Anti-malarial drug resistance to SP, a factor contributing to diminishing therapeutic efficacy, is increasing in many malaria endemic countries. All African countries have already replaced SP with artemisinin-based combination therapy as the first line treatment for uncomplicated malaria, according to the WHO guidelines. However, SP remains the recommended drug for IPTp in malaria endemic areas. In this study, the temporal trends of SP resistance molecular markers in the parasites from Kenyan pregnant women and potential epidemiological factors affecting the prevalence of the resistant markers in the pregnant women were evaluated.
The key observations from this study were: 1) a noticeable background level of SP mutations in pregnant women prior to IPTp policy adoption, 2) a significant rise in the prevalence of the drug resistance markers after IPTp adoption seemingly in parallel with an increase in the percentage of SP use in pregnancy, and 3) however an association between more SNP mutations and SP use during pregnancy only in the 1996–2000 study in univariable analysis but not in 2002–2008 and 2008–2009 study periods. These results suggest that the increase in drug-resistant parasites in the pregnant women over time could be influenced by other major factors in addition to the minor role of IPTp adoption and SP use in pregnancy. Firstly, compared to high drug pressure in large general population (children and adults) due to SP use for case management, drug pressure in the small minority of pregnant women by IPTp-SP use is relatively lower. It is most likely that the resistant parasites selected by the use of SP for case management in the large general population could serve as the potential infectious reservoirs for pregnant women, thus high prevalence of drug resistant parasites circulated in the large non-pregnant population could contribute to the observed temporal increase in resistance markers in pregnant women. Indeed, the results from our previous study, conducted in the general population of the same area, showed that the prevalence of dhfr/dhps
quintuple mutant parasites in children from the same study area increased from 29
% in 1996 to 62
% and 85
% in 2001 and 2007, respectively, after the drug policy change to SP for the treatment of uncomplicated malaria in 1998 [41
]. Secondly, IPTp-SP works by intermittently clearing asymptomatic parasitaemia and preventing new infections in pregnant women [51
] and the decreasing prevalence and intensity of infection in subsequent pregnancies also indicates the acquired immunity of the pregnant woman in preventing malaria infection as well as clearance of parasites [52
]. Recent studies from several African countries reported increased prevalence of mutant genotypes due to IPTp-SP [33
], but the increased resistant parasites did not affect the protective efficacy of IPTp [53
]. Based on the results from others [53
] and the molecular data from this study, the authors speculate that the interplay between IPTp-SP and host immunity in pregnant women could also play a role in the selection of drug resistant parasites, consequently shaping the parasite drug resistant profile in pregnant women. However, further studies are required to test this hypothesis. Lastly, the use of CTX as prophylaxis against opportunistic infection in HIV infected pregnant women, particularly the validated CTX use in the third study (2008–2009), might also contribute to the increased prevalence of SP drug resistant genotypes over time, possibly due to cross resistance between trimethoprim and pyrimethamine in P. falciparum
malaria parasites [18
]. Since multiple factors could be involved in the selection of resistant parasites in pregnant women, molecular markers when used alone may not accurately predict the failure of IPTp-SP. The assessment of the linkage between SP drug resistance molecular markers and efficacy of IPTp-SP in pregnant women in different malaria endemic areas becomes extremely important.
