Occurrence of septuple and elevated Pfdhfr-Pfdhps quintuple mutations in a general population threatens the use of sulfadoxine-pyrimethamine for malaria prevention during pregnancy in eastern-coast of Tanzania

Background Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) mutations compromise the effectiveness of sulfadoxine-pyrimethamine (SP) for treatment of uncomplicated malaria, and are likely to impair the efficiency of intermittent preventive treatment during pregnancy (IPTp). This study was conducted to determine the level of Pfdhfr-Pfdhps mutations, a decade since SP was limited for IPTp use in pregnant women in Tanzania. Methods P. falciparum genomic DNA was extracted from dried blood spots prepared from a finger prick. Extracted DNA were sequenced using a single MiSeq lane by combining all PCR products. Genotyping of Pfdhfr and Pfdhps mutations were done using bcftools whereas custom scripts were used to filter and translate genotypes into SP resistance haplotypes. Results The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in 6 (1.3%) samples. Triple PfdhfrIRNI (mutations are bolded and underlined) haplotype was dominant, contributing to 84% (number [n] = 374) of haplotypes while 446 samples were studied for Pfdhps, WT for Pfdhps (SAKAA) was found in 6.7% (n = 30) in samples. Double Pfdhps haplotype (SGEAA) accounted for 83% of all mutations at Pfdhps gene. Of 447 Pfdhfr-Pfdhps combined genotypes, only 0.9% (n = 4) samples contained WT gene (SAKAA-NCSI). Quintuple (five) mutations, SGEAA-IRNI accounted for 71.4% (n = 319) whereas 0.2% (n = 1) had septuple (seven) mutations (AGKGS-IRNI). The overall prevalence of Pfdhfr K540E was 90.4% (n = 396) while Pfdhps A581G was 1.1% (n = 5). Conclusions This study found high prevalence of Pfdhfr–Pfdhps quintuple and presence of septuple mutations. Mutations at Pfdhfr K540E and Pfdhps A581G, major predictors for IPTp-SP failure were within the recommended WHO range. Abandonment of IPTp-SP is recommended in settings where the Pfdhfr K540E prevalence is > 95% and Pfdhps A581G is > 10% as SP is likely to be not effective. Nonetheless, saturation in Pfdhfr and Pfdhps haplotypes is alarming, a search for alternative antimalarial drug for IPTp in the study area is recommended.


Background
Antimalarial drugs use and vector control are the commendable tools for global malaria prevention and control respectively [1]. However, resistance of malaria parasites to antimalarial drugs and resistance of Anopheles mosquitoes to insecticides impair the progressive fight against malaria [2]. Resistance of Plasmodium 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], for preventing pregnancy associated malaria and improve pregnancy outcomes [13].
The elevated Pfdhfr/Pfdhps protein mutations and saturation of genetic haplotypes such as quintuple (CIRNI-SGEAA) and sextuple (CIRNI-SGEGA) reported in Tanzania [10] pose risks against the effectiveness of SP in preventing placental malaria during pregnancy. In this regard, World Health Organization (WHO) recommends abandonment of IPTp-SP in settings where the Pfdhfr K540E prevalence is > 95% and Pfdhps A581G is > 10% because SP is more likely to be less effective [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 a general population to determine the status of mutations associated with SP resistance, more than one decade since SP was limited for IPTp use in pregnant women in Tanzania.

Study design
A hospital based surveillance of molecular markers for SP resistance was conducted for 4 months from April, 2019 at Kibiti Health Center (KHC) found in Eastern part of Tanzania. 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. Tanzania is found in sub-Saharan region where malaria is endemic, adopted the use of SP for malaria prevention in pregnancy women for more than one decade [12].

Study area
KHC is found in Kibiti District, Pwani region. Pwani region is one of the five regions found along the coastal of Indian Ocean. Pwani region is bordered to north by Tanga region, to the east by Dar es Salaam region and the Indian Ocean, to the south by the Lindi region, and to the west by the Morogoro region. The coastal region experiences malaria transmission throughout the year with regional prevalence ranging between 3.1 and 14.8% [16].

Study population
Patients attending clinic at KHC who presented with malaria infection related symptoms qualified for inclusion in the study. The suggestive symptoms example fever, general body weakness and headache were confirmed by the attending clinician [17]. Rapid malaria testing was performed using CareStart™ malaria HRP2/ pLDH (Pf/pan) Combo test (Access Bio, Ethiopia). Samples tested positive by rapid tests qualified for blood smear (BS) microscopy for confirmation. A total of 489 positive BS microscopy qualified for further dried blood spot preparation.

