ABSTRACT. The Malaria Evolution in South Asia (MESA) International Center of Excellence for Malaria Research (ICEMR) conducted research studies at multiple sites in India to record blood-slide positivity over time, but also to study broader aspects of the disease. From the Southwest of India (Goa) to the Northeast (Assam), the MESA-ICEMR invested in research equipment, operational capacity, and trained personnel to observe frequencies of Plasmodium falciparum and Plasmodium vivax infections, clinical presentations, treatment effectiveness, vector transmission, and reinfections. With Government of India partners, Indian and U.S. academics, and trained researchers on the ground, the MESA-ICEMR team contributes information on malaria in selected parts of India.
ABSTRACT. The Malaria Evolution in South Asia (MESA) International Center for Excellence in Malaria Research (ICEMR) was established by the US National Institutes of Health (US NIH) as one of 10 malaria research centers in endemic countries. In 10 years of hospital-based and field-based work in India, the MESA-ICEMR has documented the changing epidemiology and transmission of malaria in four different parts of India. Malaria Evolution in South Asia-ICEMR activities, in collaboration with Indian partners, are carried out in the broad thematic areas of malaria case surveillance, vector biology and transmission, antimalarial resistance, pathogenesis, and host response. The program integrates insights from surveillance and field studies with novel basic science studies. This is a two-pronged approach determining the biology behind the disease patterns seen in the field, and generating new relevant biological questions about malaria to be tested in the field. Malaria Evolution in South Asia-ICEMR activities inform local and international stakeholders on the current status of malaria transmission in select parts of South Asia including updates on regional vectors of transmission of local parasites. The community surveys and new laboratory tools help monitor ongoing efforts to control and eliminate malaria in key regions of South Asia including the state of evolving antimalarial resistance in different parts of India, new host biomarkers of recent infection, and molecular markers of pathogenesis from uncomplicated and severe malaria.
Background: Plasmodium vivax is the second most prevalent cause of malaria yet remains challenging to study due to the lack of a continuous in vitro culture system, highlighting the need to establish a biobank of clinical isolates with multiple freezes per sample for use in functional assays. Different methods for cryopreserving parasite isolates were compared and subsequently the most promising one was validated. Enrichment of early- and late-stage parasites and parasite maturation were quantified to facilitate assay planning. Methods: In order to compare cryopreservation protocols, nine clinical P. vivax isolates were frozen with four glycerolyte-based mixtures. Parasite recovery post thaw, post KCl-Percoll enrichment and in short-term in vitro culture was measured via slide microscopy. Enrichment of late-stage parasites by magnetic activated cell sorting (MACS) was measured. Short and long-term storage of parasites at either -80°C or liquid nitrogen were also compared. Results: Of the four cryopreservation mixtures, one mixture (glycerolyte:serum:RBC at a 2.5:1.5:1 ratio) resulted in improved parasite recovery and statistically significant (P<0.05) enhancement in parasite survival in short-term in vitro culture. A parasite biobank was subsequently generated using this protocol resulting in a collection with 106 clinical isolates, each with 8 vials. The quality of the biobank was validated by measuring several factors from 47 thaws: the average reduction in parasitemia post-thaw (25.3%); the average fold enrichment post KCl-Percoll (6.65-fold); and the average percent recovery of parasites (22.0%, measured from 30 isolates). During short-term in vitro culture, robust maturation of ring stage parasites to later stages (>20% trophozoites, schizonts and gametocytes) was observed in 60.0% of isolates by 48 hours. Enrichment of mature parasite stages via MACS showed good reproducibility, with an average 30.0% post-MACS parasitemia and an average 5.30 x 105 parasites/vial. Finally, the effect of storage temperature was tested, and no large impacts from short-term (7 day) or long term (7-10 year) storage at -80°C on parasite recovery, enrichment or viability was observed. Conclusions: Here, an optimized freezing method for P. vivax clinical isolates is demonstrated as a template for the generation and validation of a parasite biobank for use in functional assays.
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