This study also detected 5.3
% of the dhps
triple mutant (A437G
) (7 samples), the presence of quadruple dhps
) (1 sample), the dhfr
) mutation (1 sample), and an increase in the new mutation at dhps
(5 samples) in the third study period (2008–2009). These results suggest progressive expansion of new mutations or new genotypes in parasite populations in Kenya. Although the contribution of the new mutation dhps
(unreported prior to this study) to drug resistance is unclear, the mutations at dhfr
164 and dhps
581 or related genotypes have been associated with a high-level of SP resistance mainly in Southeast Asia and South America [55
] and, more recently, have been reported on the African continent [26
]. Due to the small sample size in the new drug resistant genotype/SNP group, no further association analyses were conducted. However, higher placental parasite density in 2008–2009 period was observed as compared to the 1996–2000. It is possible that the progressive expansion of the new genotypes could be related to the overgrowth of resistant parasites, i.e. the highly resistant parasites out-compete less fit parasites [61
]. Recent data from Tanzania suggest that the emergence of the dhps
triple mutant (A437G
) associated with the IPTp-SP use in pregnant women led to high-density parasitaemia [33
]. Taken together, the emergence of new genotypes containing mutations in dhps
436 and dhps
581, coupled with the presence of dhfr
164 mutation in pregnant women may present a challenge for the future usefulness of IPTp-SP intervention in Kenya.
A recent study conducted in Mozambique reported that IPTp-SP use increased the prevalence of resistance markers in HIV positive women and mutant infections were more prevalent in placental than peripheral samples [36
]. However, in this investigation, no statistical differences were found in the prevalence of dhps
double and dhfr/dhps
combined quintuple mutants between HIV positive and HIV negative women during all the three study periods. In the current study, there was also no significant difference in the prevalence of SP resistance markers and mean MOI between peripheral and placental-paired samples in the third study period when SP resistance was high. The discrepancy of the results between these two studies could be due to the differences in the study design, level of existing SP pressure and resistance, HIV care and other unknown factors. The Mozambique study used samples from a randomized double-blind, placebo-control trial of IPTp-SP conducted before IPTp-SP policy adoption, while the current study employed the samples collected from three different observational studies before and after IPTp-SP adoption for an extended period. However, the observation of no significant differences in mutant infections between placental and peripheral samples in the current study is consistent with the results from the studies conducted in Tanzania and Ghana, the countries with relative high levels of SP resistance [33
]. This suggests that either peripheral or placental samples can be used for monitoring drug resistant molecular markers in areas with high SP resistance level.
Overall, the prevalence of mixed infection and mean MOI decreased significantly between 1996 and 2009, but the highest parasite diversity was observed between the 2004 and 2007 period. The observed sharp increase in parasite diversity from 2004 could reflect the higher transmission intensity of the study site as the second malaria in pregnancy study was conducted from 2004 in the rural area (Siaya) where malaria transmission is relatively higher compared to Kisumu town. However, the drop in parasite diversity in 2008 followed by a significant decrease in 2009 might be related to the combination of transmission reduction by the implementation of free distribution of ITNs to pregnant women in ANC from early 2008 and SP use in pregnancy. The diversity of P. falciparum
malaria reflects acquisition of new infections and is associated with transmission intensity [65
]. A previous study conducted in Tanzania also showed that parasite diversity measured by msp2
was reduced in women with recent IPTp-SP use [33
]. Nevertheless, the observation, overall decreased parasite diversity in conjunction with increased prevalence of drug resistance molecular markers over time in this study, suggests that intra-host removal of SP drug sensitive parasite clones is present, thus purifying drug resistant parasites in pregnant women.
There were few limitations to the current study. First, the present study utilized data from three different malaria in pregnancy observational studies and these three studies varied in their original study designs. Therefore, there were some differences in patient recruitment and procedures of clinical data collection, including assessment of IPTp use and use of SP for case management, which could have affected the statistical association analysis. Additionally, the lack of baseline samples from women prior to IPTp administration and absence of information for early versus later IPTp-SP or SP use before delivery limited the comparisons to assess the selection of drug resistant parasites in pregnant women whose immunity and pharmacokinetics are altered during pregnancy. Another limitation to the current study was the lack of samples from non-pregnant women as controls from the same study periods due to original design of the three previous observational studies. It will be ideal to conduct a well-controlled longitudinal IPTp drug resistance studies in both pregnant and non-pregnant women of the same population over a period of time. Such studies will help to examine the selection of drug resistant parasites and evaluate the potential factors involved in the development of SP resistance in pregnant women.