Study site
A comprehensive drug susceptibility assessment was conducted at St. Mary’s Hospital Lacor in Gulu, Northern Uganda (Fig 1) from 2013 to 2018 [25, 26]: Oct-Nov 2013, May-Jun and Oct-Nov 2014, May-Jun and Oct 2015, Jun-Jul and Oct-Nov 2016, Jun 2017 and Jun 2018. Average temperature in the studied area is 24.6°C and average annual rainfall is about 1507 mm with two rainy seasons; a smaller peak in April-May (average rainfall 150 mm) and a heavier peak in August-September (average rainfall 234 mm) [27]. P. falciparum is the most prevalent species and is mainly transmitted by Anopheles fenestus as the major vector and An. gambiae.
Malaria control programs in the studied region include vector control by long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), artemisinin combination therapy (ACT) together with improved diagnosis, management of severe malaria, and intermittent preventive treatment of malaria during pregnancy. These control measures were performed with funding from the Global Fund, USAID/PMI, DFID, World Vision and other partners [28]. Mass distribution of LLINs was first implemented in 2009-2010, which continued until 2013-2014. Through these extensive efforts, malaria burden was effectively reduced from 72% in 2009 to 29% in 2014 [27].
Patients
Initial screening was performed using RDT (SD BIOLINE Malaria Ag P.f/Pan test, Abbott, USA) for 1575 symptomatic patients who visited St. Mary's Hospital Lacor. Inclusion criteria were: (a) patients that are P. falciparum positive by RDT and microscopy, (b) aged ≥ 6 months, and (c) with no history of taking antimalarial drug(s) within two weeks before enrollment. Patients fulfilling the inclusion criteria were enrolled after obtaining written informed consent from the patients or parent/guardian(s). For children aged 7 to 17 years, separate assent was also obtained.
Ethical approval for this study was obtained from the Lacor Hospital Institutional Research and Ethics Committee (Ref; LHIREC 021/09/13), the Uganda National Council for Science and Technology (Ref; HS 1395), and Juntendo Research and Ethics Committee (Ref; 14-169).
Sample collection and ex vivo susceptibility assay for chloroquine and lumefantrine
Blood samples of approximately 100–500 µL (< 2 years old), and 1ml (≥ 2 years old) were collected by peripheral venipuncture or finger prick and immediately transferred to the laboratory adjacent to the hospital. Thick and thin blood smears stained for 30 min with 2% Giemsa solution were used to determine parasitemia.
At every visit from 2013-2018 (a total of nine times sampling period), ex vivo drug susceptibility studies were performed. Ex vivo susceptibility was evaluated for chloroquine and lumefantrine for the samples with parasitemia ≥ 0.05% as previously reported [29]. Parasite culture was incubated in the presence of chloroquine (25–1600 nM) or lumefantrine (1.25–80 nM) at 37°C for 72 h in a gas atmosphere of 5% CO2, 5% O2 (AnaeroPack malaria culture system, Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan). Laboratory-maintained 3D7 clone was used for quality evaluation of pre-dosed drug plates. Parasite culture without antimalarials served as control. To evaluate parasite growth, thick smears were made from drug free culture after 72 h of incubation and the number of schizonts counted. If less than 5 schizonts were seen per field, the test samples included in that plate were not used for further analysis. Drug sensitivity was assessed using an enzyme-linked immunosorbent assay (ELISA) that quantifies parasite histidine-rich protein-2 (HRP-2) [30]. The effective concentration needed to inhibit P. falciparum growth by 50% (IC50) was determined by non-linear regression using an online ICEstimator software (www.antimalarial-icestimator.net) [31]. The quality of the ex vivo drug assay was evaluated based upon the level of fitness to the expected shape of the curve obtained by the inhibitory sigmoid Emax model [31] .
Pfcrt and pfmdr1 genotyping
Polymorphisms at amino acid positions 72–76 in pfcrt were determined by direct sequencing. In P. falciparum multidrug resistance-1 (pfmdr1), polymorphisms at codons 86, 184, 1034, 1042 and 1246 were determined by direct sequencing and/or restriction fragment length polymorphism (RFLP) analysis, as previously described [29, 32]. For direct sequencing, initial and nested PCR was done with PrimeSTAR Max DNA Polymerase (Takara Bio Inc., Japan) in 10 μL reaction mixture containing 1 μL of DNA template and 0.5 μM of each primer set. Excess primers and unincorporated nucleotides from the nested PCR product were enzymatically removed with ExoSAP-IT Kit (Amersham Biosciences, Buckinghamshire, UK) and direct sequence was performed (96°C for 1 min, 25 cycles of 96°C for 30s, 50°C for 30 sec and 60°C for 4 min, and a final cycle at 60°C for 1 min) with a BigDye Terminator v1.1 cycle sequencing kit in the Applied Biosystems 3130/3130xL genetic analyzer (Life Technologies, Carlsbad, California, U.S.A). Samples with overlapping peaks of at least 50% in height were considered harboring mixed genotypes.
Full sequencing of pfcrt
The full sequence of pfcrt was obtained either by whole-genome sequencing (n = 17) or target sequencing (n = 39). Whole genome sequence data was previously reported [26]. In brief, Acrodisc filters (Pall Corporation, New York, NY, USA) was used to reduce the extent of human DNA contamination from blood samples. Approximately 1–1.5 Gb of data per sample were obtained using Illumina instruments (Miseq and Hiseq2000). Single-nucleotide polymorphisms were called at all genomic positions with >80% frequency of >10 reads support.
For target sequencing, the DNA fragment of a genomic region coding for pfcrt gene was amplified by PCR with primers (Pfcrt-F: 5'-TAC TTT CCC AAG TTG TAC TGC TTC TAA GCT-3', Pfcrt-R: 5'-TTT ACC TAT TTA TCA AAA CAC CAA AAG GGA-3'), which covers the whole DNA sequence of pfcrt gene. PCR was performed with PrimeSTAR GXL DNA Polymerase (Takara Bio Inc., Japan) in 5 μL reaction mixture containing 1 μL of DNA solution and 0.25 μM of primer set. PCR conditions consisted of denaturation at 98oC for 10 s, followed by 40 cycles of amplification (98oC for 10 s, 60oC for 15 s, and 68oC for 5 min), with a final elongation period of 68ºC for 5 min. PCR products were diluted with 5 μL of pure water, electrophoresed in 2 % agarose gel and stained with ethidium bromide. The PCR products were then purified with the ExoSAP-IT reagent (Affymetrix, USA). Libraries were prepared from the purified PCR products with Nextera XT DNA Library Prep Kit (Illumina, USA). The libraries were sequenced by MiSeq (Illumina) with the paired-end method and 250 bp of read length. The reads were also used to map the pfcrt gene sequence of P. falciparum 3D7 as a reference and assembled a single contiguous sequence by CLC Genomics Workbench (Qiagen). All sequences were deposited in the DNA Data Bank of Japan (DDBJ) with accession numbers LC498195 – LC498250.
Statistical analysis
All statistical analyses were performed using R software (version 3.6.1). Data was analyzed using Kruskall Wallis test, Wilcoxon rank sum test, and Jonckheere-Terpstra test. P values < 0.05 were considered statistically significant.