A snapshot of Plasmodium falciparum malaria drug resistance markers in Sudan: a pilot study

Objectives Malaria infection is still known to be a worldwide public health problem, especially in tropical and sub-tropical African countries like Sudan. A pilot study conducted to describe the trend of P. falciparum drug resistance markers in 2017–2018 in comparison to CQ and AS/SP eras in Sudan. The Pfcrt, Pfmdr-1, Pfdhfr, and Pfdhps genes were investigated. Data deposited by the worldwide antimalarial resistance network was consulted, and the molecular markers previously reported from Sudan were analyzed. Results Drug molecular markers analysis was successfully done on 20 P. falciparum isolates. The Pfcrt K76 showed high frequency; 16 (80%). For the Pfmdr-1, 9 (45%) isolates were carrying the N86 allele, and 11 (55%) were 86Y allele. While the Y184F of the Pfmdr-1 showed a higher frequency of 184F compared to Y184; 16 (80%) and 4 (20%), respectively. In the Pfdhfr, 51I allele showed higher frequency compared to N51; 18 (90%) and 2 (10%), respectively. For S108N, 18 (90%) were 108 N and 2 (10%) were S108. In the Pfdhps, all isolates were carrying the mutant alleles; 437G and 540E. The frequency distribution of the Pfcrt, Pfmdr-1, Pfdhfr, Pfdhps was significantly different across the whole years in Sudan.


Introduction
Malaria infection is still known to be a worldwide public health problem, especially in tropical and sub-tropical African countries [1]. In Sudan, in the late 70s of the last century, a high proportion of drug resistance was reported when Chloroquine (CQ) was introduced as the first-line treatment for falciparum malaria [2]. By 2004 the malaria treatment protocol was shifted to the artemisinin-based combination treatment (ACTs); Artesunate (AS) and Sulfadoxine-Pyrimethamine (SP) as a first-line treatment against uncomplicated falciparum malaria, and artemether-lumefantrine (AL) as a secondline treatment [3]. In 2017, the malaria treatment protocol was shifted to AL for the treatment of uncomplicated falciparum malaria, and Quinine for treating the severe infections [4]. Subsequently, therapeutic efficacy studies were conducted to monitor and detect the emergence of drug-resistant malaria parasites [5][6][7][8][9]. However, another approach for the early detection of drug resistance emergence was implemented by using molecular markers to investigate the efficacy of treatments in-vitro.
P. falciparum chloroquine resistance transporter (Pfcrt) and P. falciparum multidrug resistance gene 1 (Pfmdr-1) are previously known membrane transporters associated with resistance to the drug combination of CQ and AQ or MQ and Lumefantrine (L) [10,11]. The CVIET haplotype of Pfcrt is known as the most robust CQ resistance marker in Africa [12][13][14][15]. In vitro experiments showed that N86Y and Y184F mutations in the Pfmdr-1 gene increases the inhibitory concentrations of CQ and AQ [10,12], and reduce susceptibility to MQ and L [12,13].
The need for updated molecular markers studies to investigate the frequency of falciparum malaria drugresistant is extreme. This pilot study aims to describe the trend of P. falciparum drug resistance markers in 2017-2018 in comparison to CQ and AS/SP eras in Sudan.

Materials and methods
This pilot study was conducted in Khartoum state between December 2017 and July 2018. Febrile patients (axillary temperature < 37 °C) who were diagnosed microscopically by examining Giemsa stained blood films as falciparum malaria infection were recruited. Informed consent was taken from the patients before sample collection. Participants diagnosed with P. falciparum/P. vivax co-infection and P. vivax mono-infections were excluded.

Sample collection and DNA extraction
Two ml blood samples were collected before starting the treatment and preserved into lithium heparin blood containers for DNA extraction using the Guanidine Chloride extraction method as described previously [15]. DNA was stored in − 20 °C until molecular examinations later.

Parasite genotyping and drug resistance markers assessment
The microscopic diagnosis was confirmed using the primers described previously [16]. Genotyping of the specific point mutations in the P. falciparum genome was done using the Sanger sequencing method by using the primers sets for Pfcrt, Pfmdr-1, Pfdhfr, and Pfdhps genes as described previously [17]. PCR amplicons were sequenced in both directions using the forward and reverse primers for each gene to exclude any basecalling errors that could be obtained during sequencing. Sequences were validated using GENtle software (v1.9.4) and aligned in comparison with the wildtype P. falciparum 3D7 strain reference sequences (PF3D7_0709000 for Pfcrt, PF3D7_0523000 for Pfmdr-1, PF3D7_1324800 for Pfdhfr, and PF3D7_0810800 for Pfdhps). The deduced amino acids were translated from nucleotide sequences using MEGA7 software (v7.0.26) to determine sequences mutations at the Pfcrt codon 76; Pfmdr-1 codons 86 and 184; Pfdhfr codons 51, 59, and 108; and the substitutions at the Pfdhps in codons 437 and 540. The nucleotide sequences used in this study have been deposited in the NCBI GenBank database (https :// www.ncbi.nlm.nih.gov/) under the accession numbers MT995200-MT995259.

