Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in Uganda

Moses Ocan; Fred Katabazi Ashaba; Savannah Mwesigwa; Kigozi Edgar; Moses R Kamya; Sam L Nsobya

doi:10.1371/journal.pone.0268095

. 2022 May 5;17(5):e0268095. doi: 10.1371/journal.pone.0268095

Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in Uganda

Moses Ocan ^1,^*, Fred Katabazi Ashaba ², Savannah Mwesigwa ³, Kigozi Edgar ², Moses R Kamya ^4,⁵, Sam L Nsobya ^4,⁶

Editor: Luzia Helena Carvalho⁷

PMCID: PMC9070901 PMID: 35511795

Abstract

In Uganda, Artemether-Lumefantrine and Artesunate are recommended for uncomplicated and severe malaria respectively, but are currently threatened by parasite resistance. Genetic and epigenetic factors play a role in predisposing Plasmodium falciparum parasites to acquiring Pfkelch13 (K13) mutations associated with delayed artemisinin parasite clearance as reported in Southeast Asia. In this study, we report on the prevalence of mutations in the K13, pfmdr-2 (P. falciparum multidrug resistance protein 2), fd (ferredoxin), pfcrt (P. falciparum chloroquine resistance transporter), and arps10 (apicoplast ribosomal protein S10) genes in Plasmodium falciparum parasites prior to (2005) and after (2013) introduction of artemisinin combination therapies for malaria treatment in Uganda. A total of 200 P. falciparum parasite DNA samples were screened. Parasite DNA was extracted using QIAamp DNA mini kit (Qiagen, GmbH, Germany) procedure. The PCR products were sequenced using Sanger dideoxy sequencing method. Of the 200 P. falciparum DNA samples screened, sequencing for mutations in K13, pfmdr-2, fd, pfcrt, arps10 genes was successful in 142, 186, 141, 128 and 74 samples respectively. Overall, we detected six (4.2%, 6/142; 95%CI: 1.4–7.0) K13 single nucleotide polymorphisms (SNPs), of which 3.9% (2/51), 4.4% (4/91) occurred in 2005 and 2013 samples respectively. All four K13 SNPs in 2013 samples were non-synonymous (A578S, E596V, S600C and E643K) while of the two SNPs in 2005 samples, one (Y588N) is non-synonymous and the other (I587I) is synonymous. There was no statistically significant difference in the prevalence of K13 (p = 0.112) SNPs in the samples collected in 2005 and 2013. The overall prevalence of SNPs in pfmdr-2 gene was 39.8% (74/186, 95%CI: 25.1–50.4). Of this, 4.2% (4/95), 76.9% (70/91) occurred in 2005 and 2013 samples respectively. In 2005 samples only one SNP, Y423F (4.2%, 4/95) was found while in 2013, Y423F (38.5%, 35/91) and I492V (38.5%, 35/91) SNPs in the pfmdr-2 gene were found. There was a statistically significant difference in the prevalence of pfmdr-2 SNPs in the samples collected in 2005 and 2013 (p<0.001). The overall prevalence of arps10 mutations was 2.7% (2/72, 95%CI: 0.3–4.2). Two mutations, V127M (4.5%: 1/22) and D128H (4.5%: 1/22) in the arps10 gene were each found in P. falciparum parasite samples collected in 2013. There was no statistically significant difference in the prevalence of arps10 SNPs in the samples collected in 2005 and 2013 (p = 0.238). There were more pfmdr-2 SNPs in P. falciparum parasites collected after introduction of Artemisinin combination therapies in malaria treatment. This is an indicator of the need for continuous surveillance to monitor emergence of molecular markers of artemisinin resistance and its potential drivers in malaria affected regions globally.

Introduction

Uganda is among the 29 malaria endemic countries which contributed 95% of malaria cases globally in 2019 [1]. P. falciparum is responsible for over 99% of all malaria infections in Uganda [1, 2]. In sub-Saharan Africa, artemisinin combination therapies (ACTs) remain highly efficacious in malaria treatment despite the threat of parasite resistance to both artemisinin [1, 3] and other components of the combination in South east Asia [4]. This has contributed to the current success in malaria control efforts in the region [1]. In Uganda, the recommended first-line and second-line agents in treatment of uncomplicated malaria are Artemether-lumefantrine (AL) and Dihydroartemisin-Piperaquine (DHP-PPQ) respectively [5]. A recent systematic review reported a slightly better efficacy of DHP-PPQ compared to AL in the treatment of uncomplicated malaria among children under five years in Uganda [6].

Artemisinin resistance characterized by delayed parasite clearance is currently prevalent in Southeast Asia [7, 8]. From its early detection in Cambodia [9], artemisinin resistance has spread to other areas, including Thailand, Myanmar, China, Vietnam, Bangladesh, and Lao People’s Democratic Republic [10, 11]. However, artemisinin resistance has not been detected outside China, Bangladesh and South east Asia for over a decade after it was first reported [1].

Parasite genetic background support independent emergence of K13 mutations associated with artemisinin resistance in different geographical areas in Southeast Asia [7, 8]. A study by Miotto et al., [8] reported a strong association between non-synonymous mutations fd-D193Y (ferredoxin gene), arps10-V127M (apicoplast ribosomal protein S10 gene), pfmdr2-T484I (multidrug resistance protein 2 gene), crt-I356T (chloroquine resistance transporter gene) and crt-N326S (chloroquine resistance transporter gene) and development of artemisinin resistance in P. falciparum parasites [8]. Diverse K13 mutations whose role in artemisinin resistance is not yet known are prevalent in sub-Saharan Africa [12]. With the likely threat of emergence and spread of artemisinin resistance in sub-Saharan Africa, understanding the drivers of artemisinin resistance development is critical in guiding establishment of effective control and elimination strategies to combat resistance.

In highly malaria endemic regions like sub-Saharan Africa, development and spread of artemisinin resistance could result in increased malaria related mortality. A previous study [13], projected that emergence of wide spread artemisinin resistance globally could result in more than 100,000 malaria related deaths annually. Wide spread resistance to chloroquine followed by its subsequent withdrawal from malaria treatment in sub-Saharan Africa previously resulted in more than doubling of malaria related deaths [14]. Strategies to mitigate potential emergence of artemisinin resistance and thus prolong the effective life span of these agents in sub-Saharan Africa are urgently needed as they remain crucial for malaria control efforts. This is especially the case due to the current lack of an effective malaria vaccine.

Plasmodium falciparum parasite genetic background has been reported as an important driver of artemisinin resistance development in Southeast Asia [8, 15]. Recent studies [16, 17] have reported occurrence of K13 mutations 675V (Uganda and Rwanda) and 469Y (Uganda) that are associated with delayed artemisinin parasite clearance in Southeast Asia. Surveillance to monitor prevalence of markers of a genetic background which supports the rise of K13 mutations associated with delayed artemisinin parasite clearance across malaria endemic regions is thus crucial to help inform malaria control efforts. We therefore, intended in this study to assess and compare the prevalence of fd-D193Y, aps10-V127M, pfmdr2-T484I, crt-I356T, crt-N326S and K13 mutations in P. falciparum parasites before (2005) and after- (2013) introduction of artemisinin-based combination therapies for malaria treatment in Uganda [18].

Materials and methods

Ethics statement

The protocol was reviewed and approved by Makerere University School of Biomedical Ethics Review Committee (#SBS-513) and Uganda National Council of Science and Technology (#HS168ES). Written informed consent was obtained from the parent or guardian of the children prior to enrolment into the study.

Study design, site and population

This was a cross sectional study. We used samples from two previous studies conducted by Infectious Disease Research Collaboration (IDRC); a randomized, single-blinded, longitudinal clinical trial designed to compare safety, tolerability and efficacy of three different combination antimalarial regimens for treatment of uncomplicated malaria done from November 2004 to June 2006 in Kampala [19]; and a cohort study, Program for Resistance, Immunology, Surveillance and Modeling of Malaria in Uganda (PRISM 2) conducted in three districts (Jinja, Kanungu and Tororo) from August 2011 to September 2013 [20]. In both studies malaria parasite infected blood samples were collected using ethylenediaminotetraacetic acid (EDTA) vacutainer tubes from children aged 1–10 years [19] and 6 months-10 years [20]. The two studies covered both low (Kampala, Kanungu) and high (Jinja, Tororo) malaria transmission settings in Uganda. The frozen whole blood samples were stored at the IDRC laboratory in Butabika National referral hospital and the research team accessed the samples in April 2019. All the samples were completely de-identified prior to being accessed by the research team. For our study, 100 randomly selected samples with ≥50ng/μl of DNA each collected in 2005 and 2013 by Dorsey et al., [19] and Kamya et al., [20] were included into the study. For the current study, we only used samples collected from Jinja and Kampala.

Microscopy, thick smears stained with 2% Giemsa for 30 minutes (detection of malaria parasite presence) and thin smear (malaria parasite species identification) were used for malaria diagnosis by the primary studies and were performed before sample storage [19, 20]. The thick and thin smears were prepared following a method previously described by WHO, 2004 [21]. In our current study, confirmation of the presence of P. falciparum parasites in the stored blood samples was done using malaria rapid diagnostic test (mRDT). The mRDT was done using P. falciparum histidine rich protein 2 (PfHRP-2) (Premier Medical Corporation Ltd, Gujarat, India) antigen based assay as described earlier by following the manufacturer’s guidelines [22].

DNA extraction and quantification

P. falciparum genomic DNA was extracted using QIAamp DNA mini kit (Qiagen, GmbH, Germany) following manufacturer’s guidelines. The extracted genomic DNA concentration was quantified using Nano drop spectrophotometry (Thermo Scientific, Wilmington, Delaware, USA). From the screening, 200 samples with ≥ 50 ng of P. falciparum parasite DNA were then included in the study.

PCR amplification of P. falciparum K13 -propeller gene

The primers reported in a study by Ariey et al., [23] were double checked based on 3D7 genome using NCBI primer 3 software and synthesized by Eurofins scientific (S2 Table). Amplification of the K13 propeller domain of P. falciparum DNA was done following a method by Ariey et al., [23]. Briefly, used in amplification of K13-propeller domain: 25μl reaction volume containing 12.5μl Kapa Hifi ready PCR ready mix (Roche), 1.0μl of each primer, 5.5μl of nuclease free water and 5μl of a mixture of both human and P. falciparum DNA (approximately 50ng/μL). Amplification was done in a Thermocycler under the following cycling conditions, 15 min at 95°C, then 40 cycles of 30s at 94°C, 90s at 54°C, 90s at 72°C and 10 min at 72°C.

