Abstract
Background: In clinical P. vivax, patients are highly infective to mosquitoes before they seek treatment mostly due to the earlier generation of gametocytes, relatively longer pre-patent period, and higher transmissibility resulting in increased infectivity. Periodic activation of hypnozoites leads to repeated relapsing episodes and it makes as much as 80% of all infections. To date, it is not well understood to what extent relapses contribute to the infectious reservoir. Gametocyte densities and transmissibility to mosquitoes might vary between primary and secondary infections. In our study, we have assessed the dynamics of transmissibility to mosquitoes between primary and recurrent infections.
Methodology: Self-presenting patients with signs and symptoms of malaria and confirmed of P. vivax mono- and/or mixed species infections with P. falciparum were requested to join a monthly follow-up study in Arba Minch, Ethiopia. At the time of recruitment and recurrent infections, patients were requested to donate 5mL of venous blood for direct membrane feeding experiments and molecular and serological analyses. At recruitment and recurrent infections, patients were treated according to national treatment guideline that includes chloroquine and 14-days dose of primaquine. Blood in heparinized tubes was fed immediately after collection to locally reared 4-7 days old female An. arabiensis mosquitoes that were starved for ~12 hours. Fully fed mosquitoes were transferred to clean cages after discarding unfed and partially fed mosquitoes. On day 7 after feeding, mosquitoes were dissected for oocysts detection. Parasite and gametocyte densities were quantified by expert microscopists. Molecular tests were used to quantify total parasites (18S based qPCR) and gametocytes (Pvs25 based RTqPCR).
Results: A total of 241 patients (233 P. vivax mono- and 8 mixed-species with P. falciparum) were recruited for follow-up. The median age was 14 years (range: 4 – 65) and two-third were males (63%). At presentation, 49.1% (114/232) of the patients had fever (>=37.5oC) and mean hemoglobin was 12.4 g/dL (range: 10.5 - 14.3). Treatment was administered to the patients by the health facility as per the national treatment guidelines. The study did not have a control over the type of treatment administered. At recruitment, 64.3% (155/241) of the patients received the right combination treatment (chloroquine; 3days plus primaquine; 14 days) whilst 32.0% (77/241) of them received only chloroquine for 3days. At the moment of recurrences, 74.4% (96/129) of the patients received chloroquine for 3days plus primaquine for 14 days whilst only 25.6% (33/129) of them received chloroquine for 3days. So far, 129 events of recurrent infections were detected in 84 patients out of the 241 patients (34.9%) of which the majority were 1st recurrence (n=58) with the remaining being 2nd (n=8), 3rd (n=5), and 4th (n=1). Of the total mosquito feedings done, 67.2% (174/259) were infectious to mosquitoes. The likelihood of infectivity did not vary between recruitment (67.4%, 128/190) and recurrent infections (66.7%, 23/69). Overall, almost half of the mosquitoes fed on blood from infectious individuals were infected (median, 45.3%; IQR, 25.0% – 71.9%, n=174) with no difference between feeds at recruitment (48.8%, 25.0 – 72.6, n=128) and recurrent infections (42.8%, 20.0 – 70.6%, n=46). The oocyst density was not different between recruitment (median, 8.6 oocysts/infected midgut, IQR, 3-33, n=127) and recurrent infections (6.2, 25-23.3, n=43).
Conclusion: From the current study, high proportion of P. vivax recurrent infections observed within a maximum of 8 months through the study. The recurrent infections were equally important in contributing to continued transmission with the primary infections. There is no difference between primary and recurrent infections for proportion of mosquitoes infected and density of oocysts. Molecular analysis and further follow-up of the remaining patients is continuing. This will boost the observations until April 2022.