It is unclear how the increase in resistant genotypes at delivery impacted the potential added efficacy of monthly ITPp over standard 2 dose SP in reducing preterm delivery and low birth weight infants. Although in the study sample the difference in treatment effect between the monthly and stardard regimens was not statistically significant, the observed effect sizes for duration of pregnancy and low birth weight suggested a sizable benefit in favor of the monthly regimen. It is likely that in areas of high SP resistance, the efficacy of 2 dose SP is partially compromised because of the shortening duration of post-treatment prophylaxis, and monthly dosing compensates for this by increasing the overall duration of protection relative to the 2 dose regimen. Indeed, based upon multiple studies showing that monthly SP is better at reducing the risk of low birth weight even in areas where a high proportion of parasites carry quintuple dhfr/dhps
mutants, an evidence review group convened by the WHO has recommended SP administration at every antenatal visit (WHO 2012
Importantly, the accumulation of more resistant alleles at delivery suggests increased selection of SP-resistant mutants during pregnancy, possibly as a result of IPTp. Other studies have shown conflicting results in this regard. For example, in a 2006 sampling of pregnant women in Ghana by Mockenhaupt et al. (2008)
, 73% (69/95) harbored dhfr
triple mutants in early gestation, almost none of whom had received IPTp. This percentage remained constant among delivering woman, of whom the majority had taken IPTp with SP at least once, with 73% (55/75) of placental PCR genotypes revealing triple dhfr
mutants. Thus, the administration of IPTp in pregnancy in that cohort did not seem to affect the prevalence of the dhfr
triple mutant genotype at delivery. In another study in Tanzania, the proportion of pregnant women who harbored resistant alleles at dhps
codon 581 at delivery was significantly higher in those who had received any IPTp vs. no IPTp (Harrington et al. 2009
). Among all the isolates in our study, only one parasitemia out of 149 (0.7%) harbored the dhps
581 mutation. The paucity of this mutation is consistent with other studies of pregnant women in Malawi despite heavy IPTp-SP use (Taylor et al. 2012
). Thus, selection pressures on dhfr
may vary from location to location depending on the level of resistance in the parasite population.
Of the 35 women with quintuple mutants at delivery, 24(69%) had received standard IPTp with two doses of SP, while 6(17%) had received SP monthly, and 5(14%) had received SP monthly plus two doses of azithromycin as intensive IPTp regimens. This ratio is reflective of the frequency of PCR-positive malaria in the 3 groups [33/159(21%), 10/150(6.7%), and 8/166(4.8%) in the standard, monthly SP and AZI-SP groups, respectively]. Thus, by reducing the number of women with parasitemia, presumably as a result of more successfully killing of partially resistant parasites, intensive regimens have the potential to reduce selection pressure in the population. However, no inferences can be made about whether the combination regimen (AZI-SP) protects against the development of resistance.
When parasitemia at delivery and enrollment in the same women were compared, those who were smear-positive at enrollment maintained a higher risk of P. falciparum
parasitemia at delivery (). This increased risk -- approximately two-fold for PCR outcomes and greater for smear positivity -- existed despite dosing of SP or SP and azithromycin in 95% (1251/1320) of study participants between 28–34 weeks gestation. Thirty-six women were smear positive at this third trimester visit. Of the16/36 women who had delivery samples available, only 10/16 (63%) were successfully treated with no evidence of malaria at delivery. 3/16 were smear-positive at delivery and 6/36 were PCR positive. Five of these parasitemic women were in the standard IPTp group. This is not to suggest that these women were parasitemic throughout pregnancy, as the majority of women (101/106 or 95%) who were smear-positive at enrollment showed evidence of clearance at their 28–34 week visit. Rather, these recurrent infections at delivery could be due to recrudescences; alternatively, the women may have had multiple infections as a result of residing in local transmission “hot spots” (Bousema et al. 2010
Malaria prevalence at delivery based on enrollment malaria status
There are several limitations to this study. The fixation of dhfr and dhps mutants in the delivering women does not allow us to detect an influence of the different treatment arms on the prevalence of SP resistance alleles at delivery. Since the trial did not include women who did not receive any ITPp, we also cannot rule out the possibility that the increased prevalence of drug resistant mutants at delivery was due to a general trend of increasing drug resistance over time. Finally, the limited number of malaria positive delivery samples also precludes an assessment of whether enrollment genotype influenced malaria outcome at delivery. As yet, dhfr alleles have not been correlated to SP effectiveness in pregnant women. Among other factors, the contribution of immunity to parasite clearance likely influences the association of treatment outcomes with genotypic status.
In conclusion, within a cohort of pregnant women on IPTp, we detected an increase in the prevalence of alleles associated with SP resistance from enrollment to delivery. However, few or no high-level resistance mutations (dhps581 and dhfr164) were seen. We also found that despite intensive IPTp regimens, in this area of high prevalence of SP resistant parasites, one-fifth of smear-positive pregnant women at enrollment had evidence of malaria parasites also at delivery. Our findings are consistent with a moderate level of selection of SP resistance in women receiving SP IPTp in Malawi.