For the nested PCR, 2 μl of primary PCR products were amplified under the same conditions. The PCR products were detected using 2% agarose gel electrophoresis stained with ethidium bromide. The PCR products were shipped for Sanger dideoxy sequencing at ACGT Inc. (Wheeling IL, USA) commercial sequencing center.

Amplification of P. falciparum crt, fd, arps10 and pfmdr-2 genes

Nested PCR approach was used to amplify portions of the P. falciparum crt, and pfmdr-2 genes sandwiching the genetic background mutations pfcrt N326S, and pfmdr-2 T484L. While for fd, and arps10 genes the amplicons were generated using first round polymerase chain reaction (PCR). Briefly, used in amplification, 25μl reaction volume containing 12.5μl Kapa Hifi ready PCR ready mix (Roche), 1.0μl of each primer, 5.5μl of nuclease free water and 5μl of approximately 50ng/μL of P. falciparum DNA. Amplification was done using a Thermocycler following conditions as described in S1 Table. PCR products were detected using 2% agarose gel electrophoresis stained with ethidium bromide. Primers reported in a study by Miotto et al., [8] were double checked based on the 3D7 genome using NCBI primer 3 software and synthesized by Eurofins scientific (S2 Table). The PCR products were then shipped for Sanger dideoxy sequencing at ACGT Inc. (Wheeling IL, USA) commercial sequencing center.

Sequence data analysis

Sequence data were base called using sequence analysis software Bioedit ver 5.2 and then blasted on to the NCBI sequence data base to confirm 3D7 K13 propeller, arsp10, fd, pfmdr-2 and pfcrt gene sequence identity. The sequences were analyzed using Mutation Surveyor (Soft Genetics LLC., version 5.1, State College, PA, USA) in order to determine mixed alleles based on the presence of two chromatogram peaks at one nucleotide site, and to also reduce the presence of false positive and (or) false negative mutant sites [24, 25]. Fasta files were then aligned using UGENE v.39 (Unipro) and MEGA 5.10 software to reference allele sequences PF3D7_1343700 (K13), PF3D7_1460900.1 (arps10), PF3D7_1318100 (fd), PF3D7_1447900 (pfmdr-2) and PF3D7_070900 (crt) [24, 25] for detection of single nucleotide polymorphisms. Multiplicity of infection was assessed using the peak heights, where a mixed genotype was confirmed if the minor peak was higher than a third of the major peak.

We deposited sequences for mutations detected in the K13 gene (A578S, I587I, Y588N, E596V, S600C and E643K), two sequences for mutations in the pfmdr-2 gene (Y423F, I492V) and two sequences for mutations in the arps10 gene (V127M, D128H) in the NCBI data base. The sanger traces were deposited in the GenBank and can be accessed using the accession numbers MZ668587-MZ668597 and MZ818701-MZ818770.

Statistical analysis

Data analysis was done at 95% level of significance in STATA ver 14. The prevalence of mutations was determined using proportions. Correlation analysis was done using Fisher’s exact test to assess the differences in prevalence of mutations in 2005 and 2013.

Results

Prevalence of K13 single nucleotide polymorphisms in the P. falciparum parasites

Of the 200 P. falciparum DNA samples screened for K13 mutations, 142 were successfully sequenced, 51 and 91 from 2005 and 2013 P. falciparum parasite samples respectively. Six (4.2%, 6/142) K13 SNPs were detected (Tables 1 and 2). Of these, five were non-synonymous and one was a synonymous K13 mutation, (I587I). We found four, one K13 non-synonymous mutations in the P. falciparum parasite samples collected in 2013 and 2005 respectively. K13 non-synonymous SNPs Y588N, E595V, S600C and E643K, each occurred in a single P. falciparum parasite sample. Two (2) K13 non-synonymous SNPs, A578S and E596V occurred in more than one P. falciparum parasite sample (Table 1). One K13 non-synonymous SNP Y588N occurred in a P. falciparum parasite sample collected in 2005. Five K13 non-synonymous SNPs A578S, E596V, S600C and E643K, were detected in P. falciparum parasite samples collected in 2013. There were no mixed infections identified among the samples.

Table 1. Single nucleotide polymorphisms identified in K13 gene in samples collected in Kampala and Jinja, Uganda in 2005 and 2013.

Sample identifier	Codon	Type	Reference amino acid	Mutant amino acid	Mutant locus	Reference allele	Mutant allele	n/N
K007	596^a^,^*	NS	E	V	1787	A	T	3/142
K007	643^*	NS	E	K	1927	G	A	1/142
K020	600^*	NS	S	C	1799	C	G	1/142
K065	578^b^, ^*	NS	A	S	1732	G	T^c	2/142
K120	587^e	S	I	I	1761	T	C	1/142
K120	588^d^, ^e	NS	Y	N	1762	T	A	1/142

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N: Number of samples sequenced at locus

n: Number of samples containing mutant allele

S: Synonymous SNP

NS: Non-synonymous SNP

^a Occurred in three P. falciparum parasite samples (K007, K050, K082)

^b Occurred in two P. falciparum parasite samples (K065, K070)

^c SNP has previously been identified (MalariaGen)

^d Different SNP in the same codon position has been previously reported (MalariaGen)

*SNP occurred in samples collected in 2013

^e SNP occurred in samples collected in 2005

Table 2. Prevalence of single nucleotide polymorphisms in K13, Pfmdr-2 and Pfarps 10 genes in Plasmodium falciparum samples collected in Kampala and Jinja, Uganda in 2005 and 2013.

Characteristic	Had mutation n	No mutation n	Description	Prevalence of mutation %(n/N)	Fisher’s Exact P-value	95% CI
K13 SNPs (overall)	6	136	Overall	4.2 (6/142)	0.112	1.4–7.0
	2	49	2005	3.9 (2/51)		0.4–8.2
	4	87	2013	4.4 (4/91)		2.5–12.6
K13 SNPs (2005)	1	50	Y588N	1.9 (1/51)		0.1–7.2
K13 SNPs (2005)	1	50	I587I	1.9 (1/51)		0.1–7.2
K13 SNPs (2013)	3	88	E596V	3.3 (3/91)		1.1–9.8
	1	90	E643K	1.1 (1/91)		0.2–7.6
	1	90	S600C	1.1(1/91)		0.2–7.6
	2	89	A578S	2.2(2/91)		0.5–8.5
pfmdr-2 SNPs (overall)	74	112	Overall	39.8 (74/186)	<0.001	25.1–50.4
	4	91	2005	4.2 (4/95)		1.6–10.8
	70	21	2013	76.9 (70/91)		34.0–54.4
pfmdr-2 SNPs (2005)	0	95	T484I	0(0)		-
	0	95	I492V	0(0)		-
	4	91	Y423F	4.2 (4/95)		1.6–10.8
pfmdr-2 SNPs (2013)	0	91	T484I	0(0)		-
	35	56	I492V	38.5 (35/91)		12.0–39.0
	35	56	Y423F	38.5 (35/91)		12.0–39.0
pfarps10 SNPs (overall)	2	72	Overall	2.7 (2/74)	0.238	0.3–4.2
	0	52	2005	0 (0)		-
	2	22	2013	8.3 (2/24)		1.6–19.5
pfarps10 SNPs (2005)	0	52	V127M	0 (0)		-
pfarps10 SNPs (2005)	0	52	D128H	0 (0)		-
pfarps10 SNPs (2013)	1	21	V127M	4.5 (1/22)		1.1–10.5
pfarps10 SNPs (2013)	1	21	D128H	4.5 (1/22)		1.1–10.5

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SNPs: Single Nucleotide Polymorphisms; CI: Confidence Interval, n: number of samples with a given characteristic, N = total number of samples analysed, %: Percentage

Double non-synonymous K13 mutations, E596V and E643K were both found in the same P. falciparum parasite sample. In addition, a synonymous, I587I and a non-synonymous Y588N double K13 mutations were also both present in one P. falciparum parasite sample. One K13 synonymous SNP (I587I) was found in our study.

There was no statistically significant difference in the prevalence of mutations in the K13 (p = 0.112) and arps10 (p = 0.238) genes for samples collected in 2005 and 2013. There was a statistically significant difference in the prevalence of mutations in the pfmdr-2 gene, p<0.001 among samples collected in 2005 and 2013 (Table 2).

Prevalence of genetic background mutations, pfcrt N326S, fd D193Y, arps10 V127M and pfmdr-2 T484L in P. falciparum parasites in Uganda

Sequencing of pfmdr-2, fd, pfcrt and arps10 was successful in 186, 141, 128 and 74 P. falciparum DNA samples respectively. Polymorphisms in the fd, pfmdr-2 and pfcrt genes that are markers of a genetic background where K13 mutations associated with slow artemisinin parasite clearance are likely to arise were not found in our study. The prevalence of K13 mutations was 4.2% (6/142; 95%CI: 1.4–7.0). We found one non-synonymous, Y588N (1.9%, 1/51; 95%CI: 0.1–7.2) and one synonymous K13 mutation, I587I (1.9%, 1/51; 95%CI: 0.1–7.2) in samples collected in 2005. While in P. falciparum from samples collected in 2013, we found three non-synonymous K13 mutations, E596V (3.3%, 3/91; 95CI: 1.1–9.8), E643K (1.1%, 1/91; 95%CI: 1.1–9.8) and A578S (2.2%, 2/91; 95%CI: 0.5–8.5). The overall prevalence of pfmdr-2 mutations was 39.8% (74/186; 95%CI: 25.1–50.4). In the pfmdr-2 gene, we found two non-synonymous SNPs, Y423F and I492V in P. falciparum parasite samples. The SNP Y423F occurred in 4 (4.2%, 4/95; 95%CI: 1.6–10.8), 35(38.5%, 35/91; 95%CI: 12.0–39.0) P. falciparum parasite samples collected in 2005 and 2013 respectively. The SNP, I492V occurred in 35 (38.5%, 35/91; 95%CI: 12.0–39.0) P. falciparum samples all collected in 2013. In the arps10 gene, we found two (2.7%, 2/74; 95%CI: 0.3–4.2) non-synonymous SNPs, V127M (4.5%, 1/22; 95%CI: 1.1–10.5) and D128H (4.5%, 1/22; 95%CI: 1.1–10.5) each in one P. falciparum parasite sample collected in 2013 (Table 2). The two P. falciparum parasite samples with arps10 gene mutations did not have K13 gene mutation. Two P. falciparum parasite samples collected in 2013 had mutations in both pfmdr-2 (Y423F) and K13 (A578S) genes.

