Dynamics Of Parasitemia Research Articles

Dynamics of blood stage and sporozoite-induced malarial infections in experimentally infected passerines

Complex experimental studies of vertebrate host, vector, and parasite interactions are essential in understanding virulence, but are difficult or impossible to conduct if vector species are unknown. Subinoculation of erythrocytic meronts of avian malarial parasites into susceptible hosts can avoid this problem, but this approach omits early exoerythrocytic stages, e.g. cryptozoites and metacryptozoites, that normally develop from sporozoites. A fundamental question that has remained unanswered is whether blood stage and sporozoite-induced malarial infections lead to differences in the dynamics of parasitemia in acute infections, patterns of parasite development, and host mortality. Here we demonstrate in a Carduelis spinus – Plasmodium relictum (genetic lineage pSGS1) system that experimental infections using inoculation of infected blood and using mosquito bite show similar peaks of parasitemias, but some measures of parasite development in the vertebrate host differ. Infected birds from all groups show decreased activity during the peak of parasitemia. There is no doubt that experimental infections using vectors provide the most precise information about the development of a parasite and its virulence in the host, but experimental infections using blood stages demonstrate similar parasitemias and effects on the host. These results are important for further experimental research of malarial parasites, especially studying avian Plasmodium parasites with unknown vectors.

Differences in pathogenicity and virulence of Trypanosoma brucei gambiense field isolates in experimentally infected Balb/C mice

Trypanosoma brucei gambiense (T. b. gambiense) is the major causative agent of human African trypanosomiasis (HAT). A great variety of clinical outcomes have been observed in West African foci, probably due to complex host-parasite interactions. In order to separate the roles of parasite genetic diversity and host variability, we have chosen to precisely characterize the pathogenicity and virulence of T. b. gambiense field isolates in a mouse model. Thirteen T. b. gambiense strains were studied in experimental infections, with 20 Balb/C infected mice per isolate. Mice were monitored for 30 days, in which mortality, parasitemia, anemia, and weight were recorded. Mortality rate, prepatent period, and maximum parasitemia were estimated, and a survival analysis was performed to compare strain pathogenicity. Mixed models were used to assess parasitemia dynamics, weight, and changes in Packed Cell Volume (PCV). Finally, a multivariate analysis was performed to infer relationships between all variables. A large phenotypic diversity was observed. Pathogenicity was highly variable, ranging from strains that kill their host within 9 days to a non-pathogenic strain (no deaths during the experiment). Virulence was also variable, with maximum parasitemia values ranging from 42 million to 1 billion trypanosomes/ml. Reduced PCV and weight occurred in the first two weeks of the infection, with the exception of two strains. Finally, the global analysis highlighted three groups of strains: a first group with highly pathogenic strains showing an early mortality associated with a short prepatent period; a second group of highly virulent strains with intermediate pathogenicity; and a third group of isolates characterized by low pathogenicity and virulence patterns. Such biological differences could be related to the observed clinical diversity in HAT. A better understanding of the biological pathways underlying the observed phenotypic diversity could thus help to clarify the complex nature of the host-parasite interactions that determine the resistance/susceptibility status to T. brucei gambiense.

Evaluation of a fluorescence-based method for antibabesial drug screening.

In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become routine, and the effectiveness of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness-of-fit (r(2)) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC(50)s determined by the fluorescence-based method (408 nM and 8.13 μM, respectively) and microscopy (400.3 nM and 9.4 μM, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 μM) was similar to the recently described microscopy-based value (21.7 μM) for B. bovis. Additionally, the Z' factor (0.80 to 0.90), signal-to-noise (S/N) ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay.

Strain-specific innate immune signaling pathways determine malaria parasitemia dynamics and host mortality

Malaria infection triggers vigorous host immune responses; however, the parasite ligands, host receptors, and the signaling pathways responsible for these reactions remain unknown or controversial. Malaria parasites primarily reside within RBCs, thereby hiding themselves from direct contact and recognition by host immune cells. Host responses to malaria infection are very different from those elicited by bacterial and viral infections and the host receptors recognizing parasite ligands have been elusive. Here we investigated mouse genome-wide transcriptional responses to infections with two strains of Plasmodium yoelii (N67 and N67C) and discovered differences in innate response pathways corresponding to strain-specific disease phenotypes. Using in vitro RNAi-based gene knockdown and KO mice, we demonstrated that a strong type I IFN (IFN-I) response triggered by RNA polymerase III and melanoma differentiation-associated protein 5, not Toll-like receptors (TLRs), binding of parasite DNA/RNA contributed to a decline of parasitemia in N67-infected mice. We showed that conventional dendritic cells were the major sources of early IFN-I, and that surface expression of phosphatidylserine on infected RBCs might promote their phagocytic uptake, leading to the release of parasite ligands and the IFN-I response in N67 infection. In contrast, an elevated inflammatory response mediated by CD14/TLR and p38 signaling played a role in disease severity and early host death in N67C-infected mice. In addition to identifying cytosolic DNA/RNA sensors and signaling pathways previously unrecognized in malaria infection, our study demonstrates the importance of parasite genetic backgrounds in malaria pathology and provides important information for studying human malaria pathogenesis.

