The systematic review and meta-analysis protocol has been developed following the Preferred Reporting Items for Systematic reviews and Meta-analysis Protocol (PRISMA-P) 2015 statement [28,29]. This format will also be followed when reporting the findings of the review (Additional file 1). The search and inclusion of articles will be done according to the population intervention comparator outcome (PECOS) format. This approach include: population (P), individuals infected P. falciparum parasites in Cameroon, exposure (E), use of non-artemsinin and artemisinin agents in the treatment of malaria, comparator (C), none, outcome (O), Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfk13 gene polymorphisms circulating in malaria endemic areas of Cameroon, study design (S), the review will include articles from cross-sectional studies and those reporting using P. falciparum DNA samples collected on pre-treatment day zero (D0) and during follow-ups of non-randomised and randomised clinical trial studies.
- What is the reported prevalence of falciparum drug resistance markers before and after the adoption of artemisinin-based combination therapies in Cameroon?
- What is the relationship between the ACTs (ASAQ and AL) and prevalence of falciparum drug resistance mutations (chloroquine resistance transporter mutation-K76T and multidrug resistance 1 mutation-N86Y) in Cameroon?
- What is the impact of the proportions of artesunate-amodiaquine (ASAQ), artemether-lumefantrine (AL) and sulphadoxine-pyrimethamine deployed to the different Regions on the evolution of drug resistance markers (Pfcrt K76T, Pfmdr1 N86Y, Pfdhfr N51I, Pfdhfr C59R, Pfdhfr S108N) in Cameroon?
Objectives of the systematic review
This review aims to:
- Determine the prevalence of falciparum drug resistance markers before and after the adoption of artemisinin-based combination therapies in Cameroon. The key Plasmodium falciparum anti-malarial drug resistance markers shall include: chloroquine resistance transporter (Pfcrt), multi-drug resistance 1 (Pfmdr1), dihydrofolate reductase (Pfdhfr), dihydropteroate synthase (Pfdhps), atpase 6 (Pfatp6), cytochrome b (Pfcytb) and kelch 13 (Pfk13).
- Evaluate the relationship between the efficacy of ACTs (ASAQ and AL) and prevalence of falciparum drug resistance mutations (chloroquine resistance transporter mutation K76T and multidrug resistance 1 mutation N86Y).
- Investigate the impact of the proportions of artesunate-amodiaquine (ASAQ), artemether-lumefantrine (AL) and sulphadoxine-pyrimethamine deployed to the different Regions on the evolution of drug resistance markers (Pfcrt K76T, Pfmdr1 N86Y, Pfdhfr N51I, Pfdhfr C59R, Pfdhfr S108N) in Cameroon.
Registration of the systematic review protocol
The review protocol has been registered in the International Prospective Register of Systematic Reviews (PROSPERO: http://www.crd.york.ac.uk/prospero). The current review protocol is registered with the number CRD42020162620.
An electronic systematic strategy based on the combination of key words will be used to search articles from Medline via Pubmed, EMBASE, Google Scholar, and Science Direct databases. Both interventional and observational studies will be retrieved to be included in the review. Articles will cover a period from January 1998 to date.
The reference lists of published articles will be searched for eligible studies. Authors will be contacted in the case whereby full length articles cannot be accessed. Malaria annual reports will be obtained from the Cameroon National Malaria Control Programme (NMCP) and Ministry of Public Health. In addition to published studies, unpublished Medical Doctor (MD), Master of Science (MSc) and Doctor of Philosophy (PhD) theses will be accessed for inclusion in the study.
The following Medical Search Heading (MeSH) search terms will be combined using the Boolean operators “OR” and “AND’’: “malaria”, “anti-malarial”, “drug resistance”, “Pfcrt”, “Pfmdr1”, “Pfdhfr”, “Pfdhps”, “Pfatp6’’, “Pfcytb’’, “Pfk13”, “mutations”, “gene polymorphisms”, “amino acid changes”, “Plasmodium falciparum”, “efficacy”, “artesunate-amodiaquine”, “artemether-lumefantrine”, “sulphadoxine-pyrimethamine”, “Cameroon”. The terms listed above will be used to develop the search string. The search string to be used for the identification and screening of articles in PubMed is provided as Additional file 2.
The systematic review and meta-analysis will include the following type of studies:
i) studies published from January 1998 to present; ii) studies on human participants of all ages; iii) original articles of studies that investigated either asymptomatic, uncomplicated or severe Plasmodium falciparum; iv) studies in which Polymerase Chain Reaction (PCR) genotyping of anti-malarial drug molecular resistance markers (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfcytb, Pfatp6, Pfk13) were done; v) studies written in English or French; vi) studies done within Cameroon; and vii) all multi-centric studies in which Cameroon was one of the sites or cases of imported malaria from Cameroon (Additional file 3).
The following types of studies will not be included: i) abstracts; ii) studies on in vitro, ex vivo and in vivo anti-malarial drug resistance without Deoxyribonucleic Acid (DNA) sequence genotyping; iii) genetic association studies on Pfcg2 gene; iv) studies on genetic diversity and population structure of Plasmodium falciparum without drug resistance; and v) studies on diagnostic accuracy of methods for detection of P. falciparum and mixed Plasmodium species infections.