Discussion

Plasmodium falciparum artemisinin resistance is a heritable trait with a genetic basis [7]. Genome modification studies have shown that the impact of various K13 mutations on P. falciparum artemisinin clearance and survival rates of ring stage parasites is dependent on the genetic background [26]. The risk of emergence of K13 mutations associated with delayed artemisinin parasite clearance is thus driven by specific parasite genetic background [8, 27]. In our current study polymorphisms in the fd, pfmdr-2 and pfcrt genes which support the rise of K13 mutations associated with delayed artemisinin clearance of P. falciparum parasites reported in Southeast Asia [8] were not found. However, SNPs in the pfmdr-2, pfcrt, and arps10 genes not reported in a study by Miotto et al., [8] and other previous studies in Africa [28] were detected in our current study. There is need to validate the role the identified background mutations play in artemisinin resistance development among African P. falciparum parasites.

Artemisinin resistance has been shown to arise independently in different settings in Southeast Asia [8, 11], an indicator of the role unique sets of conditions in different malaria endemic regions play in driving resistance development. In our study, we detected background mutations in the pfmdr-2, I492V (38.5%, 35/91; 95%CI: 12.0–39.0) and Y423F (2005: 4.2%, 4/95; 95%CI: 1.6–10.8; 2013: 38.5%, 35/91; 95%CI: 12.0–39.0) and arps10 (D128H; 4.5%, 1/22; 95%CI: 1.1–10.5)) genes whose role in artemisinin resistance is not known. These mutations were found in P. falciparum parasites collected in 2013 after introduction of artemisinin combination therapies for malaria treatment in Uganda [18]. There is however a need for more surveillance studies to establish P. falciparum parasite genetic background in the country. This is especially critical as previous studies done in Southeast Asia have demonstrated an association between parasite genetic background and the independent emergence of artemisinin resistance [8, 11]. A recent study in South Sudan [29] which analyzed samples collected in 2015–2017 after introduction of artemisinin agents in malaria treatment detected genetic background mutation in Pfcrt N326S gene which was previously reported in Southeast Asia and associated with artemisinin resistance. While we did not find in our samples PfcrtN326S SNP, we detected D128H after introduction of artemisinin agents in malaria treatment. Additionally, we detected a background mutation, V127M in the arps10 gene that has been shown to support development of K13 mutations associated with artemisinin resistance in Southeast Asia [8]. However, it still remains unknown what role other background mutations (arps10 D128H, pfmdr-2-Y423F, pfmdr-2-I492V) found in our study play in driving independent development and spread of artemisinin resistance among P. falciparum parasites in sub-Saharan Africa.

We detected different K13 SNPs in P. falciparum samples collected in 2005 compared to those collected in 2013 after introduction of artemisinin combination therapies in malaria treatment in Uganda. Although not statistically significant, our study found a general trend towards increase in the prevalence of K13 SNPs after introduction of artemisinin agents in malaria treatment in Uganda. A previous study by Conrad et al., [30] in Uganda showed that introduction of ACTs in malaria treatment did not increase the diversity of K13 SNPs among P. falciparum parasites despite an overall increase in the prevalence. However, the role of these mutations in artemisinin resistance remains unknown. Recent studies in Rwanda [17, 31] and Uganda [16, 32, 33] have reported presence of K13 mutations 675V and 561H (Rwanda), 675V and 469Y (Uganda) associated with delayed artemisinin parasite clearance in Southeast Asia. A recent study by Balikagala et al., [33] demonstrated delayed artemisinin clearance among parasites carrying 675V and 469Y mutations in Uganda. The discovery of these mutations in Ugandan P. falciparum parasites potentially threatens the efficacy of artemisinin combination therapies, a cornerstone in malaria treatment [1]. There is thus an urgent need to investigate the mediators driving development and spread of these mutations in the country.

Our study had some limitations, we analyzed few samples however inclusion of P. falciparum parasites collected in both high and low malaria transmission settings in the country helped improve representativeness. In addition, analysis of P. falciparum parasites collected prior to an after introduction of artemisinin combination therapies in malaria treatment in Uganda helped provide information on potential drivers of the prevalence of K13 and genetic background mutations in the country. Our study could not confirm existence of multiplicity of infection in the samples analyzed.

Conclusion

A mutation, V127M in the arps10 gene previously reported in South east Asia and associated with delayed artemisinin parasite clearance was detected in P. falciparum parasites in Uganda. The proportion of pfmdr-2 gene mutations were generally higher in P. falciparum parasites collected after introduction of artemisinin combination therapies in malaria treatment in Uganda. There were neither K13 SNPs previously implicated in artemisinin resistance in Africa and SEA, nor mutations in the associated background genes identified in this study with exception of one SNP (V127M) in the arps10 gene. There is need to conduct regular surveillance to monitor potential emergence of molecular markers of artemisinin resistance and their drivers among P. falciparum parasites in malaria affected regions globally.

Supporting information

S1 Table. PCR cycling conditions for amplification of P. falciparum DNA fragments sandwiching pfcrt N326S, fd D193Y, arps10 V127M and mdr2 T484L genetic background mutations.

(DOC)

Click here for additional data file.^{(33KB, doc)}

S2 Table. Primer sets used during amplification of Plasmodium falciparum DNA.

(DOC)

Click here for additional data file.^{(47KB, doc)}

Acknowledgments

We acknowledge the guidance of the laboratory team at the Infectious Disease Research Collaboration (IDRC)-Makerere University for their guidance.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The review has received funding from a Grant, Number D43TW010132, supported by Ofce of the Director, National Institutes of Health (OD), National Institute of Dental & Craniofacial Research (NIDCR), National Institute of Neurological Disorders and Stroke (NINDS), National Heart, Lung, and Blood Institute (NHLBI), Fogarty International Center (FIC), and National Institute on Minority Health and Health Disparities (NIMHD). Additional funding was obtained from Malaria training Grant Number D43TW010526. The funders have had no role in the design of the study and in writing of the review protocol.

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PLoS One. 2022 May 5;17(5):e0268095. doi: 10.1371/journal.pone.0268095.r001

Author response to previous submission

5 Nov 2021

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PLoS One. doi: 10.1371/journal.pone.0268095.r002

Decision Letter 0

Luzia Helena Carvalho

13 Dec 2021

PONE-D-21-33254Prevalence of arps10, fd, mdr-2 and pfkelch13 gene mutations in Plasmodium falciparum parasite population in UgandaPLOS ONE

Dear Dr. Ocan,

Thank you for submitting your manuscript to PLoS ONE. After careful consideration, we felt that your manuscript requires revision, following which it can possibly be reconsidered. As quoted by a different reviewer, major concerns were still related to study design and data presentation. According to the reviewers, the methods were not described in enough details to allow suitably skilled investigators to fully replicate and evaluate the study. In addition, a significant number of issues should be clarified and/or adjust otherwise the MS’s results may be compromised. For your guidance, a copy of the reviewers' comments was included below

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Comments:

The MS “Prevalence of arps10, fd, mdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in Uganda” by Moses Ocan et al., presents useful molecular surveillance information on the polymorphisms in the genes responsible for driving development of resistance to artemisinins.

Major comments:

1) Abstract/Results

It is difficult to follow the results description in the Abstract and Results as there is no clear demarcation between the SNP prevalence before 2005 and after 2015, because the authors report the prevalences in the pooled sample set. In my view, the analysis (in the Results and the Abstract) should be presented for each cohort separately (as a Table) and basic statistical analysis (presenting P values and confidence intervals) should be done to claim any significant difference in the point prevalences of SNPs before and after introduction of the ACTs. In the current version it is not clear if the following statement in lines 52-53 can be made “This is an indicator of the role Artemisinin combination therapies play in altering P. falciparum parasite genotype and potentially driving resistance development”. I suggest to change it emphasizing the importance of molecular surveillance in order to detect emerging resistance.

2) Introduction still needs more information on ACTs used in these areas, their efficacies and any evidence of resistance developing. Recommend to add references such as Assefa, D.G., Zeleke, E.D., Bekele, D. et al. Efficacy and safety of dihydroartemisinin–piperaquine versus artemether–lumefantrine for treatment of uncomplicated Plasmodium falciparum malaria in Ugandan children: a systematic review and meta-analysis of randomized control trials. Malar J 20, 174 (2021).

3) Information on molecular markers of artemisinin resistance in Africa needs to be added, for example, found in recent refs: Tumwebaze PK, Katairo T, Okitwi M, Byaruhanga O, Orena S, Asua V, Duvalsaint M, Legac J, Chelebieva S, Ceja FG, Rasmussen SA, Conrad MD, Nsobya SL, Aydemir O, Bailey JA, Bayles BR, Rosenthal PJ, Cooper RA. Drug susceptibility of Plasmodium falciparum in eastern Uganda: a longitudinal phenotypic and genotypic study. Lancet Microbe. 2021 Sep;2(9):e441-e449.

4) There is also a similar study (Conrad MD, Nsobya SL, Rosenthal PJ. The Diversity of the Plasmodium falciparum K13 Propeller Domain Did Not Increase after Implementation of Artemisinin-Based Combination Therapy in Uganda. Antimicrob Agents Chemother. 2019;63(10):e01234-19.) conducted in the same areas, where the authors found “No differences in diversity following implementation of ACT use were found at any of the seven sites, nor was there evidence of selective pressures acting on the locus. Our results suggest that selection by ACTs is not impacting on K13PD diversity in Uganda”. This ref and information should be discussed in Introduction and Discussion.

5) Materials and Methods. Study collection times need to be clarified.

Lines 124-125: Suggest specify enrolment time as in ref 16: “… with enrolment conducted from November 2004 to April 2005 in Kampala (16).

Ref 17 has incorrect publication year 2020 instead of 2015 in

Kamya MR, Arinaitwe E, Wanzira H, Katureebe A, Barusya C, Kigozi SP, et al. Malaria Transmission, Infection, and Disease at Three Sites with Varied Transmission Intensity in Uganda: Implications for Malaria Control. Am J Trop Med Hyg. 2020;92(5):903-12.

It should be “Malaria transmission, infection, and disease at three sites with varied transmission intensity in Uganda: implications for malaria control.

Kamya MR, Arinaitwe E, Wanzira H, Katureebe A, Barusya C, Kigozi SP, Kilama M, Tatem AJ, Rosenthal PJ, Drakeley C, Lindsay SW, Staedke SG, Smith DL, Greenhouse B, Dorsey G. Am J Trop Med Hyg. 2015 May;92(5):903-12”.

This study collection time is specified in this reference as “between August 2011 to September 2013” not “to 2018” as stated in the MS. This requires further clarification.

As only a subset from these studies being used in the present study the detailed description of how many samples from each site were included in either cohort would be beneficial.

