Saturation of Pfdhfr and Pfdhps haplotypes in general population increasingly threatens the benefits of using sulfadoxine‑pyrimethamine for intermittent preventive treatment in pregnant women in Tanzania

Background : High levels of Plasmodium falciparum resistance prompted withdrawal of sulfadoxine-pyrimethamine (SP) as the first-line treatment for uncomplicated malaria in Tanzania. However, SP was limited for intermittent preventive treatment during pregnancy (IPTp) especially where there is moderate to high malaria transmission. This study reports the patterns of P. falciparum dihydrofolate reductase ( Pfdhfr ) and dihydropteroate synthetase ( Pfdhps ) mutations. Methods: Parasite genomic DNA was extracted from dried blood spots prepared by finger prick. Batched samples (384) were sequenced in a single MiSeq lane combining all PCR products. Samples were de-plexed using the multiplexing adapters and individual CRAM files were aligned to a modified amplicon reference genome. Genotyping of Pfdhfr and Pfdhps mutations were done using bcftools as well as custom scripts to filter and translate genotypes into drug resistance haplotypes. Results: The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in only six (1.3%) samples. Triple Pfdhfr IRN I haplotype was dominant, contributing to 84% (n=374) of haplotypes. The total of 446 samples were studied for Pfdhps . WT for Pfdhps was found in 6.7% (n=30) of all samples detected. Double Pfdhps haplotype (S GE AA) accounted for 83% of mutations of the Pfdhps gene. The overall prevalence of K540E was 90.4% (n=396) while A581G was 1.1% (n=5). Additionally, 91.4% (n=447) genotypes where detected from 489 sequenced samples. Of 447 genotypes detected only 0.9% (n=4) of samples were WT (SAKAA-NCSI). Quintuple mutation, S GE AA- IRN I accounted 71.4% of concomitant Pfdhfr/Pfdhps

mutation, AG K GS -IRN I.
Conclusions : Despite the high prevalence of mutations in Pfdhfr and Pfdhps gene but the current mutations at Pfdhfr K540E and Pfdhps A581G are within the recommended WHO range, stopping IPTp-SP is recommended in areas where the Pfdhfr K540E prevalence is >95 % and Pfdhps A581G is >10 % as SP is likely to be ineffective). Nevertheless, saturation in Pfdhfr and Pfdhps haplotypes is alarming, therefore screening for alternative antimalarial drug for IPTp-SP is recommended.

Background
Antimalarial drugs use and vector control are the commendable tools for global malaria prevention and control respectively (1). However, resistance of Plasmodium to antimalarial drugs and resistance of Anopheles mosquitoes to insecticides impair the progressive fight against malaria (2). Resistance of P. falciparum to previous generations of medicines i.e. chloroquine (CQ) (3) and sulfadoxine-pyrimethamine (SP) (4) influenced the replacement of CQ with SP (5), then artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria in Tanzania (6).
Mutations in both the Pfdhfr/Pfdhps genes which greatly influence SP clinical treatment failures (11), resulted SP being unfit for first -line treatment of uncomplicated malaria in Tanzania (12). However, in 2006 the drug was limited for intermittent preventive treatment during pregnancy (IPTp-SP) in areas with moderate to high malaria transmission (13), pregnant women regardless of the presence or absence of malaria should administer at least three curative doses of SP with an interval of at least 1 month between the two doses, starting in the second trimester of pregnancy (14) to prevent pregnancy associated malaria and improve pregnancy outcomes (13).
Unfortunately, the rapid widespread of Pfdhfr/Pfdhps genes mutations and saturation of haplotypes including quintuple (CIRNI-SGEAA) and sextuple (CIRNI-SGEGA) reported in Tanzania (10) are likely to compromise effectiveness of IPTp-SP strategy. In this regard, World Health Organization (WHO) recommends stopping IPTp-SP in areas where the Pfdhfr K540E prevalence is > 95% and Pfdhps A581G is > 10% as SP is likely to be ineffective (15).
For the purpose of guiding IPTp-SP policy and inform on the current SP resistance status, routinely surveillance of SP effectiveness using molecular markers should be implemented as recommended by WHO (15). Therefore, a study was conducted in general population, more than a decade since SP was limited for IPTp use in pregnant women in Tanzania.

Methods
Study design, area, period and population 5 A hospital based surveillance of molecular markers for SP resistance was conducted between April and August 2019 at Kibiti Health Center (KHC). The study was conducted to determine the current antimalarial status of SP, more than a decade since SP were limited for IPTp for pregnant women in Tanzania. The coastal region experiences malaria transmission throughout the year with regional prevalence ranging between 3.1 and 14.8% (16). Patients attending clinic at KHC who presented with symptoms suggestive of malaria infection were recruited in the study. The symptoms such as fever, general body weakness and headache were confirmed by the attending physician (17). Malaria screening in patients were done using CareStart™ malaria HRP2/pLDH (Pf/pan) Combo test (Access Bio, Ethiopia). Then positive samples by rapid tests were subjected to blood smear (BS) microscopy for confirmation. A total of 489 dried blood samples (DBS) from patients tested positive with BS microscopy were prepared. DBS preparation and DNA extraction DBS were prepared according to MalariaGEN SpotMalaria, DBS collection protocol (18). A sterilized patient's finger was pricked, and four blood spots from each patient were prepared; two on each paper card. The blood spots were allowed to dry and placed in the desiccant sachet for storage. DNA from the DBS was extracted using QIAamp DNA Investigator Kit for isolation of total DNA from filter papers (Qiagen, Valencia, California, United States) and as described elsewhere (19).

Genotyping of Pfdhfr and Pfdhps mutations
Molecular genotyping of Pfdhfr and Pfdhps gene mutations associated with SPresistance was performed by Wellcome Sanger Institute, UK.
Briefly, targets for genotyping were identified and multiplex PCR primers were designed using a modified version of the mPrimer software (20). Primers were 6 designed to amplify products between 190-250 bp and combined into 3 pools. A two-step protocol was used to first amplify the target regions of the parasite genome, followed by a second PCR to incorporate sequencing and multiplexing adapters. Batched samples (384) were sequenced in a single MiSeq lane combining all PCR products. Samples were de-plexed using the multiplexing adapters and individual CRAM files were aligned to a modified amplicon reference genome.
Genotyping was done using bcftools as well as custom scripts to filter and translate genotypes into drug resistance haplotypes.

Haplotypes for Pfdhfr and Pfdhps mutations
The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in only 1.3% (n=6) samples. Triple Pfdhfr IRNI haplotype was dominant, contributing to 84% (n=374) of haplotypes. The total of 446 samples were studied for Pfdhps. The WT for Pfdhps was found in 6.7% (n=30) of all samples detected. The most common mutation was the change of amino acid alanine to glycine (A437G) and lysine to glutamic acid (K540E). Double Pfdhps haplotype (SGEAA) accounted for 83% of mutations of the Pfdhps gene. The overall prevalence of K540E was 90.4% (396) while A581G was 1.1% (5) ( Table 1).

Discussion
This study was conducted to determine the current SP resistance status, more than a decade since SP was limited for IPTp use in pregnant women in Tanzania (21). Moreover, high prevalence of the Pfdhfr/Pfdhps sextuple haplotype was associated with reduced birth-weight (22)(23)(24).  (15). However, rapid increase and saturation in Pfdhfr and Pfdhps mutations with spread of sextuples and septuple is of great concern. The effort to search for alternative drug (s) to replace SP for IPTp should be increased.

Competing interests
The authors declare that they have no competing interests.

Availability of data
The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Consent for publication
Not applicable

Funding
This study was funded by Swedish International Development Cooperation Agency