75/day; PMR48 26.6, 95% CI: 22.2–31.8), but significantly higher (P < 0.001) than in sporozoite studies (0.47/day, 95% CI: 0.43–0.50/day; PMR48 8.6, 95% CI: 7.3–10.1).ConclusionsParasite growth rates were similar across different IBSM studies and higher than infections induced by sporozoite.

Quantification of host-mediated parasite clearance during blood-stage Plasmodium infection and anti-malarial drug treatment in mice

A major mechanism of host-mediated control of blood-stage Plasmodium infection is thought to be removal of parasitized red blood cells (pRBCs) from circulation by the spleen or phagocytic system. The rate of parasite removal is thought to be further increased by anti-malarial drug treatment, contributing to the effectiveness of drug therapy. It is difficult to directly compare pRBC removal rates in the presence and absence of treatment, since in the absence of treatment the removal rate of parasites is obscured by the extent of ongoing parasite proliferation. Here, we transfused a single generation of fluorescently-labelled Plasmodium berghei pRBCs into mice, and monitored both their disappearance from circulation, and their replication to produce the next generation of pRBCs. In conjunction with a new mathematical model, we directly estimated host removal of pRBCs during ongoing infection, and after drug treatment. In untreated mice, pRBCs were removed from circulation with a half-life of 15.1 h. Treatment with various doses of mefloquine/artesunate did not alter the pRBC removal rate, despite blocking parasite replication effectively. An exception was high dose artesunate, which doubled the rate of pRBC removal (half-life of 9.1 h). Phagocyte depletion using clodronate liposomes approximately halved the pRBC removal rate during untreated infection, indicating a role for phagocytes in clearance. We next assessed the importance of pRBC clearance for the decrease in the parasite multiplication rate after high dose artesunate treatment. High dose artesunate decreased parasite replication ∼46-fold compared with saline controls, with inhibition of replication contributing 23-fold of this, and increased pRBC clearance contributing only a further 2.0-fold. Thus, in our in vivo systems, drugs acted primarily by inhibiting parasite replication, with drug-induced increases in pRBC clearance making only minor contributions to overall drug effect.

Disease Severity and Effective Parasite Multiplication Rate in Falciparum Malaria.

Patients presenting with severe falciparum malaria in a Bangladeshi tertiary hospital had higher total parasite burden, estimated by parasitemia and plasma PfHRP2, than uncomplicated malaria patients despite shorter fever duration. This suggests that higher parasite multiplication rates (PMR) contribute to causing the higher biomass found in severe disease. Compared with patients without a history of previous malaria, patients with previous malaria carried a lower parasite biomass with similar fever duration at presentation, suggesting that host immunity reduces the PMR.

Genotypic and phenotypic characterization of G6PD deficiency in Bengali adults with severe and uncomplicated malaria

BackgroundControl of malaria increasingly involves administration of 8-aminoquinolines, with accompanying risk of haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Few data on the prevalence and genotypic basis of G6PD deficiency are available from Bangladesh, where malaria remains a major problem in the South (Chittagong Division). The aim of this study was to determine the prevalence of G6PD deficiency, and associated G6PD genotypes, in adults with falciparum malaria in southern Bangladesh.MethodsG6PD status was assessed via a combination of fluorescent spot testing (FST) and genotyping in 141 Bengali patients admitted with falciparum malaria to two centres in Chittagong Division from 2012 to 2014. In addition, an analysis of genomic data from 1000 Genomes Project was carried out among five healthy Indian subcontinent populations.ResultsOne male patient with uncomplicated malaria was found to have G6PD deficiency on FST and a genotype associated with deficiency (hemizygous Orissa variant). In addition, there were two female patients heterozygous for deficiency variants (Orissa and Kerala-Kalyan). These three patients had a relatively long duration of symptoms prior to admission compared to G6PD normal cases, possibly suggesting an interaction with parasite multiplication rate. In addition, one of 27 healthy local controls was deficient on FST and hemizygous for the Mahidol variant of G6PD deficiency. Examination of 1000 Genomes Project sequencing data across the Indian subcontinent showed that 19/723 chromosomes (2.63%) carried a variant associated with deficiency. In the Bengali from Bangladesh 1000 Genomes population, three of 130 chromosomes (2.31%) carried deficient alleles; this included single chromosomes carrying the Kerala-Kalyan and Orissa variants.ConclusionsIn line with other recent work, G6PD deficiency is uncommon in Bengalis in Bangladesh. Further studies of particular ethnic groups are needed to evaluate the potential risk of wide deployment of primaquine in malaria control efforts in Bangladesh.

Changes in Serological Immunology Measures in UK and Kenyan Adults Post-controlled Human Malaria Infection.

Background: The timing of infection is closely determined in controlled human malaria infection (CHMI) studies, and as such they provide a unique opportunity to dissect changes in immunological responses before and after a single infection. The first Kenyan Challenge Study (KCS) (Pan African Clinical Trial Registry: PACTR20121100033272) was performed in 2013 with the aim of establishing the CHMI model in Kenya. This study used aseptic, cryopreserved, attenuated Plasmodium falciparum sporozoites administered by needle and syringe (PfSPZ Challenge) and was the first to evaluate parasite dynamics post-CHMI in individuals with varying degrees of prior exposure to malaria.Methods: We describe detailed serological and functional immunological responses pre- and post-CHMI for participants in the KCS and compare these with those from malaria-naïve UK volunteers who also underwent CHMI (VAC049) (ClinicalTrials.gov NCT01465048) using PfSPZ Challenge. We assessed antibody responses to three key blood-stage merozoite antigens [merozoite surface protein 1 (MSP1), apical membrane protein 1 (AMA1), and reticulocyte-binding protein homolog 5 (RH5)] and functional activity using two candidate measures of anti-merozoite immunity; the growth inhibition activity (GIA) assay and the antibody-dependent respiratory burst activity (ADRB) assay.Results:Clear serological differences were observed pre- and post-CHMI by ELISA between malaria-naïve UK volunteers in VAC049, and Kenyan volunteers who had prior malaria exposure. Antibodies to AMA1 and schizont extract correlated with parasite multiplication rate (PMR) post-CHMI in KCS. Serum from volunteer 110 in KCS, who demonstrated a dramatically reduced PMR in vivo, had no in vitro GIA prior to CHMI but the highest level of ADRB activity. A significant difference in ADRB activity was seen between KCS volunteers with minimal and definite prior exposure to malaria and significant increases were seen in ADRB activity post-CHMI in Kenyan volunteers. Quinine and atovaquone/proguanil, previously assumed to be removed by IgG purification, were identified as likely giving rise to aberrantly high in vitro GIA results.Conclusions: The ADRB activity assay is a promising functional assay that warrants further investigation as a measure of prior exposure to malaria and predictor of control of parasite growth. The CHMI model can be used to evaluate potential measures of naturally-acquired immunity to malaria.