Prevalence of PfDHFR and PfDHPS Mutations Associated with Drug Resistance Among Pregnant Women Receiving IPTp-SP at

Background: Prevention and treatment of malaria during pregnancy is crucial in dealing with maternal mortality and adverse fetal outcomes. WHO’s recommendation to treat all pregnant women with sulphadoxine-pyrimethamine (SP) through antenatal care structures was implemented in Kenya in the year 1998 but concerns about its effectiveness in preventing malaria in pregnancy has arisen due to the spread of parasites resistant to SP. We aimed to determine the prevalence of SP resistance markers in Plasmodium falciparum parasites isolated from pregnant women seeking antenatal care at Msambweni County Referral Hospital, located in coastal Kenya, between the year 2013 and 2015. Methods: This hospital-based study included 106 malaria positive whole blood samples for analysis of SP resistance markers within the PfDHFR gene (codons 51,59 &108) and PfDHPS gene (codons 437 & 540). The venous blood collected from all pregnant women was tested for malaria via light microscopy, then thereafter separated into plasma and red cells and stored in a -86⁰ freezer for further studies. Archived red blood cells were processed for molecular characterization of SP resistance markers within the PfDHFR gene and PfDHPS using real time PCR platform. Results: All samples had at least one mutation in the genes associated with drug resistance; polymorphism prevalence of PfDHFR51I, 59R and 108N was at 88.7%, 78.3% and 93.4%, respectively, while PfDHPS polymorphism accounted for 94.3% and 91.5% at 437G and 540K, respectively. Quintuple mutations (at all the five codons) conferring total SP resistance had the highest prevalence of 86%. Quadruple mutations were observed at a frequency of 10.4%, and 24.5% had a

heterogeneous outcome with both wildtype and mutant genotypes in the genes of interest.
Conclusion: The data suggest a high prevalence of Pf genetic variations conferring resistance to SP among pregnant women, which may explain reduced efficacy of IPTp treatment in Kenya. There is need for extensive SP resistance profiling in Kenya to inform IPTp drug choices for successful malaria prevention during pregnancy.

Background
Malaria is a significant public health problem in sub-Saharan Africa and remains a major contributor to morbidity and mortality in the African continent (1). The World Health Organization (WHO) has reported that Africa carries the highest burden of malaria: 92% of the malaria cases and 93% of malaria deaths worldwide (2). 99.7% of the cases are caused by Plasmodium falciparum with pregnant women being a particularly vulnerable population (3) especially those carrying first pregnancies at a young maternal age (4). Malaria in pregnancy contributes to maternal anemia leading to spontaneous abortion, stillbirth, premature birth and low birth weight (3).
WHO has recommended an intermittent preventative treatment for pregnant women (IPTp) interventions using sulphadoxine-pyrimethamine (SP) drug, that Kenya implemented in the year 1998. Pregnant women then received at least two doses of SP given from the second trimester of pregnancy, which was later revised in 2009 to a monthly dose, to be administered during their antenatal clinic (ANC) visits (5).
IPTp prophylactic treatment has quickly been countered by the rise of P. falciparum (Pf) parasites resistant to SP, resulting in a loss in sensitivity to the SP drug. This resistance is attributed to single nucleotide polymorphism (SNP) mutations within the DHFR and DHPS genes that are target sites for the sulphadoxine and pyrimethamine active components of the drugs, which are most effective when working in synergy (6). In East Africa, the prevalence of these mutations is high, reaching near 100% in some regions (7,8,9,10), thus raising concerns on the efficacy of the drug in preventing malaria in pregnancy.
The SP drug is still considered by practitioners to be effective in clearing the parasites in pregnant women, despite the high levels of Pf resistance that have been reported. To clarify this issue, the WHO has recommended that more studies be carried out to investigate the prevalence of Pf SP resistance molecular markers in parasites in the context of IPTp (11). Malaria is the leading cause of morbidity in Kwale County with a prevalence of 37.7% in comparison to other disease morbidities such as diarrhea, influenza, and respiratory diseases among others, which account for 4.6, 16.4, 5, and 3.1 per cent of disease burden in the county, respectively (12).
This study investigated the prevalence of SP resistance molecular markers in parasites isolated from blood samples collected from the pregnant women receiving

Study area and sample collection
This descriptive cross-sectional study involved pregnant women seeking antenatal care at the Msambweni County Referral Hospital between the year 2013 and 2015.
There were a total of 763 pregnant women enrolled in the study who visited the antenatal clinic, and all were tested for malaria using light microscopy (13).
Whatman filter paper was used to prepare dried blood spots (DBS) from the archived red blood cell samples which were then individually reserved in coded plastic bags with silica desiccant beads. The DBS were transported at room temperature to Centre for Biotechnology Research and Development (CBRD) within the Kenya Medical Research Institute (KEMRI) in Nairobi, Kenya for further molecular analysis.

