This work highlights the applicability of culture adapted field isolates of
P. falciparum in anti-gametocyte drug discovery. Here, we present a simple technique to produce gametocytes in high yield from gametocyte producing field isolates, useful for gametocytocidal drugs screening applications. To date, limited data is available involving field isolates for directly testing gametocytocidal activity. This might be because very few isolates can reproducibly generate gametocytes in high yield in vitro and also show a gradual loss of gametocyte producing potential in continuous culture [
23]. This makes it difficult to study the process of gametocytogenesis [
23,
24] and to perform gametocytocidal drug screening due to the dependency on cryopreserved stabilates with minimum passage in order to preserve gametocyte production phenotype [
13]. The two field isolates, RKL-9 and JDP-8 used in present study did not show any significant reduction in gametocyte production potential in vitro for at least 6 months in asexual culture. This gives these isolates an additional advantage over clonal lines such as 3D7 in which ability to form gametocytes wanes in as little as 2 weeks [
25]. Moreover, gametocyte production potential in RKL-9 and JDP-8 appeared to be stable after multiple freeze–thaw cycles. Other studies also reported no loss in gametocyte production upon maintenance of isolates in asexual culture for 18 months [
22] and also after cryopreservation [
13,
26,
27]. In vitro gametocyte production potential of parasite is strain specific [
28], exhibited in response to ‘nonspecific’ stress in the form of environmental stimuli [
22,
23,
29]. However, the definition of ‘stress’ as well as other triggers involved in pathway shift towards gametocytogenesis in
P. falciparum are not precisely clear [
30]. The stress on the parasite is not regulated by a single component but might be a collective contribution of multitude of factors, such as high parasite load, and decrease with haematocrit [
23,
31]. Studies suggest that some clones show more preference towards production of gametocytes [
26,
29,
32,
33] than others under similar conditions as a result of which gametocyte production in some isolates is upregulated [
34,
35]. This is also evident from data reported here, as out of 15 culture adapted field isolates, only 2 (RKL-9 and JDP-8) were able to reproducibly produce > 2% gametocytes in vitro. Moreover, asexual stages cultivated from both RKL-9 and JDP-8 were found to be chloroquine resistant. Production of higher gametocytaemia in vitro (and in vivo) by drug resistant parasites may be correlated with tendency to spread the resistant mutation as a part of parasite’s survival strategy [
34,
36]. However, current study was not designed in that context and separate studies involving more number of field isolates are needed to be carried out before a link between drug resistance and in vitro gametocytogenesis can be established.
Herein, gametocytocidal activity of methylene blue which is primarily used for treatment of methemoglobinemia is evaluated amongst Indian field isolates of
P. falciparum. In present study as well as other in vitro studies carried out using standard laboratory strains, methylene blue was able to effectively target gametocytes, especially relatively less metabolizing stage V [
8‐
13,
28]. However, IC
50 values obtained for methylene blue were inconsistent across all these studies (varied from 29.5 nM in [
10] to 490 nM in [
8] and 106.44 nM (late stage mean IC
50, present study)]. In spite of differences in drug efficacies in multiple studies (might be due to variation in culture parameters including length of drug exposure, type of screening assay, and difference in parasite strain used [
28]), methylene blue was effective in killing gametocytes across all these studies. In the present work, morphological alterations induced by methylene blue are described. These alterations comprise of shrinkage, distortions and membrane deformations clearly representing unhealthy gametocytes. However, it is difficult to directly correlate morphology with viability. Therefore, viability and infectivity of these morphologically deformed gametocytes remains to be evaluated. Moreover, primaquine is a gametocytocidal drug having in vivo activity [
5] but data reported here identifies it as non-gametocytocidal. This disparity between efficacy data highlights the absence of any metabolic activation in vitro because of lack of liver enzymes activity required for generation of active metabolites of primaquine [
37]. However, identity of the metabolites and mode of action of primaquine is not fully elucidated [
38]. Although, primaquine was not expected to show any significant potency in vitro but was added in present study to validate earlier studies [
8,
9] and also served as an additional negative control for methylene blue other than DMSO. A major advantage that methylene blue confers over its alternatives is that, it is the only registered non 8-aminoquinoline having late stage gametocytocidal activity, which is inexpensive and currently, a suitable alternative to primaquine. So evaluation of gametocytocidal activity of methylene blue amongst Indian field isolates of
P. falciparum has utmost importance. However, more evidence is needed to ascertain a dose that is safe for both G6PD deficient and G6PD non-deficient population as well as effective for stopping transmission. The study sets the stage for further basic and clinical research required for consideration of methylene blue as a gametocytocidal adjunct along with standard ACT in India and developing recommendations for future use.