Laura Chery

Severe adult malaria is associated with specific PfEMP1 adhesion types and high parasite biomass

Significance The clinical presentation of severe malaria differs between children and adults, but the factors leading to these differences remain poorly understood. Here, we investigated parasite virulence factors in adult patients in India and show that specific endothelial protein C receptor (EPCR)-binding parasites are associated with severe adult malaria and act together with parasite biomass in patient hospitalization and disease severity. We found substantial differences in EPCR binding activity from severe malaria isolates. However, even parasite domains that partially obstructed the interaction between EPCR and its ligand activated protein C were sufficient to interfere with activated protein C-barrier protective activities in human brain endothelial cells. Thus, restoration of EPCR functions may be a key target for adjunctive malaria drug treatments. The interplay between cellular and molecular determinants that lead to severe malaria in adults is unexplored. Here, we analyzed parasite virulence factors in an infected adult population in India and investigated whether severe malaria isolates impair endothelial protein C receptor (EPCR), a protein involved in coagulation and endothelial barrier permeability. Severe malaria isolates overexpressed specific members of the Plasmodium falciparum var gene/PfEMP1 (P. falciparum erythrocyte membrane protein 1) family that bind EPCR, including DC8 var genes that have previously been linked to severe pediatric malaria. Machine learning analysis revealed that DC6- and DC8-encoding var transcripts in combination with high parasite biomass were the strongest indicators of patient hospitalization and disease severity. We found that DC8 CIDRα1 domains from severe malaria isolates had substantial differences in EPCR binding affinity and blockade activity for its ligand activated protein C. Additionally, even a low level of inhibition exhibited by domains from two cerebral malaria isolates was sufficient to interfere with activated protein C-barrier protective activities in human brain endothelial cells. Our findings demonstrate an interplay between parasite biomass and specific PfEMP1 adhesion types in the development of adult severe malaria, and indicate that low impairment of EPCR function may contribute to parasite virulence.

Urban Malaria: Understanding its Epidemiology, Ecology, and Transmission across Seven Diverse ICEMR Network Sites

A major public health question is whether urbanization will transform malaria from a rural to an urban disease. However, differences about definitions of urban settings, urban malaria, and whether malaria control should differ between rural and urban areas complicate both the analysis of available data and the development of intervention strategies. This report examines the approach of the International Centers of Excellence for Malaria Research (ICEMR) to urban malaria in Brazil, Colombia, India (Chennai and Goa), Malawi, Senegal, and Uganda. Its major theme is the need to determine whether cases diagnosed in urban areas were imported from surrounding rural areas or resulted from transmission within the urban area. If infections are being acquired within urban areas, malaria control measures must be targeted within those urban areas to be effective. Conversely, if malaria cases are being imported from rural areas, control measures must be directed at vectors, breeding sites, and infected humans in those rural areas. Similar interventions must be directed differently if infections were acquired within urban areas. The hypothesis underlying the ICEMR approach to urban malaria is that optimal control of urban malaria depends on accurate epidemiologic and entomologic information about transmission.

Malaria evolution in South Asia: Knowledge for control and elimination

The study of malaria parasites on the Indian subcontinent should help us understand unexpected disease outbreaks and unpredictable disease presentations from Plasmodium falciparum and Plasmodium vivax infections. The Malaria Evolution in South Asia (MESA) research program is one of ten International Centers of Excellence for Malaria Research (ICEMR) sponsored by the US National Institutes of Health. In this second of two reviews, we describe why population structures of Plasmodia in India will be characterized and how we will determine their consequences on disease presentation, outcome and patterns. Specific projects will determine if genetic diversity, possibly driven by parasites with higher genetic plasticity, plays a role in changing epidemiology, pathogenesis, vector competence of parasite populations and whether innate human genetic traits protect Indians from malaria today. Deep local clinical knowledge of malaria in India will be supplemented by basic scientists who bring new research tools. Such tools will include whole genome sequencing and analysis methods; in vitro assays to measure genome plasticity, RBC cytoadhesion, invasion, and deformability; mosquito infectivity assays to evaluate changing parasite-vector compatibilities; and host genetics to understand protective traits in Indian populations. The MESA-ICEMR study sites span diagonally across India and include a mixture of very urban and rural hospitals, each with very different disease patterns and patient populations. Research partnerships include government-associated research institutes, private medical schools, city and state government hospitals, and hospitals with industry ties. Between 2012 and 2017, in addition to developing clinical research and basic science infrastructure at new clinical sites, our training workshops will engage new scientists and clinicians throughout South Asia in the malaria research field.

Brief Report: Reticulocyte preference and stage development of Plasmodium vivax isolates

Plasmodium vivax, the most widely distributed human malaria parasite, is restricted to reticulocytes, limiting its asexual proliferation. In recent years, cases of severe and high-level P. vivax parasitemia have been reported, challenging the assumption that all isolates are equally restricted. In this article, we analyze the reticulocyte preference of a large number of Indian P. vivax isolates. Our results show that P. vivax isolates significantly vary in their level of reticulocyte preference. In addition, by carefully staging the parasites, we find that P. vivax schizonts are largely missing in peripheral blood, with the presence of schizonts in circulation correlating with a high reticulocyte preference.