Molecular genotyping
Genomic DNA was extracted from the DBS using a QIAamp DNA mini blood kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. The malaria microscopy results were then confirmed for Pf using a Singleplex real-time qPCR assay carried out on an Applied Biosystems™ 7500 Fast Real-time PCR machine using a primer and probe set previously published (14) with each sample denatured at 95⁰C for 10 seconds and cycled 45 times with each cycle consisting of 95⁰C for 15 seconds and 55⁰C for 60 seconds (15) A modified Multiplex real time PCR assay described elsewhere (15) was performed with the same thermocyclic conditions using two hydrolysis probes for each codon.
The wildtype strain was detected by a FAM labeled probe while the mutant strain was detected by a HEX labeled probe and each probe was tagged to a Black hole quencher. These probes differentiated single nucleotide polymorphisms (SNPs) within the PfDHFR gene and PfDHPS genes that are associated with SP resistance, targeting three SNPs within the PfDHFR gene at codon 51I, 59 R and 108N that confer resistance to pyrimethamine and two SNPs within the PfDHPS gene at codon 437G and 540E that confer resistance to sulphadoxine. PCR was carried out in a 25µl final volume containing 12.5µl of Agpath-ID™ One- Step RT-PCR Kit, 5µl of DNA template, forward and reverse primers at various concentrations shown in table 4 and both mutant and wildtype probes at a final concentration of 0.2uM (15) P. falciparum strain 3D7 and Dd2 were used as controls for wildtype and mutant strains respectively.

Statistical analysis
Characteristics of women were presented as means with standard deviations (SD) and as proportions. The prevalence of malaria parasites and mutations were expressed as a proportion with their respective 95% confidence interval (95% CI), while parasite load as median and its interquartile range (IQR). Comparisons of different factors for significant difference was done using t-test for quantitative variables while Chi square was used for binary variables, with a p-value of <0.05 considered significant. Statistical analysis was conducted using Stata version 12.0 software (StataCorp, 4905 Lakeway Drive, College Station, Texas 77845 USA).

Results
The majority of 763 pregnant women screened for malaria in this study were married (72%) and had up to primary level of education (81%). The mean (± SD) age and gestation were 26 (± 6.4) years and 23 (± 5.2) weeks respectively. The median (IQR) number of visits with dispensed folic tablets and SP-IPTp were three (3)(4) and three (2)(3)(4) respectively. The majority of women (98%) had at least one dose of IPTp, while 88% received the WHO recommended IPTp doses (≥2). Malaria parasites were detected in 135 pregnant women, yielding a prevalence of 17.7 % (95% CI: 15.1-20.6). Young age, marital status and first pregnancy were significantly associated with malaria parasite infection (Table 1). SP-IPTp was not associated with decreased odds of malaria infection (p = 0.251). Of the 132 single women in the study, 32 (24%) were infected with malaria compared to 88 (16%) of married women. Therefore, single women (who were also likely to be younger) had increased odds of malaria infection compared to married women (OR = 1.7; 95%CI: . Similarly, primigravidae were 1.7 times as likely to be detected with malaria parasites compared to multigravidae (OR = 1.7; 95%CI: 1.1-2.5).
Placentas from malaria infected women had a significantly lower mean weight (518.3±96.74g) compare to that of women negative for malaria (550.3±98.86g), p<0.01. However, malaria infection was not associated with mean placenta length, width or height (Table 1). Of the 568 women with hemoglobin levels, the mean Hb Of the 135 pregnant women who tested positive for malaria, only 84 had archived blood samples available and these were included in P. falciparum genetic analysis. These women had a mean age: 23.7±6.14 years and 32 (38%) were primigravidae.
The mean baseline Hb was 9.5 (± 2.31) g/dL ( Table 2). From these women, 106 of the blood samples (70 blood samples collected at first antenatal care visit and 36 collected at delivery) were all confirmed to contain P. falciparum parasites by real time PCR assay and were included in the mutation analyses. The median parasite load was 2760 (1200-7133) parasites/µL of blood.
All samples were successfully genotyped yielding prevalences of PfDHFR gene and PfDHPS SNP mutations that ranged from 83-100% (Table 3a). Of the 106 genotypes, 94 (88.7%) harbored PfDHFR gene mutant allele 51I, thus conferring a change in amino acid from asparagine (N) to isoleucine (I). Five samples (4.7%) had a wildtype allele at codon 51N and six samples (5.6%) had mixed outcomes of both wild-type and mutant alleles at this codon (Table 3a) Only eight and four women had quadruple and triple genotypes, respectively.