Previous reports on drug resistance markers in Sudan
Data deposited by the worldwide antimalarial resistance network (WWARN) (https ://www.wwarn .org/) was consulted, and the molecular markers previously reported from Sudan were identified, collected, and analyzed to compare between past and present frequency of malaria drug resistance mutations. Data sets included SP molecular surveyors (https ://www.wwarn .org/sp-molec ularsurve yor) and ACT partner drug molecular surveyors (https ://www.wwarn .org/track ing-resis tance /act-partn er-drug-molec ular-surve yor). Numbers and drug molecular marker genotypes of P. falciparum isolates included in the historical literature review data set analyzed in this study are presented in Additional file 1.

Statistical analysis
The statistical analysis was done using the statistical Package for Social Sciences (SPSS, v20.0). One-way ANOVA test was used to calculate the least significance difference of frequency distribution in the molecular markers. Pearson correlation was used to investigate the association between the different drug resistance markers. P value ≤ 0.05 was considered statistically significant.

Molecular genotyping results
In this pilot study, a total of 28 malaria parasite isolates were genotyped, of them, 2 and 6 isolates were excluded since were P. falciparum/P. vivax coinfections and P. vivax infections, respectively. The remaining 20 isolates were confirmed by PCR as P. falciparum mono-infections.
The frequency distribution of the Pfcrt and Pfmdr-1, Pfdhfr, and Pfdhps mutations was significantly different across the whole years in Sudan. An illustrated statistical significance and insignificance of the frequency distribution of P. falciparum multidrug resistance markers between the different years' intervals are described in Additional file 2 Tables S1-S5.

Discussion
The reported frequency of Pfcrt K76 allele in this study was significantly high compared to previous years, especially during the CQ deployment years in Sudan [9]. During the AS/SP era; since 2004, Pfcrt K76 allele had increased. This increase can be suggested due to limited or ambient exposure of P. falciparum parasite to CQ or might be due to an increase in AL pressure. Similar results were reported from Malawi, and Tanzania supporting the increase of Pfcrt K76 allele [18][19][20][21]. While, in Zambia, complete disappearance of Pfcrt 76 T allele from the examined isolates was reported [22].
The Pfdhfr IN haplotype and Pfdhps GE haplotype significantly increased in 2017-2018 where both haplotypes were showing high frequency; 90% and 100%, respectively. This increase could be due to the enduring pressure of AS/SP since 2007 [23]. This phenomenon was observed in all countries where AS/SP was used to treat uncomplicated malaria such as Nigeria and Equatorial Guinea; where the prevalence of mutant haplotypes reached more than 90% [24][25][26].
The prevalence of Pfmdr-1 NF haplotype in 2017-2018 is noted among 35% of the study samples. Comparison between previous years for the frequency distribution of Pfmdr-1 double haplotypes showed a statistically significant difference between all years and 2008 when at that time the recommended malaria treatment was AS/ SP, and since 2009 the use of AL is significantly increased due to malpractice in drug use, such as usage of incorrect dosage and insufficient information stated to patients about the prescribed treatment which may lead to the increase in resistance and recurrent infections rates [27]. Previously, the development of molecular markers for AL resistance was thought to be difficult because there were no known resistant lab lines can be used as resistance controls. Meanwhile, investigating the Pfmdr-1 NFSND haplotype could be the role evolution for the developed mutations because it allows a longer survival rate of the parasite [11,28].
In the present study, Pfmdr-1 86Y allele, Pfdhfr IN haplotype, and Pfdhps GE haplotype were constituting the majority of the studied samples; 90%, which is also similar to a previous study conducted in Sudan; where all the investigated parasite isolates were carrying the Pfmdr-1 86Y allele, Pfdhfr IN haplotype, and Pfdhps 540E [23].
The prevalence of Pfdhps double haplotype GE detected in this study could hinder the effect of SP if used as intermittent preventive therapy during pregnancy (IPTp). Although, SP as IPTp was not implemented and there is no information about the use of SP during pregnancy in Sudan [29]. The presence of Pfdhfr IRN in combination with Pfdhps GE haplotypes forming the quintuple mutant haplotype confers a high risk for treatment failure in malaria-infected children and nonpregnant adults who receive SP as a seasonal malaria chemoprevention treatment (SMC-SP) [30]. However, previous studies indicated that IPTp-SP is still efficacious in areas with a high prevalence of resistant P. falciparum parasite [31]. Nevertheless, the increased resistance rate might compromise the implication of IPTp-SP [32][33][34].

Conclusion
This study describes the distribution of P. falciparum multidrug resistance markers throughout Sudan. The study provides a baseline data of the status of these markers which could be very useful for the malaria control program for establishing surveillance system to monitor the emergence of malaria drug resistance for more effective treatment protocol and successful control of the disease.

Limitations
• The lack of clinical and background information, particularly, the previously used drug before the blood sampling substantially can affect the prevalence of the alleles. Therefore, complete clinical history information is needed and can be very useful in future studies.
Additional file 1: Table S1. Numbers and drug molecular markers genotypes of P. falciparum isolates from Sudan 1989 -2018.
Additional file 2: Table S1. Statistical significance of Pfcrt K76T frequency distribution across the different previous studies conducted in Sudan. Table S2. Statistical significance of Pfmdr-1 N86Y frequency distribution across the different previous studies conducted in Sudan. Table S3. Statistical significance of Pfdhfr N51I and S108N double haplotype frequency distribution across the different previous studies conducted in Sudan.