Reticulocyte Preference and Stage Development of Plasmodium vivax Isolates

Plasmodium vivax, the most widely distributed human malaria parasite, is restricted to reticulocytes, limiting its asexual proliferation. In recent years, cases of severe and high-level P. vivax parasitemia have been reported, challenging the assumption that all isolates are equally restricted. In this article, we analyze the reticulocyte preference of a large number of Indian P. vivax isolates. Our results show that P. vivax isolates significantly vary in their level of reticulocyte preference. In addition, by carefully staging the parasites, we find that P. vivax schizonts are largely missing in peripheral blood, with the presence of schizonts in circulation correlating with a high reticulocyte preference.

Improved light microscopy counting method for accurately counting Plasmodium parasitemia and reticulocytemia.

Even with the advances in molecular or automated methods for detection of red blood cells of interest (such as reticulocytes or parasitized cells), light microscopy continues to be the gold standard especially in laboratories with limited resources. The conventional method for determination of parasitemia and reticulocytemia uses a Miller reticle, a grid with squares of different sizes. However, this method is prone to errors if not used correctly and counts become inaccurate and highly time‐consuming at low frequencies of target cells. In this report, we outline the correct guidelines to follow when using a reticle for counting, and present a new counting protocol that is a modified version of the conventional method for increased accuracy in the counting of low parasitemias and reticulocytemias. Am. J. Hematol. 91:852–855, 2016.

Infection of laboratory colonies of Anopheles mosquitoes with Plasmodium vivax from cryopreserved clinical isolates.

Plasmodium vivax is the most geographically widespread malaria parasite. Unique features of transmission biology complicate P. vivax control. Interventions targeting transmission are required for malaria eradication. In the absence of an in vitro culture, transmission studies rely on live isolates from non-human primates or endemic regions. Here, we demonstrate P. vivax gametocytes from both India and Brazil are stable during cryopreservation. Importantly, cryopreserved gametocytes from Brazil were capable of infecting three anopheline mosquito species in feedings done in the United States. These findings create new opportunities for transmission studies in diverse locales.

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays

ABSTRACT Plasmodium vivax chloroquine resistance has been documented in nearly every region where this malaria-causing parasite is endemic. Unfortunately, P. vivax resistance surveillance and drug discovery are challenging due to the low parasitemias of patient isolates and poor parasite survival through ex vivo maturation that reduce the sensitivity and scalability of current P. vivax antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for P. vivax parasites. We next performed a medium screen for formulations that enhance ex vivo survival. Finally, we optimized an isotopic metabolic labeling assay for measuring P. vivax maturation and its sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume ex vivo isolates by an average of >40-fold. The use of Iscoves modified Dulbeccos medium for P. vivax ex vivo culture approximately doubled the parasite survival through maturation. Coupling these with [3H]hypoxanthine metabolic labeling permitted sensitive and robust measurements of parasite maturation, which was used to measure the sensitivities of Brazilian P. vivax isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the P. vivax isolate sensitivities to antimalarials for resistance surveillance and drug discovery.

Dynamics of Plasmodium vivax sporogony in wild Anopheles stephensi in a malaria-endemic region of Western India

BackgroundIn global efforts to track mosquito infectivity and parasite elimination, controlled mosquito-feeding experiments can help in understanding the dynamics of parasite development in vectors. Anopheles stephensi is often accepted as the major urban malaria vector that transmits Plasmodium in Goa and elsewhere in South Asia. However, much needs to be learned about the interactions of Plasmodium vivax with An. stephensi. As a component of the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA), a series of membrane-feeding experiments with wild An. stephensi and P. vivax were carried out to better understand this vector-parasite interaction.MethodsWild An. stephensi larvae and pupae were collected from curing water in construction sites in the city of Ponda, Goa, India. The larvae and pupae were reared at the MESA ICEMR insectary within the National Institute of Malaria Research (NIMR) field unit in Goa until they emerged into adult mosquitoes. Blood for membrane-feeding experiments was obtained from malaria patients at the local Goa Medical College and Hospital who volunteered for the study. Parasites were counted by Miller reticule technique and correlation between gametocytaemia/parasitaemia and successful mosquito infection was studied.ResultsA weak but significant correlation was found between patient blood gametocytaemia/parasitaemia and mosquito oocyst load. No correlation was observed between gametocytaemia/parasitaemia and oocyst infection rates, and between gametocyte sex ratio and oocyst load. When it came to development of the parasite in the mosquito, a strong positive correlation was observed between oocyst midgut levels and sporozoite infection rates, and between oocyst levels and salivary gland sporozoite loads. Kinetic studies showed that sporozoites appeared in the salivary gland as early as day 7, post-infection.ConclusionsThis is the first study in India to carry out membrane-feeding experiments with wild An. stephensi and P. vivax. A wide range of mosquito infection loads and infection rates were observed, pointing to a strong interplay between parasite, vector and human factors. Most of the present observations are in agreement with feeding experiments conducted with P. vivax elsewhere in the world.

Distinct genomic architecture of Plasmodium falciparum populations from South Asia.

Previous whole genome comparisons of Plasmodium falciparum populations have not included collections from the Indian subcontinent, even though two million Indians contract malaria and about 50,000 die from the disease every year. Stratification of global parasites has revealed spatial relatedness of parasite genotypes on different continents. Here, genomic analysis was further improved to obtain country-level resolution by removing var genes and intergenic regions from distance calculations. P. falciparum genomes from India were found to be most closely related to each other. Their nearest neighbors were from Bangladesh and Myanmar, followed by Thailand. Samples from the rest of Southeast Asia, Africa and South America were increasingly more distant, demonstrating a high-resolution genomic-geographic continuum. Such genome stratification approaches will help monitor variations of malaria parasites within South Asia and future changes in parasite populations that may arise from in-country and cross-border migrations.