Ajeet Kumar Mohanty1, Charles de Souza2, Deepika Harjai2, Prathamesh Ghavanalkar2, Mezia Fernandes3,4, Anvily Almeida3,4, Jayashri Walke3,4, Suresh Kumar Manoharan3,4, Ligia Pereira3,4, Rashmi Dash3,4, Anjali Mascarenhas3,4, Edwin Gomes3, Thanyapit Thita5, Laura Chery4, Anupkumar R Anvikar6, Ashwani Kumar2,7, Neena Valecha6, Pradipsinh K Rathod4, Rapatbhorn Patrapuvich8. 1. Field Unit, National Institute of Malaria Research, Campal, Goa, 403001, India. ajitbiotech1@gmail.com. 2. Field Unit, National Institute of Malaria Research, Campal, Goa, 403001, India. 3. Goa Medical College and Hospital, Bambolim, Goa, 403202, India. 4. Department of Chemistry, University of Washington, Seattle, WA, 98195, USA. 5. Drug Research Unit for Malaria (DRUM), Center of Excellence in Malaria Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand. 6. National Institute of Malaria Research (ICMR), Sector 8, Dwarka, New Delhi, 110077, India. 7. ICMR-Vector Control Research Centre, Medical Complex, VCRC Road, Indra Nagar, Priyadarshini Nagar, Puducherry, 605006, India. 8. Drug Research Unit for Malaria (DRUM), Center of Excellence in Malaria Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand. rapatbhorn.pat@mahidol.edu.
Abstract
BACKGROUND: Efforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivax patient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here. METHODS: Membrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively. RESULTS: Laboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load. CONCLUSIONS: Optimized conditions for the production of high-quality P. vivax sporozoite-infected An. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.
BACKGROUND: Efforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivaxpatient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here. METHODS: Membrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively. RESULTS: Laboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load. CONCLUSIONS: Optimized conditions for the production of high-quality P. vivax sporozoite-infectedAn. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.