In a setting where the clinical efficacy of SP was above 95%, direct feeding assays showed that P. falciparum gametocytes after SP treatment had very low infectivity to A. gambiae mosquitoes. This reduced infectivity was seen during two consecutive transmission seasons and was demonstrated both by the numbers of mosquitoes that were oocyst-positive and the density of oocysts in the midguts of positive mosquitoes. To examine the possibility of a systematic bias in our experiment we repeated the same procedures with gametocytes carriers after chloroquine treatment. These experiments were performed contemporaneously with the second year of SP feeding experiments. We found that mosquitoes fed on chloroquine-treated gametocyte carriers had higher oocyst positivity and higher oocyst density per midgut, suggesting that the direct feeding procedures themselves did not account for the low infectivity.
Although these findings are at odds with some recent studies that suggested that SP increases gametocyte infectivity (Targett et al., 2001
; Hallett et al., 2006
), they are in agreement with older literature that found that pyrimethamine and related drugs decreased gametocyte infectivity to the vector (Shute and Maryon, 1954
; Jeffery, 1963
; Chutmongkonkul et al., 1992
; Hogh et al., 1998
; Robert et al., 2000
). This difference is most likely because, as was done in the older studies, we used direct feeding methods, allowing mosquitoes to feed directly on the skin of gametocytemic volunteers, while the recent studies have all used membrane feeding, which involves withdrawing venous blood into anticoagulated tubes and passing it through a series of tubes to feed to mosquitoes through a membrane (Hogh et al., 1998
; Drakeley et al., 1999
; Robert et al., 2000
; Targett et al., 2001
; Govere et al., 2003
; Bharti et al., 2006
; Chotivanich et al., 2006
; Hallett et al., 2006
). Membrane feeding and direct feeding have several differences: membrane feeding is performed through an artificial membrane (not human skin) and the gametocyte-containing blood is typically washed to replace the host plasma with non-immune sera or other surrogates for human serum (Toure et al., 1998
; Bonnet et al., 2000
). This washing step removes the drug and any host immunity components that would be present in the patients’ blood and would be taken by the mosquito together with the blood-meal. Moreover, the temperature perturbations and other manipulations and time taken to do membrane feeds may have other effects on infectivity rates. Although the membrane feeding is a cleaner experimental procedure for comparative studies as it reduces or eliminates potential effects of drugs, immunity and other host factors that could impact on transmission, direct feeding is the natural route of transmitting malaria gametocytes to the mosquito vector, and is presumably the most predictive of infectivity under natural conditions.
Our results show that post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population, consistent with drug selection favoring transmission of resistant gametocytes, as has been reported previously (Mendez et al., 2002
; Sowunmi and Fateye, 2003
; Hallett et al., 2006
). Although there was no asexual parasitemia in the post-treatment samples analyzed, remaining DNA from recently killed or persistent sub-patent asexual parasites could have been amplified by PCR (Thera et al., 2005
; Ladeia-Andrade et al., 2009
), confounding our results. To examine this possibility, we amplified the DHFR gene using a mutation-specific PCR method that has a lower limit of detection of approximately 250 parasites/μl (Plowe et al., 1995
), well above the threshold for detection of asexual parasites by microscopy. Indeed, during these experiments, samples with fewer than 75 gametocytes per microliter yielded no PCR product. Previous studies showed that drug-resistant parasites appear to have an advantage in differentiating into gametocytes (Mendez et al., 2002
) and being infectious to the vector.
The DHPS 540 mutation, which had not previously been found in Mali and which was absent in the pre-treatment asexual population, was present in the gametocyte population. This suggests that the parasites carrying that mutation were present at a level too low to be detected by our PCR method and subsequently expanded to reach detectable levels and differentiated into gametocytes after the elimination of the more sensitive strains by the drug treatment. We have previously shown very rapid selection of asexual stage parasites in response to pyrimethamine treatment (Doumbo et al., 2000
), and the same appears to occur with gametocytes.
Evidence from field observations and from experimental in vitro systems indicates that many factors can affect the rate of differentiation of asexual blood parasites into the gametogenesis pathway (Carter and Miller, 1979
; Dyer and Day, 2000
). Factors including genetic differences (Day et al., 1993
; Alano et al., 1995
), host anemia (Von Seidlein et al., 2001
), immunological responses (Smalley et al., 1981
) and stress (drugs) (Sokhna et al., 2001
) can all induce gametogenesis. The time course for the appearance of gametocytes harboring the resistance-conferring mutations post-SP treatment was consistent with an SP treatment effect, with the greatest prevalence of the majority of mutations occurring between days 3 and 28 following SP treatment, peaking on day 7. These results are consistent with a study from Colombia that demonstrated that an increasing number of DHFR mutations was associated with longer parasite clearance time and gametogenesis (Mendez et al., 2002
An increased duration and density of gametocyte carriage after SP treatment has been suggested to be an early indicator of drug resistance. This increased gametocytemia among patients who carry drug-resistant P. falciparum
would fuel the spread of resistance even before treatment failure rates increase significantly (Mendez et al., 2002
; Barnes et al., 2008
). The true impact of treatment on transmission may vary as a function of the drugs’ pharmacodynamics and intrinsic gametocidal effects. Our data suggest that increases in gametocyte carriage in the treated population may not always translate into increased malaria transmission.
Although SP is still highly efficacious in Mali when molecular correction is used to account for re-infections, this study together with our recent publication (Tekete et al., 2009
) show an upward trend in SP treatment failure and resistance in the area. This trend, together with the significant increase in the prevalence of the DHFR/DHPS quadruple mutant genotype and the appearance of the quintuple mutant genotype in the gametocyte population, signal that SP resistance is spreading in the country.
This study suggests that an intervention that causes increased gametocyte carriage may not necessarily translate into increased malaria transmission. The mere presence of gametocytes, whether measured by microscopy or by detection of gametocyte-specific RNA (Schneider et al., 2006a
), might not signal infectivity. A better understanding of the mechanisms involved in differences in infectivity of P. falciparum
gametocytes will shed light on the spread of antimalarial drug resistance in the field. Robust and sensitive assays that can accurately predict infectivity to mosquitoes are needed to guide the assessment of interventions aimed at blocking malaria transmission, an essential component of ongoing malaria control efforts and plans for possible elimination campaigns.