Data on selected antimalarial drug resistance markers in Zambia

This article describes data on selected resistance markers for antimalarial drugs used in Zambia. Antimalarial drug resistance has hindered the progress in the control and elimination of malaria. Blood samples were collected during a cross-sectional household survey, conducted during the peak malaria transmission, April to May of 2017. Dried blood spots were collected during the survey and transported to a laboratory for analysis. The analysed included polymerase chain reaction (PCR) followed by high resolution melt (HRM) for mutations associated with Sulfadoxine-pyrimethamine resistance in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps) genes. Mutations associated with artemether-lumefantrine resistance in falciparum multi-drug resistance gene 1 (Pfmdr1) were also assessed using PCR and HRM analysis, whereas the P. falciparum Kelch 13 (PfK13) gene was assessed using nested PCR followed by amplicon sequencing.

resistance in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps) genes. Mutations associated with artemetherlumefantrine resistance in falciparum multi-drug resistance gene 1 ( Pfmdr1 ) were also assessed using PCR and HRM analysis, whereas the P. falciparum Kelch 13 ( PfK13 ) gene was assessed using nested PCR followed by amplicon sequencing.
© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) Table   Subject Parasitology Specific subject area Antimalarial drug resistance Type of data Table  Image Chart Graph Figure  How data were acquired DNA was extracted and Polymerase chain reaction followed by high resolution melt on a LightScanner 384 system (BioFire Diagnotics, inc., Salt Lake USA) and sequencing using ABI 3500XL Genetic Analyser (Applied Biosystems, Foster, USA) were used for analysis. Data format Raw Analyzed Parameters for data collection Genomic DNA was extracted from Dried blood spots were collected during the survey. The extracted DNA was analysed using Photo-induced electron transfer-PCR (PET-PCR) for malaria positivity and species identification. Only Plasmodium falciparum positive samples were analysed for resistance markers Description of data

Value of the Data
• This data is important for the monitoring of antimalarial drug resistance.
• This data can guide policy makers on the resistance pattern of the currently used antimalarial. • The data can be used for further studies on resistance makers especially in systematic review and meta-analysis. • The data add to the body of information of mutations in the Pfdhfr and Pfdhps genes of SP, Pf mdr-1 related to lumefantrine sensitivity and Pfkelch 13 related to artemether resistance.

Data Description
The data set ( https://data.mendeley.com/datasets/zfk9brr5d9/1 ) consist of results obtained from HRM-PCR technic from samples collected from the Western and Southern Provinces. The results were from an analysis of three gene Pfdhfr (51, 59, 108 and 164), Pfdhps (436, 437, 540 and 581) and Pfmdr (86,184 and 1246). The wild type is indicated as '1', while the mutant '0'. The data shows wild type, mutant and mixed infections. The

DNA extraction
Genomic DNA was extracted from 6 mm DBS punches, approximately 13.8 μl whole blood, using a Qiagen DNA mini kit (Qiagen, Hilder, Germany) and eluted in 100 μl elution buffer. The puncher was cleaned after every sample by dipping in 70% ethanal and flaming. The extraction process for RDT positive and negative samples was different. RDT-positive samples were extracted individually, while RDT-negatives were extracted in pools of 10, and pools that came out positives were deconvoluted and re-extracted individually. The extracted DNA was stored at 4 °C for immediate analysis.

PET-PCR analysis
Samples were run using PET-PCR and PCR-HRM. PET-PCR was performed on a LightCycler LC 480 real-time PCR machine (Roche, Basel, Switzerland), as described in 2013 by Lucchi et al [2] . To amplify Plasmodium 18S ribosomal RNA the primer shown in Table 2 were used. Briefly, 5μl of DNA template was amplified in a 20 μl reaction volume containing 1x of the Taqman 2X environmental master mix (Applied Biosystems, Life Technologies LTD, Warrington, UK) as follows: 95 °C for 15 min, followed by 45 cycles of 95 °C for 20 s, 60 °C for 40 s and 72 °C for 30 s. Samples were tested in duplicate and recorded positive if both duplicate samples had a cycle threshold (CT) value < 40.

HRM analysis for Pfdhfr, Pfdhps and Pfmdr 1
Pre-amplification: The PCR-HRM analysis started with a pre-amplification process to enhance the template concentrations. The pre-amplification was performed on all P. falciparum positive samples. A pre-amplification master (PreAmp Master Mix, Life Technologies, Inc, Grand Island, NY, USA) was used with a mixture of primers for the assays that were run. DNA from samples with a CT value of > 35 was pre-amplified in a 10 μl reaction volume and the ones with CT <     Table 3 . Specific controls for wild type or mutant genes were included for each assay. The amplification conditions were as follows: 95 °C denaturation for 2 min, 50 cycles of 94 °C for 5 s and 66 °C for 30 s, and a premelt cycle of 5 s each at 95 °C and 37 °C. The product was heated from 40 °C to 90 °C on the Lightscanner system and the change in fluorescence was recorded as the samples melted incrementally. The following assays were run Pfdhfr (N51I, C59R, I164L and S108N); Pfdhps (S436F, A437G, K540E/N and A581G); and Pfmdr (N86F, Y184F and D1246Y. The annealing temperature for all assays was 66 °C with the exception of two assays S108N and D1246Y that were run at 63 °C [ 3 , 4 ].

Kelch-13 propeller domain amplification and sequencing
Amplification of the Kelch 13 Propeller gene was performed using nested PCR and primers in Table 4 were used. For both primary and nested reactions, the total reaction volumes were 25 μL and 50 μL respectively. The reaction contained 1x final concentration buffer, 2.5 mM MgCl 2 , 20 nM of each dNTPs and 1.25 U Taq® polymerase (Solis BioDyne, Estonia), 250 nM of each primer, with 2.5 template (25 μL reaction volume) and 5 μL template (50 μL reaction volume). Sequencing of Pfk13 was performed in South Africa at Inqaba Biotechnology industries. The labelled products were cleaned using the ZR-96 DNA sequence Clean-up Kit (Catalogue No. D4053, Zymo research). The cleaned products were then analysed using the Applied Biosystems ABI 3500XL Genetic Analyser (Themofisher).

Data Analysis
Allele prevalence was analysed using Stata version 13 (College Station, TX, USA). Any sample that contained a mixed result (i.e. presence of both wild-type and mutant alleles) was scored as a mutant. The graph was prepared in excel. Multiple nucleotide sequence alignments were analysed by MacVector (Cambridge, UK) using the 3D7 PfK13 sequence (GenBank accession no. XM001350122) as a reference to detect point mutations in the gene.

Ethics Statement
Ethical clearance was sought from the Regional Committee for Medical and Health Research Ethics-Western Norway Ref no. 2016/1393/REK Vest (Norway) and from the University of Zambia Biomedical Research Ethics Committee (UNZABREC) (Zambia) Ref no. 010-05-16. This study was an analysis of samples from a larger study, that was assessing progress made in malaria control in Zambia, thus ethical clearance for the larger study was earlier obtained from UNZABREC, ref no. 007-03-14. Permission to use the Ministry of Health data was obtained from the National Health Research Authority. All data analysed were anonymized. Consent was obtained during the data collection from the participants involved in the study.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.