Lost in translation? Bridging the preclinical / clinical divide

Establishing the relevance of preclinical data to the clinical situation is a problem that has plagued drug development in the majority of therapeutic areas. Typically a drug development cascade relies on preclinical data assuming the in vitro and in vivo data are consistent and predict some elements of clinical efficacy. Quantitative translation from preclinical to clinical is model based and requires PK (pharmacokinetics) and PK/PD (pharmacokinetic/pharmacodynamics) models linking drug exposure with effect. The first modeling step undertaken for anti-malaria drug development at MMV has been to develop a model capable of linking preclinical in vitro to preclinical in vivo data to: • Derive efficacy parameters for scaling including additional parameters that put them in context (parasite growth rates and maximum rates of killing) • Determine the relationship and consistency between the in vitro and preclinical in vivo data. This approach, whilst using data from a broader range of compounds and mechanisms, does assume that preclinical data is relevant replicating, at least partially, the clinical disease. The in vitro data characterized the effects of fixed concentrations of compound on the reduction of P falciparum parasite concentrations and estimated IC50s. The in vivo assessment in SCID mice determined the compound’s dose-dependent reduction of P falciparum parasite concentrations and estimated an ID90. The in vivo data was modelled using a nonlinear mixed effects PK/PD model in Nonmem and its parameter estimates compared to those from the in vitro data. The modeling was performed in a step-wise approach where a PK model was fitted to concentration time course data from a single dose in order to simulate the time courses used in the efficacy study (4 doses, 24 hours apart). The PK/PD model was then fitted to the observed parasitemia data using the simulated concentrations. The PD model estimated baseline parasitemia, rate constants for parasite growth and death, and the concentration dependent moderation of parasite death. The model determined that the parasite concentration expanded by approximately 3-4 times every 48 hours (in contrast to approximately 10 times in humans). For the two compounds modeled to date, model-estimated IC50 values from the in vivo data matched the in vitro estimates of IC50. The model, using the estimated parameters was also able to accurately simulate the decline in parasitemia and subsequent recrudescence following a single dose in the SCID P falciparum model providing some mechanistic validation. This modeling is at an early phase requiring more data, preclinical and clinical, for validation including evaluating methods for the prediction of clinical PK. The agreement between in vivo and in vitro parameter estimates is encouraging and will be confirmed as Agreement between in vitro and in vivo parameters is encouraging as is the model’s ability to capture parasitemia dynamics. A simple approximation of this model is being evaluated for use in assessing MMV’s preclinical candidates. In addition to confirming the model’s preclinical utility, clinical data is being sought to test whether the model is equally able in predicting drug effects on P falciparum in humans, confirming the relevance of preclinical data.

Dynamics of parasitemia of malaria parasites in a naturally and experimentally infected migratory songbird, the great reed warbler Acrocephalus arundinaceus

Little is known about the development of infection of malaria parasites of the genus Plasmodium in wild birds. We used qPCR, targeting specific mitochondrial lineages of Plasmodium ashfordi (GRW2) and Plasmodium relictum (GRW4), to monitor changes in intensities of parasitemia in captive great reed warblers Acrocephalus arundinaceus from summer to spring. The study involved both naturally infected adults and experimentally infected juveniles. The experiment demonstrated that P. ashfordi and P. relictum lineages differ substantially in several life-history traits (e.g. prepatent period and dynamics of parasitemia) and that individual hosts show substantial differences in responses to these infections. The intensity of parasitemia of lineages in mixed infections co-varied positively, suggesting a control mechanism by the host that is general across the parasite lineages. The intensity of parasitemia for individual hosts was highly repeatable suggesting variation between the host individuals in their genetic or acquired control of the infections. In future studies, care must be taken to avoid mixed infections in wild caught donors, and when possible use mosquitoes for the experiments as inoculation of infectious blood ignores important initial stages of the contact between the bird and the parasite.

Plasmodium malariae blood-stage dynamics.

We examine the dynamics of parasitemia, fever, and gametocytemia reflected in the preintervention charts of 180 malaria-naive U.S. neurosyphilis patients infected with the USPHS strain of Plasmodium malariae, for malariatherapy, focusing on the 84 charts for which more than 35 days of patency preceded intervention and daily records encompassed 92% or more of the duration of each infection. Inoculum size did not influence any outcome variable. Fevers (days with temperatures > or =101 F) followed patterns that fit recognized brood structures more often than did our approximations of merogony cycles (via local peaks in parasitemia), but neither closely fit textbook quartan patterns. There were no discernable patterns in gametocytemia. Successful transmission to mosquitoes increased following subcurative drug treatment but did not depend on detectable gametocytemia.