Historical literature review and molecular analysis of malaria drug resistance markers of Plasmodium falciparum field-isolates from Sudan.

Background Malaria infection is still known to be a worldwide public health problem, especially in tropical and sub-tropical African countries like Sudan. The fight against malaria is still taking place due to many factors. One of these factors is the presence of Plasmodium falciparum drug resistant parasites. This study is aiming at studying the P. falciparum drug resistance markers and analyzing the historical literature on these markers in Sudan. Methods A descriptive cross-sectional healthcare-centers based study conducted in Khartoum state between December 2017 and July 2018. Febrile patients diagnosed with P. falciparum malaria infection were recruited. Two ml blood samples were collected prior to start treatment. Genotyping of the specific point mutations in the P. falciparum genome was done using Sanger sequencing method for the Pfcrt, Pfmdr-1, Pfdhfr, and Pfdhps genes. Data deposited by the worldwide antimalarial resistance network 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. One-way ANOVA test was used to calculate the least significance of frequency distribution in the molecular markers collected from the previous reports from Sudan in comparison to this study. Pearson correlation was used to investigate the association between the different drug resistance markers. Results Drug molecular markers analysis was successfully done on the 20 P. falciparum isolates. the Pfcrt K76 showed the highest frequency; 16 (80%). Pfcrt 76T was 4 (20%). For the Pfmdr-1 marker, 9 (45%) isolates were carrying the N86 allele and 11 (55%) were 86Y allele. While the Y184F of the Pfmdr-1 showed higher frequency of 184F compared to Y184; 16 (80%) and 4 (20%), respectively. Concerning the double Pfmdr-1 haplotype, NY haplotype was 2 (10%), NF was 7 (35%), YF was 9 (45%), and YY was 2 (10%). In the Pfdhfr , 51I allele showed higher frequency compared to N51; 18 (90%) and 2 (10%),

showing increased prevalence each year. All studied genes were showing increase prevalence of the mutant alleles and reduction of the wildtype alleles. In this study, the GE mutant haplotype was prevalent in all the studied samples. Frequency distribution of the Pfcrt K76T and Pfmdr-1 N86Y alleles, Pfmdr-1 ; N86Y and Y184F, Pfdhfr ; N51I and S108N, and Pfdhps ; A437G and K540E double haplotypes was significantly different across the whole years in Sudan. Conclusion This study describes the distribution of P. falciparum multidrug resistance markers throughout Sudan providing a solid baseline data of the status of these markers which could be very useful for the malaria control program not only for establishing surveillance system that monitor the change in and/or the emergence of malaria drug resistance but it will also offer a guidance for the evidence-base decisionmaking regarding the treatment protocol national and regional wise.

Background
Malaria infection is still known to be a worldwide public health problem, especially in tropical and subtropical African countries [1]. Based on the WHO estimations in 2019, malaria infections reached 228 million cases with 405 thousand related-deaths [1]. In Sudan, anti-malarial resistance among Plasmodium falciparum had emerged since 1955 investigated by Abbot, who firstly reported the phenomenon of drug-resistant in Wad Madani when using Amodiaquine-hydrochloride (AQ) and treatment (ACTs). The combination of Artesunate (AS) and Sulfadoxine-Pyrimethamine (SP) was then adopted as the first-line treatment against uncomplicated falciparum malaria, and artemetherlumefantrine (AL) was considered as a second-line treatment [11]. Therapeutic Efficacy Studies (TES) were conducted to monitor and detect the emergence of drug-resistant malaria parasites. Another approach for the early detection of the emergence of drug resistance was implemented by using molecular markers in order to investigate the efficacy of treatments in vitro. Once

Methods
A descriptive cross-sectional healthcare-centers based study conducted in Khartoum, Sennar, River Nile, Ad Damazin and Gedaref states between December 2017 and July 2018. Febrile patients (axillary temperature < 37 C) who were microscopically diagnosed with P. falciparum malaria infection were recruited to participate in the study after obtaining informed consent to collect the blood sample.
Participants diagnosed with P. falciparum/ P. vivax co-infection and P. vivax mono-infections were excluded from the study. Two ml blood samples were collected prior to start treatment. No follow up data were obtained from patients after treatment. Blood samples were preserved into lithium heparin anticoagulation blood containers and shipped to the biotechnology lab, faculty of pharmacy at The Ahfad University for women for DNA extraction and molecular genotyping of drug resistance markers.