The Preferred Reporting Items for Systematic reviews and Meta-analysis (PRISMA) flow chart  will be adopted in the identification and screening for inclusion of articles. Articles identified from searches of the computerised databases will be screened for eligibility based on title and abstract. Ineligible articles and duplicates will eventually be removed. Full-length articles of the selected studies will be read to confirm for fulfilling of the inclusion criteria before data extraction begins. Two independent reviewers (Peter Thelma Ngwa Niba-PTNN and Lesley Ngum Ngum-LNN) will screen the titles and abstracts to identify potentially eligible studies and data from full-length articles that fulfil the inclusion criteria will be extracted. Discrepancies will be resolved by mutual consent or by independent review from the third researcher (Akindeh Mbuh Nji-AMN). The whole process will be supervised by Wilfred Fon Mbacham (WFM) and Michael Alifrangis (MA).
Data extraction procedure
The Strengthening Reporting of Genetic Association studies (STREGA) guideline  will be used in developing a data extraction form for extracting data from the included articles. Data extraction process will focus on the types of study design (observational versus interventional), year the studies were conducted, study site, sample size, age of participant, genotyping method, genotyping accuracy, drug resistance gene, type of amino acid changes, and prevalence of mutations (Additional file 4).
Studies (observational or interventional) published multiple times in similar topics by the same authors will be diligently screened to avoid duplication of data. These studies will be differentiated based on primary variables (anti-malarial drug resistance markers and frequency of single nucleotide polymorphisms) containing the datasets of interest.
Mixed genotypes will be considered as mutants during data collation on frequency of mutations derived from different studies. The Microsoft Excel 2010 (Microsoft Corporation, Redmond, Washington, United States of America) shall be used to design the data extraction sheet. The database in Microsoft Excel will be piloted and validated before completion of the review process.
Methodological quality assessment and sensitivity analysis
The quality of the reviewed studies will be assessed through sensitivity analysis, which classifies the included studies into low and high qualities according to modified Jadad scale for randomised controlled trials (RCTs)  and the strengthening the reporting of observational studies in epidemiology (STROBE) statement for observational studies . Modified Jadad scale assesses the quality of a trial with the range from 0 to 8 (randomisation and its appropriate use, blinding and its appropriate use, withdrawals and dropouts, description of inclusion and exclusion criteria, assessment of adverse effects, and description of statistical analysis). The score for the modified Jadad scale range of 0-3 represents low or poor quality and score ranges of 4–8 represents good to excellent quality. The observational studies will be categorised as low quality with a score under 75% of the STROBE checklist and high quality with a score over 75% of the STROBE checklist. Two reviewers will independently assess the quality of the methodology of included studies.
Assessment of publication bias
The risk of publication bias in the included articles will be assessed using the asymmetry of funnel plot and Egger’s regression test with P<0.05. This will be done at both the study and outcome levels. The funnel plot contains the standard error on the y-axis and proportion on the x-axis.
Data will be managed in the Zotero standalone software version 5.0.56 (Corporation for Digital Scholarship, Vienna, Virginia, United States). Eligible articles will be imported into the software and duplicates removed.
Data analysis, heterogeneity assessment and data interpretation
Quantitative syntheses will be done using the “metaphor” and “meta” commands in the R statistical software package version 3.5.2 (supported by the R Foundation for Statistical Computing, Vienna, Austria). The heterogeneity of the included studies will be evaluated using the Cochran’s Q and the I2 statistics. The random effects model will be used as standard in the determination of heterogeneity between studies . The I2 values will be expressed in percentages. Heterogeneity will be classified as low, moderate and high, with upper limits of 25%, 50% and 75% for I2, respectively .
Forest plots will be used to present the data on pooled prevalence of mutations in anti-malarial drug resistance genes. Sub-group analyses shall be also done to demonstrate the aggregated prevalence of Pfcrt K76T, Pfmdr1 N86Y, Pfdhfr IRN haplotype and Pfk13 gene mutations.
Frequency distribution tables will be used to demonstrate the evolution of key anti-malarial drug resistance markers in Cameroon from 1998 to present. The Pearson Chi square test in the International Business Machines Statistical Software Package for Social Sciences (IBM SPSS) version 20.0 software package (IBM Corporation, Armonk, New York, United States of America) will be used to establish the evolution of drug resistance markers over time. Furthermore, the relationship between the efficacy of ACTs (ASAQ and AL) and anti-malarial drug resistance makers (Pfcrt 76T and Pfmdr1 86Y) will be assessed on line graphs. The correlation coefficient (r) will be used to assess the relationship between efficacy of ACTs (AL and ASAQ) and prevalence of Pfcrt 76T and Pfmdr1 86Y mutants over time after checking for normality using the Shapiro-Wilk test. In addition, the relationship between proportions of anti-malarial drugs (ASAQ, AL and SP) deployed in Cameroon and prevalence of drug resistance markers (Pfcrt 76T, Pfmdr1 86Y and Pfdhfr IRN) from 2006 to 2017 will also be explored using line graphs and r. The level of significance will be set at p<0.05 at 95 % confidence interval.