6) Line 134 “For our study, 100 P. falciparum infected blood samples each collected in 2005 and 2015 by Dorsey et al., (16) and Kamya et al., (17), respectively were randomly selected for inclusion. In addition, only samples with ≥50ng/µl of DNA were included in the study.”

The statement needs to be rephrased as samples were not exactly collected in 2005 or 2015. This needs to be addressed throughout the entire text.

How many samples from each site were included and how randomization was achieved?

Also suggest to change to “100 randomly selected samples with ≥50ng/µl of DNA were included into the study” .

6). Line 155: It is not clear if the primers for K13 amplification were designed by the authors or just synthesized based on previously published ones by Ariey et al. 2014? Did authors designed the primers for other genes SNPs?

7) Supplement A includes the tables also included in S1 and S2 supplementary files, which is redundant.

8) Lines 193 and 218” Authors refer to carrying “amplicon sequencing”, perhaps, meaning “PCR amplicon”, which is a little bit confusing since this term is typically used for deep amplicon sequencing, where the libraries are generated from a PCR product and sequenced to identify rare mutations. In this study it appears that authors have carried out Sanger sequencing of PCR products. If that is the case, I suggest, that the authors remove “amplicon” and refer to their sequencing methodology as “Sanger sequencing of PCR products”. It also follows, that it is not possible to determine the multiplicity of infections by analysing the Sanger sequencing chromatograms peaks (e.g., of K13 genes). It would be possible to determine the dominant alleles, but this method lacks sensitivity and unable to determine minor alleles (as outlined in Zhong, D., Koepfli, C., Cui, L. et al. Molecular approaches to determine the multiplicity of Plasmodium infections. Malar J 17, 172 (2018). Analysis of highly polymorphic markers (genes or microsattelites) is used to estimate MOI.

9) Lines 302… The statement in conclusions is correct but without statistical analysis can be misleading: “The proportions of pfkelch13, arsp10, and mdr-2 gene mutations were higher in P. falciparum parasites collected a decade after introduction of artemisinin combination therapies in malaria treatment in Uganda.”

Minor comments:

1) For consistency, the genes’ abbreviations need to be the same throughout the text, for an example mdr-2 or pfmdr2. There are typos in arps10 gene name in lines 85 and 109.

2) Likewise, aminoacid abbreviations used in some cases are 3-letter ones whereas in others are 1-letter abbreviation -needs to be consistent throughout the text.

2). It is more appropriate to use single nucleotide polymorphism (SNP) term rather than mutations, for an example when referring to K13 polymorphisms.

3). Ref 13 and 27 are the same.

4) Lines 96 “Wide spread chloroquine resistance followed by its subsequent withdrawal…” suggest to change to “Wide spread of resistance to chloroquine followed by its subsequent withdrawal…”

Line 148: add GmbH and the country information to Qiagen company reference in the following sentence: “P. falciparum genomic DNA was extracted using QIAamp DNA mini kit (Qiagen, GmbH, Germany).

Reviewer #2: Reviewer’s comments:

The authors describe the prevalence of Pfkelch 13 gene mutations and other background genetic mutations thought to associate with artemisinin resistance. The author utilised available samples at their disposal to answer the research question. While I appreciate the efforts of the authors, I shall request that the following corrections be made before acceptance:

#1- The author should state clearly if the mutations found in the Pfkelch 13 gene were among associated to artemisinin resistance. This should be clear as from abstract through discussion.

#2- The author should so kindly introduce some statistical analysis like fishers’ exact test or chi square to compare the proportion of mutations before and after introduction of ACT in Uganda in his samples. This is important!!!

Line 42#- the sentence should be corrected.

Line 81-82# I went through the reference provided by the author and it appears that it didn’t support the statement. The author should find an appropriate reference to that statement.

Line 217# . Could the author please make a clear table showing the mutations that occurred in 2005 and that which occurred in 2015. The table is not interesting in its current form and does not represent the line of thoughts in the paper.

Line 254 -225#- The author seems to contradict his basis for accessing the background mutations. In line 81-86, the author mentioned that the background mutations were associated with artemisinin resistance in different geographical areas in Southeast Asia, but here he presents that their role in artemisinin resistance is not known. I suggest that the authors should think around this and rephrase accordingly.

Line 298-300# - The author should support the statement with statistics.

Line 295-300#- The author should fit in one or two implications of his findings in the conclusion to make a bit stronger.

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Reviewer #1: No

Reviewer #2: Yes: Ikegbunam Moses (PhD)

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PLoS One. 2022 May 5;17(5):e0268095. doi: 10.1371/journal.pone.0268095.r003

Author response to Decision Letter 0

5 Jan 2022

RESPONSE TO THE REVIEWER’S COMMENTS

Reviewer #1: Comments:

Response: Thanks for the comment

Reviewer #1: Comments: Major comments:

1) Abstract/Results

Response: Re-analysis of the data has been done to reflect prevalence of mutations in the different years. For K13 SNPs overall prevalence is 4.2% (6/142) while in 2005 (1.4%, 2/142) and 2013 (2.8%, 4/142). For the pfmdr-2 mutations, overall prevalence is 39.8% (74/186) while in 2005 (2.2%, 4/186) and in 2013 (37.6%, 70/186). For arps10 mutations, overall prevalence is 2.7% (2/74) while in 2013 (2.7%, 2/74) and this mutation was not detected among samples collected in 2005. There was no significant relationship in the prevalence of mutations in the K13 (p=0.087) and arps10 (p=0.146) genes for samples collected in 2005 and 2013. There was a significant relationship in the prevalence of mutations in the pfmdr-2 gene, p<0.001 among samples collected in 2005 and 2013.

Detailed results on this analysis is presented in Table 4 in the revised manuscript.

This revision has been effected in the revised manuscript.

Reviewer #1: Comments: 2) Introduction still needs more information on ACTs used in these areas, their efficacies and any evidence of resistance developing. Recommend to add references such as Assefa, D.G., Zeleke, E.D., Bekele, D. et al. Efficacy and safety of dihydroartemisinin–piperaquine versus artemether–lumefantrine for treatment of uncomplicated Plasmodium falciparum malaria in Ugandan children: a systematic review and meta-analysis of randomized control trials. Malar J 20, 174 (2021).

Response: Thanks for the comment, the requested information is mentioned in the first paragraph of the manuscript. However, we have added further information on the different ACTs used in Uganda. In Uganda, the recommended first-line and second-line agents in treatment of uncomplicated malaria are Artemether-lumefantrine (AL) and Dihydroartemisin-Piperaquine (DHP-PPQ) respectively (MoH, 2011). A recent systematic review reported a slightly better efficacy of DHP-PPQ compared to AL in the treatment of uncomplicated malaria among children under five years in Uganda (Assefa et al., 2021).

This has been incorporated in the revised manuscript.

Reviewer #1: Comments: 3) Information on molecular markers of artemisinin resistance in Africa needs to be added, for example, found in recent refs: Tumwebaze PK, Katairo T, Okitwi M, Byaruhanga O, Orena S, Asua V, Duvalsaint M, Legac J, Chelebieva S, Ceja FG, Rasmussen SA, Conrad MD, Nsobya SL, Aydemir O, Bailey JA, Bayles BR, Rosenthal PJ, Cooper RA. Drug susceptibility of Plasmodium falciparum in eastern Uganda: a longitudinal phenotypic and genotypic study. Lancet Microbe. 2021 Sep;2(9):e441-e449.

Response: According to a number of studies, K13 mutations in Africa are very diverse and its probable that each study is likely to find slightly different K13 mutations. A statement on the diversity of K13 mutations in sub-Saharan Africa is provided in the earlier version of the manuscript and we think that this is sufficient information based on the current evidence around K13 mutations in Africa. Additionally, a paper by Conrad et al., 2019, reports no increase in the diversity of K13 mutations however, like our current study the article finds a general increase in the prevalence of K13 mutations in the period after introduction of ACTs in malaria treatment in Uganda. A statement on the relationship between introduction of ACTs and the diversity of K13 mutations has been incorporated in the revised manuscript.

This information has been incorporated in the revised manuscript

Reviewer #1: Comments 5) Materials and Methods. Study collection times need to be clarified.

Lines 124-125: Suggest specify enrolment time as in ref 16: “… with enrolment conducted from November 2004 to April 2005 in Kampala (16).

Response: Yes, sorry for the lack of clarity on this. We picked samples for our current study from those which were collected in 2005. This statement has been incorporated in the revised manuscript.

Reviewer #1: Comments: Ref 17 has incorrect publication year 2020 instead of 2015 in

It should be “Malaria transmission, infection, and disease at three sites with varied transmission intensity in Uganda: implications for malaria control.

Response: Thanks for the comment. This has been corrected in the revised manuscript.

Reviewer #1: Comments: Kamya MR, Arinaitwe E, Wanzira H, Katureebe A, Barusya C, Kigozi SP, Kilama M, Tatem AJ, Rosenthal PJ, Drakeley C, Lindsay SW, Staedke SG, Smith DL, Greenhouse B, Dorsey G. Am J Trop Med Hyg. 2015 May;92(5):903-12”.

This study collection time is specified in this reference as “between August 2011 to September 2013” not “to 2018” as stated in the MS. This requires further clarification.

Response: Thanks for the comment. We are sorry for the lack of clarity on this. The dates for sample collections have been corrected. For our study we analyzed samples collected in 2013. This has been clarified in the revised manuscript.

Reviewer #1: Comments: As only a subset from these studies being used in the present study the detailed description of how many samples from each site were included in either cohort would be beneficial.

Response: In our current study only samples from Jinja and Kampala were used. Of which 100 samples each from Jinja and Kampala were picked.

This has been provided in the revised manuscript.

Reviewer #1: Comments: 6) Line 134 “For our study, 100 P. falciparum infected blood samples each collected in 2005 and 2015 by Dorsey et al., (16) and Kamya et al., (17), respectively were randomly selected for inclusion. In addition, only samples with ≥50ng/µl of DNA were included in the study.”

The statement needs to be rephrased as samples were not exactly collected in 2005 or 2015. This needs to be addressed throughout the entire text.

Response: Thanks for the comment, a study by Dorsey et al., (16), a single-blind randomized clinical trial was conducted between November 2004 and June 2006. In our current study, were accessed 100 blood samples collected in 2005. For a study by Kamya et al., (17), the study was conducted from August 5, 2011 to September 30, 2013. We accessed 100 blood samples collected in 2013. This has been corrected in the revised manuscript.

Reviewer #1: Comments: How many samples from each site were included and how randomization was achieved?

Also suggest to change to “100 randomly selected samples with ≥50ng/µl of DNA were included into the study” .