DNA extraction
The genomic DNA was extracted using the Guanidine Chloride extraction method described previously by Ciulla et al. (1988), with minor modifications [27]. In brief, 1 ml from each blood sample was placed into 2.5 ml tube and washed using RBCs washing buffer; and then nucleated and parasite cells were lysed using cell lysis buffer. 1 ml of 6 M Guanidine chloride solution and 10 micro of proteinase K enzyme were added and incubated over night at 37 o C. The following day, 2 ml Chloroform were added to the mixture and centrifuged at 14000 rpm for 10 min; 3 layers separation were formed. The upper layer containing the parasite and human DNA was transferred into new 2 ml tubes; and 1 ml absolute ethanol was added to precipitate the DNA. Precipitated DNA was then washed with 70% ethanol and allowed to dry for 1 hour before being dissolved with 200 micro distilled water. Purity and concentration of the extracted DNA was measured using Nanodrop 1000 spectrophotometer (USA).
DNA was stored in -20 o C until molecular examinations later.

Parasite genotyping and assessment of multiple infection
To confirm the microscopic diagnosis, the protocol and primers sets described previously by Snounou et al. (1993) were used [28]. Whereas, for assessment of multiple infection, primers and PCR conditions used were according to the previously described protocol [29].

Drug resistance markers assessment
Genotyping of the specific point mutations in the P. falciparum genome was done using Sanger sequencing method using the primers sets for the Pfcrt, Pfmdr-1, Pfdhfr, and Pfdhps genes as described previously [30]. Primers sets used in this study were described in table 1. PCR amplification conditions were set to 95 o C for 5 min as initial denaturation followed by 35 cycles of 95 o C for 1 min, 55 o C for 1 min (for Pfcrt, Pfmdr-1) or 58 o C for 1 min (for Pfdhfr and Pfdhps), and 72 o C for 1 min, followed by final extension step at 72 o C for 10 min. All thermal conditions were performed in MJ thermocycler PCR machine (USA). PCR amplicons were then sequenced in both directions using the forward and reverse primer for each gene to exclude any base calling 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

Previous reports on drug resistance markers
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-molecular-surveyor), and ACT partner drug molecular surveyors (https://www.wwarn.org/tracking-resistance/act-partner-drugmolecular-surveyor). 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 Supplementary file 1.

Statistical analysis
The statistical analysis was done using the statistical Package for Social Sciences (SPSS, v20.0). Oneway ANOVA test was used to calculate the least significance difference (LSD) of frequency distribution in the molecular markers collected from the previous reports from Sudan in comparison to this study.
Pearson correlation was used to investigate the association between the different drug resistance markers. P value ≤ 0.05 was considered significant.

The molecular genotyping of the field-isolated P. falciparum and multiclonal infections assessment
In this small-scale 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, P. vivax infection, respectively.
Concerning the Pfdhps drug molecular marker, all the 20 (100%) isolates were carrying the mutant alleles; 437G and 540E. the Pfdhps haplotype present was the double GE haplotype only ( Table 2).
No statistically significant correlation was found for the Pfcrt, Pfmdr-1, Pfdhfr, and Pfdhps. However, a statistically significantly positive correlation was observed for Pfmdr-1 and the combined Pfdhfr and  (Fig. 1).