DBS preparation and DNA extraction
Dried Blood Spots (DBS) were prepared as per Malaria-GEN SpotMalaria, DBS collection protocol [18]. Briefly, a well sterilized middle patient's finger was pricked, and 4 blood spots from each patient were dropped on a separate circle of the filter paper; two on each paper card. The spotted blood were dried and put in the desiccant sachet for storage. DNA from the DBS was extracted following QIAamp DNA Investigator Kit for isolation of total DNA from filter papers (Qiagen, Valencia, CA, USA) and as described by Oyola et al.,2016 [19].

Genotyping of Pfdhfr and Pfdhps mutations
Molecular genotyping of Pfdhfr and Pfdhps gene mutations associated with SP-resistance was performed by Wellcome Sanger Institute, UK as previously described but customized for Pfdhfr and Pfdhps mutations [20].
To summarize, targeted genotypes were spotted and multiplex PCR primers were designed using mPrimer software [21] and were run according to Wellcome Sanger Institute protocol (Goncalves, manuscript in preparation; https://www.malariagen.net/projects/spotmalaria). Primers were made to amplify products between 190 and 250 bp and combined into 3 pools. A multi steps protocol was used to first amplify the target regions of the P. falciparum genome, then PCR to combine sequencing and multiplexing adapters. About 384 samples were sequenced in one MiSeq lane by combining all PCR products. Samples were de-plexed using the multiplexing adapters and individual CRAM files were aligned to a modified amplicon reference genome for further analysis.

Statistical analysis
Statistical Package for Social Sciences version 25 (SPSS Software, Chicago Inc., USA) was used to import laboratory data from the Microsoft Excel sheet (Redmond, WA) for data analysis and interpretation. Genotyping was done using bcftools and custom scripts to filter and translate genotypes into Pfdhfr and Pfdhps resistance haplotypes. Haplotypes were expressed as frequencies and percentages.

Haplotypes for Pfdhfr and Pfdhps
The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in only 1.3% (number [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 ( Table 1). The overall prevalence of K540E was 90.4% (n = 396) while A581G was 1.1% (n = 5).

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.
In this study, triple Pfdhfr IRNI haplotype mutation was dominant, contributing to 84% of all Pfdhfr haplotypes whereas double Pfdhps haplotype (SGEAA) accounted for 83% of mutations of the Pfdhps haplotypes. Compared to study by Baraka et al.,2015 [10], Pfdhfr IRNI haplotype were predominant in all sites with significantly higher frequencies at Muheza (93.3%) compared to Muleba (75.0%) and Nachingwea districts (70.6%). In this regard, there was 13.4% increase in Pfdhfr IRNI haplotype prevalence when Nanchingwea (70.6%) and Kibiti district (84%) both found along the coastal region were compared. Additionally, the previous study found that double Pfdhps haplotype SGEAA was significantly high at Muheza (27.2%) and Muleba (20.8%) while none (0%) was detected at Nachingwea while the current study at Kibiti district detected 83% Pfdhps SGEAA mutations. Meaning that, from 2015 to 2019 there is an increase of 0 to 83% Pfdhps SGEAA mutation in the coastal region, respectively. WHO malaria report of 2019 has clearly mentioned pregnant women as the most affected group and use of effective IPTp should be highly implemented to combat risks associated with pregnancy malaria [26]. On top of that, a recent study by Mikomangwa et al., [27] concluded that pregnant woman who was malaria positive had 11 times more risk of giving birth to low birth weight (LBW) child when compared to those who tested malaria negative.
Nevertheless, Pfdhfr K540E and Pfdhps A581G, major predictors for IPTp-SP failure, had the prevalence of 90.4% for Pfdhfr K540E and 1.1% for Pfdhps A581G. This guarantee the continued use of IPTp-SP in the study area. WHO recommends stoppage of SP for IPTp if Pfdhfr K540E prevalence > 95% and Pfdhps A581G > 10% [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) for IPTp in the study areas should be prioritized. The current study was conducted in the general population, hence limited data on clinical, birth outcomes and malaria infection status in pregnant women in relation to SP resistance genotypes.

Conclusions
This study found high prevalence of Pfdhfr-Pfdhps quintuple mutations. Mutations at Pfdhfr K540E and Pfdhps A581G, major predictors for IPTp-SP failure were within the recommended WHO range. Abandonment of IPTp-SP is recommended in areas where the Pfdhfr K540E prevalence is > 95% and Pfdhps A581G is > 10% because SP is likely to be not effective. Saturation in Pfdhfr and Pfdhps haplotypes is alarming, a search for alternative antimalarial drug for IPTp, the study area in particular is recommended.