Response: A study by Kamya et al., (17) was part of a larger PRISM 2 study which was conducted in three districts of Jinja, Kanungu and Tororo while the study by Dorsey et al., (16) was conducted only in Kampala. For our current study, only 100 samples each from Jinja and Kampala were used. This has been incorporated in the revised manuscript.

Reviewer #1: Comments: 6). Line 155: It is not clear if the primers for K13 amplification were designed by the authors or just synthesized based on previously published ones by Ariey et al. 2014? Did authors designed the primers for other genes SNPs?

Response: Thanks for the comment. For K13 gene, we synthesized the primers based on previously published primers in a study by Ariey et al., 2014. For pfmdr-2, arps10, fd and pfcrt genes, the primers published in a study by Miotto et al., were double checked using NCBI primer 3 software. The primers reported in a study by Miotto et al., were then synthesized. All the primers were synthesized by Eurofins scientific. This has been corrected in the revised manuscript.

Reviewer #1: Comments :7) Supplement A includes the tables also included in S1 and S2 supplementary files, which is redundant.

Response: Thanks for the observation, ‘Supplement A’ has been removed/deleted from the manuscript files in the revised submission.

Reviewer #1: Comments: 8) Lines 193 and 218” Authors refer to carrying “amplicon sequencing”, perhaps, meaning “PCR amplicon”, which is a little bit confusing since this term is typically used for deep amplicon sequencing, where the libraries are generated from a PCR product and sequenced to identify rare mutations. In this study it appears that authors have carried out Sanger sequencing of PCR products. If that is the case, I suggest, that the authors remove “amplicon” and refer to their sequencing methodology as “Sanger sequencing of PCR products”.

Response: Thanks for the comments, this has been corrected in the revised manuscript. The statement now reads, ‘Sanger sequencing of PCR products’.

Reviewer #1: Comments: It also follows, that it is not possible to determine the multiplicity of infections by analysing the Sanger sequencing chromatograms peaks (e.g., of K13 genes). It would be possible to determine the dominant alleles, but this method lacks sensitivity and unable to determine minor alleles (as outlined in Zhong, D., Koepfli, C., Cui, L. et al. Molecular approaches to determine the multiplicity of Plasmodium infections. Malar J 17, 172 (2018). Analysis of highly polymorphic markers (genes or microsattelites) is used to estimate MOI.

Response: The limitation of analyzing Sanger sequencing chromatogram peaks in determining multiplicity of infections has been included in the revised manuscript. We have performed statistical analysis of the data to support the statement in the conclusion.

Minor comments:

Reviewer #1: Comments: 1) For consistency, the genes’ abbreviations need to be the same throughout the text, for an example mdr-2 or pfmdr2. There are typos in arps10 gene name in lines 85 and 109.

Response: Thanks for the comment, this has been adjusted in the revised manuscript.

Reviewer #1: Comments: 2) Likewise, aminoacid abbreviations used in some cases are 3-letter ones whereas in others are 1-letter abbreviation -needs to be consistent throughout the text.

Response: This has been corrected in the revised manuscript.

Reviewer #1: Comments: 2). It is more appropriate to use single nucleotide polymorphism (SNP) term rather than mutations, for an example when referring to K13 polymorphisms.

Response: This has been adjusted in the revised manuscript

Reviewer #1: Comments: 3). Ref 13 and 27 are the same.

Response: Thanks for the comment, this has been corrected in the revised manuscript.

Reviewer #1: Comments: 4) Lines 96 “Wide spread chloroquine resistance followed by its subsequent withdrawal…” suggest to change to “Wide spread of resistance to chloroquine followed by its subsequent withdrawal…”

Response: Thanks, this has been changed to “Wide spread of resistance to chloroquine followed by its subsequent withdrawal…”, in the revised manuscript.

Reviewer #1: Comments: Line 148: add GmbH and the country information to Qiagen company reference in the following sentence: “P. falciparum genomic DNA was extracted using QIAamp DNA mini kit (Qiagen, GmbH, Germany).

Response: Thanks for the comment, this has been revised to “P. falciparum genomic DNA was extracted using QIAamp DNA mini kit (Qiagen, GmbH, Germany) in the corrected manuscript.

Reviewer #2: Reviewer’s comments:

#1- The author should state clearly if the mutations found in the Pfkelch 13 gene were among associated to artemisinin resistance. This should be clear as from abstract through discussion.

Response: Thanks for the comments, in our manuscript this information is clearly stated in the discussion section. The K13 mutations found in our study have not among those that have been validated to be associated with artemisinin resistance.

Reviewer #2: Reviewer’s comments: #2- The author should so kindly introduce some statistical analysis like fishers’ exact test or chi square to compare the proportion of mutations before and after introduction of ACT in Uganda in his samples. This is important!!!

Response: Thanks, we have performed statistical analysis and have incorporated the findings in the results section of the manuscript.

Reviewer #2: Reviewer’s comments Line 42#- the sentence should be corrected.

Response: Thanks, we have corrected the sentence.

Reviewer #2: Reviewer’s comments: Line 81-82# I went through the reference provided by the author and it appears that it didn’t support the statement. The author should find an appropriate reference to that statement.

Response: Thanks for the comment, we have provided an alternative reference in the revised manuscript.

Reviewer #2: Reviewer’s comments: Line 217#. Could the author please make a clear table showing the mutations that occurred in 2005 and that which occurred in 2015. The table is not interesting in its current form and does not represent the line of thoughts in the paper.

Response: Thanks for the comment, the mutations that occurred in 2005 and 2015 have been clearly indicated in the table in the revised manuscript.

Reviewer #2: Reviewer’s comments: Line 254 -225#- The author seems to contradict his basis for accessing the background mutations. In line 81-86, the author mentioned that the background mutations were associated with artemisinin resistance in different geographical areas in Southeast Asia, but here he presents that their role in artemisinin resistance is not known. I suggest that the authors should think around this and rephrase accordingly.

Response: Thanks for this observation however, this statements are not contradictory. A study by Miotto et al., 2011 confirmed the role of certain background mutations; fd-D193Y (ferredoxin gene), arps10-V127M (apicoplast ribosomal protein S10 gene), pfmdr2-T484I (multidrug resistance protein 2 gene), crt-I356T (chloroquine resistance transporter gene) and crt-N326S (chloroquine resistance transporter gene) in supporting the development of K13 mutations associated with artemisinin resistance. In our study we found only one mutation arsp10-V127M in one parasite sample collected in 2013 that had been previously reported in a study by Miotto et al. However, different background mutations from those reported by Miotto et al., were found in other genes and this are the mutations which the statement is referring to. This is because the role of the different background mutations that we found in our study is not known as they have not been confirmed/validated. This has been clarified in the revised manuscript.

Reviewer #2: Reviewer’s comments: Line 298-300# - The author should support the statement with statistics.

Response: Thanks, we have performed statistical analysis and provided the results to support the statement.

Reviewer #2: Reviewer’s comments: Line 295-300#- The author should fit in one or two implications of his findings in the conclusion to make a bit stronger.

Response: Thanks, this has been effected in the revised manuscript.

Attachment

Submitted filename: RESPONSE TO THE REVIEWER_dec21.doc

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PLoS One. doi: 10.1371/journal.pone.0268095.r004

Decision Letter 1

Luzia Helena Carvalho

31 Jan 2022

PONE-D-21-33254R1Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in UgandaPLOS ONE

Dear Dr. Ocan,

Thank you for submitting your manuscript to PLoS ONE. After careful consideration, we feel that your manuscript will likely be suitable for publication if the authors revise it to address specific points raised now by the reviewer. According to the reviewer, there are some specific areas where further improvements would be of substantial benefit to the readers. For your guidance, a copy of the reviewers' comments was included below.

Please submit your revised manuscript by Mar 17 2022 11:59PM If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: The manuscript has been substantially improved since last revision. I believe the data presented in the paper are important and worthy of publication, but the paper still requires some work.

Below are some comments, which I believe still need to be addressed.

Major comments

(Lines numbers are based on numbers in the pdf version containing track changes.)

1). Authors have now added Table 4 to the Results section that shows the breakdown in prevalence of mutations in years 2005 and 2013, which is appreciated. However, there are errors in calculations for the 2005 and 2013 prevalence for all molecular markers. In this table the overall frequency of SNPs (for both years 2005 and 2013) is calculated correctly as total number of SNPs divided by total number of samples analysed for both years. However, when the authors calculate the prevalence in years 2005 or 2013, separately, the authors also use the total number of samples in the denominator, rather than using the number of samples from the respective year. This is not correct.

For an example, for SNP prevalence in K13 gene the total number of samples in which K13 gene was successfully sequenced was 142, of which 51 samples were collected in 2005 and 91 were collected in 2013. For overall (2005 and 2013) prevalence of K13 SNPs authors use the following calculation:

[6 SNPs)/142]*100%=4.2%, which is correct. However, for the year 2005 the prevalence is calculated as (2/142)* 100%=1.4% and for 2013 as (4/142)* 100%=2.8%, which is not correct. It should be calculated as (2/51)*100%= 3.9% and (4/91)*100%=4.4% for 2013, respectively. The same considerations and corrections would apply to other molecular markers analysed in the study (pfmdr-2 and arps10). Consequently, the contingency analysis for each SNP for 2005 and 2013 should be done with P-values calculated using exact Fisher’s test with 95% confidence intervals also recalculated for all markers/SNPs. This would also may change the conclusions reached in the paper and mentioned in the abstract.

I also suggest to include the number of samples analysed for every molecular marker gene for each year in the table 4 for ease of understanding as shown below:

Characteristic Molecular Marker/SNPs Collection Year Prevalence, % (n/N) P-value 95% CI

Pfkelch13 Overall 4.2 (6/142)

2005 1.4 3.9 (2/51)

2013 2.8 4.3 (4/91)

2). Lines: 37 (abstract), 128, 232, 236, 241, 280, 283, 285, 294. In these lines there are still references to 2015, which is incorrect as authors have confirmed that it should be 2013, the year when samples were collected. Could authors please check this for consistency throughout the text.

3). Lines 233-239: When describing the SNPs in Pfkelch13 gene, authors should refer to Table 3 initially, and then to Table 4 when describing numbers and prevalence in years 2005 and 2013. I also could not find Tables 1 and 2 in the main Text. Could authors please check and change the Table numbers if this is the case.

4). Authors might want to consider changing the titles for the Table 3 “Single nucleotide polymorphisms identified in pfkelch13 gene in samples collected in Kampala and Jinja, Uganda in 2005 and 2013.” Also, in footnotes in Table 3 it would be good to add the year when polymorphism was observed in those samples.