The trend in P. falciparum multidrug resistance from 1989 to 2018 in Sudan
For the Pfcrt, the T allele that confers CQ resistant was at higher frequency during 2000-2001 (89.6%), however, T mutant allele frequency started to dropdown reaching up to 43.9% in 2016 and bottomed at 20% in 2018. In this study, the frequency of the K76 wildtype allele was higher compared to all previous years; 80%. Whereas, the N86Y mutation of the Pfmdr-1 was extremely flocculation during the past years (Fig. 2).
The prevalence of the Pfmdr-1 double haplotype N86Y and Y184F was only obtained from 4 previous studies. Results of Pfmdr-1 double haplotype in comparison with this study showed an increase in the NY mutant haplotype in 2017-2018 compared to 2016; 45% and 31.5%, respectively. While noted a quite reduction of the NY and YY wildtype haplotypes throughout all years (Fig. 4).
Frequency distribution of the Pfcrt K76T and Pfmdr-1 N86Y alleles, Pfmdr-1; N86Y and Y184F, Pfdhfr; N51I and S108N, and Pfdhps; A437G and K540E double haplotypes was significantly different across the whole years in Sudan. an illustrated statistical significance and insignificance of frequency distribution of P. falciparum multidrug resistance markers between the different years intervals is described in supplementary file 2; (tables S1-S5).

Discussion
Studying the molecular markers to monitor the prevalence of drug resistance is extremely beneficial especially for public health programs that aim to reduce the prevalence of malaria infections because drug-resistant parasites are the major threat to achieve success malaria control/elimination. In this study we aimed to investigate the fluctuation in the P. falciparum multidrug resistance markers in Sudan during CQ, AS/SP and ACT eras. In the current small-scale study, the reported frequency of Pfcrt K76 wildtype allele in the filed-isolates was very high compared to all previous years, especially during the years of CQ deployment in Sudan [16]. However, since AS/SP era, post-CQ in 2004, Pfcrt K76 allele tends to increase. This could be occurred due to limited exposure of the P. falciparum parasite to the CQ or the complete absence of the pressure since infections with P. vivax or P. falciparum /P. vivax coinfection is been treated with combination of AL and PQ [1]. However, this increase in Pfcrt K76 might be due to the increase of AL pressure. Although, in this study the quintuple NFSND haplotype of the Pfmdr-1 is not completely studied, but this is also considered alarming since the presence of NF haplotype could be accompanied with the SND haplotype, and consequently leading to AL resistance since parasites carrying the NFSND haplotype reported to survive 15-fold higher AL concentrations [22]. Previously, 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, but with the ability to predict with the aid of the . But difference obtained in this study is that most of the isolates were carrying Pfcrt K76 wildtype allele. As this is in accordance to stop the use of CQ in malaria treatment. [41]. However, previous studies indicated that IPTp-SP is still efficacious in some areas with high prevalence of resistant P. falciparum parasite [42]. Nevertheless, the increased resistance rate might compromise the implication of IPTp-SP [43][44][45].
In this study, a small frequency of R59 mutation was observed, this could be attributed to the small sample size investigated particularly that the R59 mutation is quite known throughout most of the African countries. However, comparing to previously reported studies in Sudan, this frequency is similar, as suggested previously, this could be due to high frequency of Pfdhfr 51I and 108N, and Pfdhps 437G and 540E mutations lead to a genetical cost-effectiveness competitions between the multi-drugs-resistance mutations within the parasites for determination of the effective combination of mutations that increase the parasite survival rate [46].

Conclusions And Recommendations
In addition to studying the molecular markers of malaria multi-drug resistance in the field collected isolates of P. falciparum we reviewed and summarized the previously published reports about malaria drugs resistance in Sudan. This study describes the distribution of P. falciparum multidrug resistance markers throughout Sudan. It also provides a solid baseline data of the status of these markers which could be very useful for the malaria control program not only for establishing surveillance system that monitor the change in and/or the emergence of malaria drug resistance but it will also offer a guidance for the evidence-base decision-making regarding the treatment protocol national and regional wise. Nevertheless, a country-wide monitoring and evaluation program for the early detection of the drug-resistance is extremely needed for more effective treatment protocol and a successful control of the disease. Committee, National University-Sudan. Oral and written informed consents were obtained from participants' or parents or guardians in case of children.

Consent for publication
Not applicable Availability of data and material All datasets used and analysed in this study are available in the manuscript. Sequences analysed in this study were not submitted into the NCBI database.

Supplementary Files
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