Historical literature review and molecular analysis of malaria drug resistance markers of Plasmodium falciparum field-isolates from Sudan.
Authors: Mohamed Nouh S. , Abdelbagi Hanadi , Osman Hussam A. , Ahmed Abdallah E. , Yousif Alaa M. , Edris Yusraa B. , Osman Eman Y. , Elsadig Aahd R. , Siddig Emmanuel E. , Mustafa Madinna , Mohammed Ammar A. , Ali Yousif , Osman Maha M. , Ali Mohamed S. , Omer Rihab A. , Ahmed Ayman , Sibley Carol H. 
Published: February 2020

Abstract
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%), respectively. Whereas for C59R, C59 was 18 (90%), and 59R was 2 (10%). For S108N, 18 (90%) for 108N and 2 (10%) for S108. The triplet haplotype ICN of the Pfdhfr ; was the most frequent haplotype; 16 (80%). Concerning the Pfdhps , all the 20 (100%) isolates were carrying the mutant alleles; 437G and 540E. the Pfdhps haplotype present was the double GE haplotype only. No statistically significant correlation was found for the Pfcrt , Pfmdr-1 , Pfdhfr , and Pfdhps . Historical reports on P. falciparum multidrug resistant collected from 1989 to 2016 showed extreme fluctuation. High prevalence of Pfcrt 76T allele was observed in Khartoum throughout all years of previous studies, while in Gedaref Pfcrt 76T 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 decision-making regarding the treatment protocol national and regional wise.