Also, a suggestion for Table 4 title would be “Prevalence of single nucleotide polymorphisms in pfkelch13, Pfmdr-2 and Pfarps 10 genes in Plasmodium falciparum samples collected in Kampala and Jinja, Uganda in 2005 and 2013”, instead of “Table 4: Distribution of the P. falciparum parasite mutations by year of sample collection, 2005 and 2013 (K13, n=142; pfmdr-2, n =186; arps10, n = 74)”.

I suggest to remove the word “different” from description of SNPs in pfmdr-2 and arps 10 genes, because it includes the SNPs at 484 and 127 in those genes reported by Miotto et al., 2015. The explanation in the text would be sufficient.

5) Abstract:

“In Uganda, Artemether-Lumefantrine and Artesunate are recommended for uncomplicated and 31 severe malaria respectively, but are currently threatened by parasite resistance”-I think the introductory sentence should be reworded as at present the data do not support resistance threat for this combination in Africa.

Also, suggest to replace “transcriptional” with “epigenetic” factors, implying changes in transcription.

In the abstract the actual SNPs, which were analysed in the present study¬ in the molecular markers genes should be stated.

The sentence “Overall, prevalence of SNPs in pfmdr-2 gene was 39.8% (74/186), of this 2.2% (4/186), 37.6% (70/186) occurred in 2005 and 2013 samples respectively” is misleading as it only refers to 2 “other” SNPs Y423F and I492V (not mentioned as Southeast Asian background mutations), but not to the SNP at pfmdr-2 T424I SNP reported by (Miotto et al., 2015). This need to be clarified in the abstract.

The prevalence numbers for all SNPs need to be recalculated with the number of samples analysed in the respective year used in the denominator. These may change the conclusion “There were more pfkelch13, arps10 and pfmdr-2 SNPs gene mutations in P. falciparum parasites collected a decade (2015) after introduction of Artemisinin combination therapies in malaria treatment.”

6). I believe that Discussion needs to be amended to reflect the following points:

1) The majority of samples before and after introduction of the ACTs are the wild type with only few SNPs identified in a very low fraction of samples (needs to be recalculated and presented). Importantly, they were different SNPs detected after introduction of ACTs, compared to those detected prior to, albeit all occurred at very low frequencies before and after introduction of the ACT.

2) As the K13 SNPs detected before and after introduction of ACTs are different it has to be stressed when making a comparison of K13 SNPs and conclusion that we see “There were more pfkelch13, arps10 and pfmdr-2 SNPs gene mutations in P. falciparum parasites collected a decade (2015) after introduction of Artemisinin combination therapies in 64 malaria treatment”. (lines 62-64) and in the discussion (Lines 320-322): “Additionally, our study found a general increase in the prevalence of K13 SNPs in samples collected after (2013) introduction of ACTs in malaria treatment in Uganda. It is likely that statistical analysis of the data would support this conclusion.

3) Importantly, there were no SNPs that were previously reported in Uganda “Balikagala et al., (34) demonstrated delayed artemisinin clearance among parasites carrying 675V and 469Y mutations in Uganda” (line 328-329) found in this study. These findings and their implications for the efficacy of the ACTs and potential for the resistance should be discussed further.

4) Absence of mutations in pfmdr-2 (T484I) and arps10 (V127M) in Africa was previously noted by Miotto et al 2015, with conclusion that these background mutations are specific to Southeast Asia. These findings have been subsequently confirmed by other recent studies that can be added to the discussion (Diakité, Seidina A S et al. “A comprehensive analysis of drug resistance molecular markers and Plasmodium falciparum genetic diversity in two malaria endemic sites in Mali.” Malaria journal vol. 18,1 361. 12 Nov. 2019, doi:10.1186/s12936-019-2986-5). Also findings on pfmdr-2 SNPs from Hussien M at al., Antimalarial drug resistance molecular makers of Plasmodium falciparum isolates from Sudan during 2015-2017. PLoS One. 2020 Aug 20;15(8)) should be discussed.

5) There were no mutations in Pfcrt N326S observed in the present study, whereas in Hussian et al there were 46.3% 96/207 samples carrying this SNP. This needs to be discussed.

6) Discussion on the importance in rise of other mutations in pfmdr-2 I492V 18.8? (35/?) 12.0 -39.0 Y423F 18.8 ?(35/?) needs to be added as these SNPs are the only ones that significantly more prevalent in 2013 (needs to be supported by stat. analysis).

Minor comments

Line 192: abbreviation PCR can be used as it has been previously defined. The same can be said about using SNP abbreviation throughout the text after it was first defined.

Lines 198: “double checked based on the 3D7 genome…” replace were tested

Line 234: (4.2%, 6/142) K13 single nucleotide polymorphisms (SNPs) were detected (Table 4). Of these, five (5) were non-synonymous and one (1) was a synonymous K13 mutation, (I587I).

Lines 235-236: four (54) (error), one (1) K13 non-synonymous mutations in the P. falciparum parasite samples collected 236 in 2015 and 2005 respectively.

In some sentences throughout the text numbers are duplicated in brackets in others are not. Please use consistent convention in the paper. Also, when listing SNPs it would be better to use their aa position numbers in the ascending order. For an example,

Line 238-241: One (1) K13 non-synonymous SNP Y588N occurred in a P. falciparum parasite sample collected in 2005. Five (5) K13 non-synonymous SNPs A578S, E596V, S600C and E643K and were detected in P. falciparum parasite samples collected in 2015.

Also, please use the same order (ascending) in the Table 4.

Line 276-278: Polymorphisms in the fd, pfmdr -2 and pfcrt genes that are markers of a genetic background where pfkelch13 mutations associated with slow artemisinin parasite clearance are likely to arise previously reported in Southeast Asia (Miotto, 20160 were not found in our study

Line 277: Pf mdr – 2 replace with pfmdr-2. Also, in some instances authors use pfkelch 13 and others K13 gene name. Please, use the same name throughout.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Attachment

Submitted filename: Reviewers comments MC 20220126.docx

Click here for additional data file.^{(19.8KB, docx)}

PLoS One. 2022 May 5;17(5):e0268095. doi: 10.1371/journal.pone.0268095.r005

Author response to Decision Letter 1

9 Feb 2022

RESPONSE TO REVIEWER’S COMMENTS ON MANUSCRIPT PONE-D-21-33254R1

We thank the reviewers for the comments on our manuscript, addressing this comments have improved the manuscript. Here below is the summary of our responses to the comments raised by the reviewers.

Comment Reviewer #1: The manuscript has been substantially improved since last revision. I believe the data presented in the paper are important and worthy of publication, but the paper still requires some work.

Below are some comments, which I believe still need to be addressed.

Major comments

(Lines numbers are based on numbers in the pdf version containing track changes.)

Response: Thanks for the observation, this has been addressed in the revised manuscript

Comment Reviewer #1: 1). Authors have now added Table 4 to the Results section that shows the breakdown in prevalence of mutations in years 2005 and 2013, which is appreciated. However, there are errors in calculations for the 2005 and 2013 prevalence for all molecular markers. In this table the overall frequency of SNPs (for both years 2005 and 2013) is calculated correctly as total number of SNPs divided by total number of samples analysed for both years. However, when the authors calculate the prevalence in years 2005 or 2013, separately, the authors also use the total number of samples in the denominator, rather than using the number of samples from the respective year. This is not correct.

I also suggest to include the number of samples analysed for every molecular marker gene for each year in the table 4 for ease of understanding as shown below:

Characteristic Molecular Marker/SNPs Collection Year Prevalence, % (n/N) P-value 95% CI

Pfkelch13 Overall 4.2 (6/142)

2005 1.4 3.9 (2/51)

2013 2.8 4.3 (4/91)

Response: Thanks for the comments, the re-analysis has been done using Fisher’s Exact test and the results are presented in Table 4. We have included the requested numbers in the revised table.

Comment Reviewer #1: 2). Lines: 37 (abstract), 128, 232, 236, 241, 280, 283, 285, 294. In these lines there are still references to 2015, which is incorrect as authors have confirmed that it should be 2013, the year when samples were collected. Could authors please check this for consistency throughout the text.

Response: Thanks for the observation. We have corrected the year to 2013 all through the revised manuscript.

Comment Reviewer #1: 3). Lines 233-239: When describing the SNPs in Pfkelch13 gene, authors should refer to Table 3 initially, and then to Table 4 when describing numbers and prevalence in years 2005 and 2013. I also could not find Tables 1 and 2 in the main Text. Could authors please check and change the Table numbers if this is the case.

Response: Tables 1 and 2 are referenced in the main text under the methods section but provided as additional material. Revision has been made on reference to Table 3 first when describing the Pfkelch13 gene.

Comment Reviewer #1: 4). Authors might want to consider changing the titles for the Table 3 “Single nucleotide polymorphisms identified in pfkelch13 gene in samples collected in Kampala and Jinja, Uganda in 2005 and 2013.” Also, in footnotes in Table 3 it would be good to add the year when polymorphism was observed in those samples.

Response: This correction has been effected in the revised manuscript

Comment Reviewer #1: 5) Abstract:

Also, suggest to replace “transcriptional” with “epigenetic” factors, implying changes in transcription.

In the abstract the actual SNPs, which were analysed in the present study¬ in the molecular markers genes should be stated.

Response: The introductory sentence is appropriate and current data does support resistance threat to artemisinin agents in Africa as shown in a study by Balikagala et al., 2021. This sentence has been maintained in the revised manuscript.

The word ‘transcriptional’ has been replaced with ‘epigenetic’ in the revised manuscript.

The sentence “Overall, prevalence of SNPs in pfmdr-2 gene was 39.8% (74/186), of this 2.2% (4/186), 37.6% (70/186) occurred in 2005 and 2013 samples respectively is appropriate. However, we have removed the word ‘overall’ in the revised manuscript.

The prevalence of all the SNPs has been recalculated however, the direction of the findings remained the same and thus the conclusion “There were more pfkelch13, arps10 and pfmdr-2 SNPs gene mutations in P. falciparum parasites collected a decade (2015) after introduction of Artemisinin combination therapies in malaria treatment.” Has been maintained in the revised manuscript.

Comment Reviewer #1: 6). I believe that Discussion needs to be amended to reflect the following points:

5) There were no mutations in Pfcrt N326S observed in the present study, whereas in Hussian et al there were 46.3% 96/207 samples carrying this SNP. This needs to be discussed.

Response: Thanks for the comment, the discussion has been amended to reflect the suggestions. The role the different background mutations identified from the genes need to be validated among the African Plasmodium falciparum parasites.

Minor comments

Comment Reviewer #1: Line 192: abbreviation PCR can be used as it has been previously defined. The same can be said about using SNP abbreviation throughout the text after it was first defined.