Discussion
Malaria prevalence of 18% found in this study was higher than 10% and 13% observed among pregnant women in another Kenyan coastal region (16) and the lake region in Tanzania (17), respectively, but significantly lower than 31% documented in the Kenyan lake regions (16). Therefore, the high burden of malaria in pregnancy remains of public health concern. As observed in this study and elsewhere (18-20), younger women in their first pregnancy are at greatest risk of infection and should be targeted with preventive and early treatment interventions.
IPTp-SP is an important prophylactic therapy recommended for prevention of malaria in high endemic African regions to reduce morbidity/mortality among pregnant women and adverse pregnancy outcomes (2).The emerging high Pf resistance to the SP is likely to render IPTp-SP prophylactic intervention ineffective. Similar to our findings, other studies have reported high prevalence (78%-97%) of quintuple PfDHFR/PfDHPS haplotype mutations in western Kenya (10,21). Studies elsewhere have documented quadruple mutations (65%) in Equatorial Guinea (22) and 48% in DRC (23), and triple mutations (92%) in Gabon (24) and 61-71% in Burkina Faso (25) as the most prevalent. Similar to India, where double mutation was the most prevalent (26,27), a study in Brazil reported double mutation as most prevalent but did not find quintuple or quadruple mutations (28).
Findings from this study demonstrated very high prevalence of SNPs at the two important genes that confer SP resistance. The high prevalence of 89% observed at PfDHFR51I in this study was similarly to (85%-100%) documented in western Kenya and elsewhere (10,(21)(22)(23)(24)29), but contrary to the 21% reported in India (26).
PfDHFR polymorphism at 51I, 59 R and 108N combined mutations of 89% observed in this study was similar to 89-97% documented in western Kenya among pregnant women (10,21). Proportion comparable to these have been reported elsewhere: 97% in Uganda (8); 87% in Equatorial Guinea (22); 98% in Cameroon (29), and 93% in Senegal (30). These frequencies were higher compared to 48% detected in DRC (23) and 54 -74% in Burkina Faso (25,31). The high frequency (94%) of PfDHPS gene double mutation (437G and 540E) reported in this study concurs with findings from other studies in East Africa that reported 90-99% and 99% in western Kenya (10,21) and Uganda (8) respectively. However, double mutation in this gene are reportedly rare occurring with a prevalence of 4% in Equatorial Guinea (22), 1% in Burkina Faso (25),and 2% in DRC (23). An average mutation prevalence of 71.3% was seen at codon 540E in the coastal regions of Tanzania which was low when compared to the country prevalence of 92.4% (9).
Several studies have demonstrated that the differing degrees of antimalarial drug resistance are dependent upon the number and combination of mutations present (32). The PfDHFR/PfDHPS quintuple mutant, in either mixed or pure form, is the most clinically relevant molecular marker for SP resistance. In the East African region, the prevalence of molecular markers of SP resistance has been increasing since the emergence of the first resistance-conferring mutations in the 1950s (9). Therefore, continuous molecular surveillance will allow early detection of drug resistance susceptibility and high mutation prevalence within the PfDHFR and PfDHPS gene of the Pf parasite that reduce SP drug effectiveness as a prophylactic treatment for malaria in pregnancy (25). These finding show that there is high prevalence of PfDHFR/PfDHPS haplotype mutations believed to confer resistance to SP, the drug of choice for malaria prophylactic treatment in pregnant women. Our data aligned with findings in other parts of Kenya and other tropical regions in defining high prevalence of SP resistance markers within circulating Pf isolates.

Conclusion
Results from this study suggest that coastal Kenya has high prevalence of PfDHFR triple mutation and PfDHPS double mutation that could potentially undermine the efficacy of SP drug for prophylactic treatment among pregnant women. Despite growing evidence of high prevalence of genetic mutation within the PfDHFR and PfDHPS genes associated with SP drug resistance, it is still the recommended drug for IPTp in Kenya and sub-Saharan Africa regions which carry a disproportionately high burden of malaria (2). There is therefore urgent need for development of safe

AVAILABILITY OF DATA AND MATERIALS
The dataset analyzed for this study is available from the corresponding author on reasonable request.

COMPETING INTERESTS
Authors declare no conflict of interest.