Upregulation of gametocytogenesis in anti-malarial drug-resistant Plasmodium falciparum

Abstract

The deadliest form of human malaria is primarily caused by the protozoan parasite Plasmodium falciparum. These parasites establish pathogenicity in the human host with a very low number of sexual forms or gametocytes, which are transmitted to the mosquitoes. Several studies have reported that exposing artemisinin-sensitive P. falciparum rings to a low concentration of dihydroartemisinin (DHA) results in dormancy, and the artemisinin-induced dormant (AID) forms are recovered into normal growth stages after 5-20 days. In this study, artemisinin-resistant P. falciparum parasites were tested for the development of AID forms and their recovery. Interestingly, it was found that exposure of an asynchronous culture of artemisinin-resistant P. falciparum IPC 5202 to DHA, a line carrying a mutation in the PfK13 gene that is linked to artemisinin resistance, also results in dormancy. Both the ring and some late stages of these AID forms recovered after 10-15 days. Furthermore, a high proportion of the recovered dormant forms developed into sexual forms or gametocytes after 3-4 weeks, which is almost a 7-8 times higher rate of conversion of asexual to sexual forms (gametocytes) or the malaria transmissible forms. In contrast, only early ring forms of artemisinin-sensitive parasites recovered slowly, and additional exposure of these parasites to artemisinin resulted in complete clearance within a week. This is in contrast to the resistant parasites exposed to a second dose of artemisinin, which resulted in a very high rate of dormancy and recovery into sexual forms or gametocytes.

Keywords: Dihydroartemisinin; Gametocytes; Plasmodium falciparum; Protozoan; Resistance.

Fig. 1

Artemisinin-Induced Dormant (AID) forms of an artemisinin-resistant P. falciparum line. P. falciparum parasite PfIPC5202 was cultured in vitro in the absence of (a) or exposure to DHA (b) for 6 h, washed and allowed to grow for another 16 h in the absence of DHA. Giemsa-stained blood smears were prepared and photographed using a phase contrast microscope. a shows various asexual stages such as rings, trophozoites and schizonts, b depicts the AID forms marked by a red arrow (color figure online)

Fig. 2

Upregulation of gametocytogenesis in AID forms: P. falciparum IPC 5202 was exposed to 700 nM of DHA for 16 h and the culture was maintained for 5 days. On day 5, the culture was divided into four equal portions. One portion, Bar #3 (P700-S700-6h), was treated with a second dose of 700 nM of DHA for 6 h, a second portion, Bar #4 (P700-S700-24h), was treated with a second dose of 700 nM of DHA for 24 h, a third portion, Bar #5 (P700-S100-70h) was treated with 100 nM of DHA for 70 h, and a fourth portion, Bar #2 (P700S0) was maintained without any further treatment of a second dose of DHA.. These cultures were incubated at 37 °C for 20-40 days with daily media changes. The gametocyte development was monitored every other day and final gametocytemia was calculated when they reached stage IV or V. As a control, a standard gametocyte culture was maintained for P. falciparum IPC 5202 without any added DHA, Bar #1 (no DHA). P-first dose of DHA; S-second dose of DHA