Response: Thanks, this has been corrected in the revised manuscript

Comment Reviewer #1: Lines 198: “double checked based on the 3D7 genome…” replace were tested

Response: Thanks, this has been revised in the corrected manuscript

Comment Reviewer #1: Line 234: (4.2%, 6/142) K13 single nucleotide polymorphisms (SNPs) were detected (Table 4). Of these, five (5) were non-synonymous and one (1) was a synonymous K13 mutation, (I587I).

Response: This has been corrected in the revised manuscript.

Comment Reviewer #1: Lines 235-236: four (54) (error), one (1) K13 non-synonymous mutations in the P. falciparum parasite samples collected 236 in 2015 and 2005 respectively.

Response: Thanks for the observation, this has been corrected in the revised manuscript.

Comment Reviewer #1: Line 238-241: One (1) K13 non-synonymous SNP Y588N occurred in a P. falciparum parasite sample collected in 2005. Five (5) K13 non-synonymous SNPs A578S, E596V, S600C and E643K and were detected in P. falciparum parasite samples collected in 2015.

Also, please use the same order (ascending) in the Table 4.

Response: Thanks for the comment, this has been corrected in the revised manuscript.

Comment Reviewer #1: Line 276-278: Polymorphisms in the fd, pfmdr -2 and pfcrt genes that are markers of a genetic background where pfkelch13 mutations associated with slow artemisinin parasite clearance are likely to arise previously reported in Southeast Asia (Miotto, 20160 were not found in our study

Response: Thanks for the observation, the citation, Miotto et al., has been included into the sentence in the revised manuscript.

Comment Reviewer #1: Line 277: Pf mdr – 2 replace with pfmdr-2. Also, in some instances authors use pfkelch 13 and others K13 gene name. Please, use the same name throughout.

Response: Thanks for the comment, this has been corrected in the revised manuscript.

Attachment

Submitted filename: RESPONSE TO REVIEWER.doc

Click here for additional data file.^{(38KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0268095.r006

Decision Letter 2

Luzia Helena Carvalho

14 Mar 2022

PONE-D-21-33254R2Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in UgandaPLOS ONE

Dear Dr. Ocan,

Thank you for resubmitting your manuscript for review to PLoS ONE. After careful consideration, we feel that your manuscript will likely be suitable for publication if it is revised to address major points raised by the reviewer. While the subject of the MS was of interest, the authors did not properly address relevant topics raised during the peer review process.At this time, we strongly recommend that the authors include the modifications requested by the reviewer. For your guidance, a copy of the reviewers' comments was included below.

Please submit your revised manuscript by March 25. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have partially addressed some of my previously mentioned points. However, I believe that the paper still requires further improvements and authors should address the following points raised in my previous comments (please see below).

1). The Abstract/Results/Discussion (as previously mentioned)

The aim of the paper was stated in the Introduction (lines 113-116) as “We therefore, intended in this study to assess and 114 compare the prevalence of fd-D193Y, aps10-V127M, pfmdr2-T484I, crt-I356T, crt-N326S and 115 K13 mutations in P. falciparum parasites before (2005) and a decade (2013) after introduction of 116 artemisinin-based combination therapies for malaria treatment in Uganda (18)”. Therefore, the SNPs prevalences in those years should be calculated correctly as previously mentioned.

In the present revision authors have provided the break-down in numbers of samples analyzed in 2005 and 2013 and calculated the respective prevalence for each collection time, which are now shown in Table 4. However, the authors have not amended or changed the data in the Abstract and in the Results sections for the prevalence values for 2005 or 2013 to reflect that, and still used the total number of samples analysed for both years overall in the denominator. As the primary aim of this paper is to compare the prevalence of the SNPs in samples collected in 2005 and in 2013 (after introduction of ACTs), the respective prevalence should be calculated correctly. When reporting (throughout the paper), the prevalence values should be accompanied by the fraction (n/N) showing the number of samples with SNPs (n) and N number of samples analysed in this time period, as well as 95% confidence interval (CI). Also, when comparing these values, the authors should state if there is (or not) a statistically significant difference between the prevalence values in 2005 and 2013 (with P-value), which are calculated based on these numbers.

Also, if authors could use the prevalence values in the Discussion rather than just mentioning a particular detected SNP, so the readers could put it in a context and appreciate the significance of the findings.

Please see below the specific examples (line numbers are as in the “Clean” version).

Lines 47-55 (Abstract) and throughout the Results section authors should amend the abstract and the result with the SNP prevalence data for 2005 and 2013 years respectively, where the number of SNPs is divided by the number of samples analysed in that particular time period (i.e. either in 2005 or in 2013), not the combined overall number of samples to match the “Prevalence” data shown in Table 4.

2). I suggest Tables 1 and 2 should be renamed S1 and S2 as they are in the Supplementary material not in the main text and renumber Tables 3 and 4.

3). Table 4. Please use consistent gene names nomenclature in the Table title and in the Table (i.e. Pfkelch 13 or K13).

Column 2 and 3 have (%) in the columns’ headings but don’t actually show these percentages. These columns can be combined with the column “Prevalence, % (n)” if the number of samples (N) analysed per actual time period can be added “Prevalence, % (n/N)” as previously suggested.

4). Results section.

Lines 269 The sentence about the SNPs in pfmdr2 (“There was a significant relationship in the prevalence of mutations in the pfmdr-2 gene….”) should be included in the next section, as this section is about SNPs in K13 gene. Also, could authors state if there is a statistically significant difference between the prevalence values instead of "significant relationship")

Throughout the Results section the actual prevalence values from Table 4 should be used with confidence intervals and P-values for the prevalence values for 2005 and 2013, respectively. It is very important as it allows readers to appreciate the statistical analysis of the data. Also, if authors also could list codons in the ascending order when mentioning SNPs throughout the text (for example in line 278).

Discussion

If authors could use the prevalence values and sample numbers throughout the Discussion as well when commenting on identified SNPs and also compare with those in the referenced published literature.

As the aim of the paper was to detect if the background mutations identified by Miotto et al.in SEA are present in Uganda, it would be more logical to start the Discussion with the findings related to this primary aim and then continue discussing the secondary (also important) findings. I would suggest to rearrange the paragraphs in the Discussion and start the discussion with the text shown in lines 309-319.

319: “Plasmodium falciparum artemisinin resistance is a heritable trait with a genetic basis (7). Genome modification studies have shown that the impact of various K13 pfkelch13 mutations on P. falciparum artemisinin clearance and survival rates of ring stage parasites is dependent on the genetic background (27). The risk of emergence of K13pfkelch13 mutations associated with delayed artemisinin parasite clearance is thus driven by specific parasite genetic background (8, 314 28). In our current study polymorphisms in the fd, pfmdr-2 and pfcrt genes which support the rise of K13 mutations associated with delayed artemisinin clearance of P. falciparum parasites reported in Southeast Asia (8) were not found. However, SNPs in the pfmdr-2, pfcrt, and arps10 genes not reported in a study by Miotto et al.,(8) and other previous studies in Africa (29) were detected in our current study. There is need to validate the role the identified background mutations play in artemisinin resistance development among African P. falciparum parasites.”

Lines 299-302: In the following sentences it is not clear what is actually “similar”: “A recent study in South Sudan (26) which analyzed samples collected in 2015-2017 after introduction of artemisinin agents in malaria treatment detected genetic background mutation in Pfcrt N326S gene which was previously reported in Southeast Asia and associated with artemisinin resistance. This is similar to a finding from our current study where we detected a background mutation, V127M in the arps10 gene that has been shown to support development of K13 mutations associated with artemisinin resistance in Southeast Asia(8)”

Add the following “While we did not find Pfcrt N326S SNP in our samples, we detected…”

“… after introduction of artemisinin agents in malaria treatment detected genetic background mutation in Pfcrt N326S gene which was previously reported in Southeast Asia and associated with artemisinin resistance. While we did not find Pfcrt N326S SNP in our samples, we detected a background mutation, V127M in the arps10 gene that has been also shown to support development of K13 mutations associated with artemisinin resistance in Southeast Asia(8)”.

Line 320: This statement is ambiguous: “We detected different K13 SNPs in P. falciparum samples collected both prior (2005) and after (2013) introduction of artemisinin combination therapies in malaria treatment in Uganda.

Suggest to change the sentence to indicate that K13 SNPs identified in the present study are different to those previously implicated in artemisinin resistance in Africa and SEA. For example, “In our samples collected in 2005 and 2013 we detected K13 SNPs different to those previously implicated in artemisinin resistance (ref). or "We detected different K13 SNPs in P. falciparum samples collected in 2005 compared to those collected in 2013 after introduction of artemisinin combination therapies in malaria treatment in Uganda".

Line 322: Our findings are similar to a recent study in Uganda that reported K13 mutations in the P. falciparum 323 parasites (30)- Needs clarification similar in what way?

Line 323-324. “Additionally, our study found a general increase in the prevalence of K13 SNPs in samples collected after (2013) introduction of ACTs in malaria treatment in Uganda”.

This statement is bit misleading since the is no statistically significant differences in the prevalences of these SNPs before and after introduction of ACTs.

The similar statement in Conclusions (below) can also be misinterpreted with the vagueness of the term “generally”. While it is correct in case of novel SNPs in pfmdr2, differences in SNPs for K13 and arsp10 were not statistically significant. Conclusions, should also reflect the findings related to the primary aim of the study and state that there were neither K13 SNPs previously implicated in artemisinin resistance in Africa and SEA, nor mutations in the associated background genes identified in this study with exception of one SNP in arps10 gene.

Line 331 “The proportions of K13, arsp10, and pfmdr-2 gene mutations were generally higher in P. falciparum parasites collected after introduction of artemisinin combination therapies in malaria treatment in Uganda

Other comments.

Line 34: Spaces are needed between P. and falciparum

Lines 36,56, 115: Saying it is “a decade” between 2005 and 2013 is not accurate-it is 8 years.

Line 257 fcrt replace for pfcrt

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

PLoS One. 2022 May 5;17(5):e0268095. doi: 10.1371/journal.pone.0268095.r007

Author response to Decision Letter 2

30 Mar 2022

RESPONSE TO REVIEWER’S COMMENTS ON MANUSCRIPT PONE-D-21-33254R2

We are grateful to the reviewers for the comments raised on our manuscript. The comments have been addressed and here below is a summary of the point-by-point response to the different questions.

Comment, Reviewer #1: The authors have partially addressed some of my previously mentioned points. However, I believe that the paper still requires further improvements and authors should address the following points raised in my previous comments (please see below).

1). The Abstract/Results/Discussion (as previously mentioned)

Response:

Thanks for this observation, actually the calculations were made as previously requested except that we forgot to remove the denominator ‘142’ and place the correct denominator used in the analysis, 51 (for 2005) and 91 (for 2013) for K13 mutations. This error has been corrected in the revised manuscript.

Comment: In the present revision authors have provided the break-down in numbers of samples analyzed in 2005 and 2013 and calculated the respective prevalence for each collection time, which are now shown in Table 4. However, the authors have not amended or changed the data in the Abstract and in the Results sections for the prevalence values for 2005 or 2013 to reflect that, and still used the total number of samples analysed for both years overall in the denominator.

Response:

Thanks for this observation, this was an editorial error as we forgot to adjust the denominators in the revised manuscript despite the fact that the analysis were done following the adjusted denominators as previously guided. We have adjusted the analysis as requested in the revised manuscript.

Comment: As the primary aim of this paper is to compare the prevalence of the SNPs in samples collected in 2005 and in 2013 (after introduction of ACTs), the respective prevalence should be calculated correctly. When reporting (throughout the paper), the prevalence values should be accompanied by the fraction (n/N) showing the number of samples with SNPs (n) and N number of samples analysed in this time period, as well as 95% confidence interval (CI). Also, when comparing these values, the authors should state if there is (or not) a statistically significant difference between the prevalence values in 2005 and 2013 (with P-value), which are calculated based on these numbers.

Response:

Thanks for the comment, this analysis are clearly stated in table 4. We have further provided the descriptive summaries for the different mutations in the results section of the revised manuscript.

Comment: Also, if authors could use the prevalence values in the Discussion rather than just mentioning a particular detected SNP, so the readers could put it in a context and appreciate the significance of the findings.

Response:

Thanks for the comment, this has been incorporated in the revised manuscript.

Comment: Please see below the specific examples (line numbers are as in the “Clean” version).

Response: This is noted

Comment: Lines 47-55 (Abstract) and throughout the Results section authors should amend the abstract and the result with the SNP prevalence data for 2005 and 2013 years respectively, where the number of SNPs is divided by the number of samples analysed in that particular time period (i.e. either in 2005 or in 2013), not the combined overall number of samples to match the “Prevalence” data shown in Table 4.

Response: Thanks, this has been effected in the revised manuscript

Comment: 2). I suggest Tables 1 and 2 should be renamed S1 and S2 as they are in the Supplementary material not in the main text and renumber Tables 3 and 4.

Response: The information in the tables S1 and S2 in the supplementary material is different from the information in tables 1 and 2 in the main text. We therefore, think the current numbering is appropriate and has been maintained in the revised manuscript.

3). Table 4. Please use consistent gene names nomenclature in the Table title and in the Table (i.e. Pfkelch 13 or K13).

Response: This has been noted and corrected in the revised manuscript

Comment: Column 2 and 3 have (%) in the columns’ headings but don’t actually show these percentages. These columns can be combined with the column “Prevalence, % (n)” if the number of samples (N) analysed per actual time period can be added “Prevalence, % (n/N)” as previously suggested.

Response: This has been noted however, from your previous comment you requested for a clear separation of number of samples which had and those which did not have mutations and we effected. We find it confusing to now request for merging the numbers again. We have however, taken note of the comment and made adjustments in table 4 as indicated in the revised manuscript.

4). Results section.

Comment: Lines 269 The sentence about the SNPs in pfmdr2 (“There was a significant relationship in the prevalence of mutations in the pfmdr-2 gene….”) should be included in the next section, as this section is about SNPs in K13 gene. Also, could authors state if there is a statistically significant difference between the prevalence values instead of "significant relationship")

Response;

This is noted and has been effected in the revised manuscript

Comments: Throughout the Results section the actual prevalence values from Table 4 should be used with confidence intervals and P-values for the prevalence values for 2005 and 2013, respectively. It is very important as it allows readers to appreciate the statistical analysis of the data. Also, if authors also could list codons in the ascending order when mentioning SNPs throughout the text (for example in line 278).

Response: Thanks for the comment, this has been effected in the revised manuscript

Discussion

Comment: If authors could use the prevalence values and sample numbers throughout the Discussion as well when commenting on identified SNPs and also compare with those in the referenced published literature.

Response: Thanks however, we this as a repetition of the results which are clearly stated in the results section. We have however, effected the adjustment as requested.

Comment: As the aim of the paper was to detect if the background mutations identified by Miotto et al.in SEA are present in Uganda, it would be more logical to start the Discussion with the findings related to this primary aim and then continue discussing the secondary (also important) findings. I would suggest to rearrange the paragraphs in the Discussion and start the discussion with the text shown in lines 309-319.

Response: This is noted and has been effected in the revised manuscript.

Comment: Lines 299-302: In the following sentences it is not clear what is actually “similar”: “A recent study in South Sudan (26) which analyzed samples collected in 2015-2017 after introduction of artemisinin agents in malaria treatment detected genetic background mutation in Pfcrt N326S gene which was previously reported in Southeast Asia and associated with artemisinin resistance. This is similar to a finding from our current study where we detected a background mutation, V127M in the arps10 gene that has been shown to support development of K13 mutations associated with artemisinin resistance in Southeast Asia(8)”

Add the following “While we did not find Pfcrt N326S SNP in our samples, we detected…”

Response: Thanks for the comment. This has been effected in the revised manuscript

Comment: Line 320: This statement is ambiguous: “We detected different K13 SNPs in P. falciparum samples collected both prior (2005) and after (2013) introduction of artemisinin combination therapies in malaria treatment in Uganda.

Response: The comment is noted, we have made the suggested adjustments in the revised manuscript.

Comment: Line 322: Our findings are similar to a recent study in Uganda that reported K13 mutations in the P. falciparum 323 parasites (30)- Needs clarification similar in what way?

Response: Thanks for the comment, this sentence has be deleted from the revised manuscript.

Comment: Line 323-324. “Additionally, our study found a general increase in the prevalence of K13 SNPs in samples collected after (2013) introduction of ACTs in malaria treatment in Uganda”.

This statement is bit misleading since the is no statistically significant differences in the prevalences of these SNPs before and after introduction of ACTs.

Response: The statement has been corrected in the revised manuscript. Although not statistically significant, there is an observed trend towards increase in the prevalence of K13 mutations following introduction of artemisinin agents in malaria treatment.

Comment: The similar statement in Conclusions (below) can also be misinterpreted with the vagueness of the term “generally”. While it is correct in case of novel SNPs in pfmdr2, differences in SNPs for K13 and arsp10 were not statistically significant. Conclusions, should also reflect the findings related to the primary aim of the study and state that there were neither K13 SNPs previously implicated in artemisinin resistance in Africa and SEA, nor mutations in the associated background genes identified in this study with exception of one SNP in arps10 gene.

Response: This is noted and has been corrected in the revised manuscript

Comment: Line 331 “The proportions of K13, arsp10, and pfmdr-2 gene mutations were generally higher in P. falciparum parasites collected after introduction of artemisinin combination therapies in malaria treatment in Uganda

Response: This has been revised to refer to only pfmdr-2 gene mutation in the revised manuscript.

Other comments.

Comment: Line 34: Spaces are needed between P. and falciparum

Response: This has been corrected

Comment: Lines 36,56, 115: Saying it is “a decade” between 2005 and 2013 is not accurate-it is 8 years.

Response: Thanks for the comment, this has been corrected in the revised manuscript

Comment: Line 257 fcrt replace for pfcrt

Response: This has been corrected in the revised manuscript.

Attachment

Submitted filename: RESPONSE TO REVIEWER.doc

Click here for additional data file.^{(38.5KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0268095.r008

Decision Letter 3

Luzia Helena Carvalho

18 Apr 2022

PONE-D-21-33254R3Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in UgandaPLOS ONE

Dear Dr. Ocan,

Thank you for submitting your manuscript for review to PLoS ONE. After careful consideration, we feel that your manuscript will likely be suitable for publication if the authors revise it to address critical points raised by the reviewer. According to reviewer, there are some specific areas where further improvements would be of substantial benefit to the readers. A copy of the reviewers’ comments was included for your information.

Please submit your revised manuscript by Jun 02 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: Authors have addressed almost all of the comments. I believe that the MS can be accepted for publication after the following minor comments are addressed-please see below. The version with Track changes is used for line references.

Line 47: Authors inserted “…and arps10 (p=0.238)”, which is repetitive with the same statement below in lines 56-57 “…There was no statistically significant difference relationship (p=0.238) in the prevalence of arps10 SNPs..”. Suggest to delete “and arps10 (p=0.238)” in line 47.

Line 250: Table 2 in Column “Prevalence of mutation, %(n/N)” “N” is actually not shown, while it is stated in the column heading and in the footnote to the table. As I previously, suggested, N (the denominator) needs to be added for every number in this column consistent with the column’s heading. It would make it easier to comprehend the data and consistent with the format of data shown in the text.

Line 114: “South east Asia” please change for Southeast or South East Asia.

Line 141-142 “…Confirmation of the presence of P. falciparum parasites in the stored(?) blood samples was done by two techniques namely malaria rapid diagnostic test (mRDT) and microscopy”. Were RDT and microscopy done before samples were frozen or after samples were thawed after storage. While RDTs are possible to do on frozen samples I am not sure if the quality slides could be produced after the blood is lysed upon thawing. If authors could clarify this statement.

Line 205: “Correlation analysis was done using Fisher’s exact test to assess the relationship between mutations and year of sample collection” Suggest to change it to “.. using Fisher’s exact test to assess the differences in the prevalence of mutations in 2005 and 2013.”

Line 213 “…(4.2%, 6/142) K13 SNPs were detected (Table 2)”. Suggest to replace with “… SNPs were detected (Tables 1 and 2)” to reference the Tables in their order of appearance.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. 2022 May 5;17(5):e0268095. doi: 10.1371/journal.pone.0268095.r009

Author response to Decision Letter 3

20 Apr 2022

RESPONSE TO REVIEWER’S COMMENTS ON MANUSCRIPT PONE-D-21-33254R3

Comments, Reviewer #1: Authors have addressed almost all of the comments. I believe that the MS can be accepted for publication after the following minor comments are addressed-please see below. The version with Track changes is used for line references.

Response: Thanks for the comments that have helped improve the manuscript, we are grateful.

Comment, Reviewer #1: Line 47: Authors inserted “…and arps10 (p=0.238)”, which is repetitive with the same statement below in lines 56-57 “…There was no statistically significant difference relationship (p=0.238) in the prevalence of arps10 SNPs..”. Suggest to delete “and arps10 (p=0.238)” in line 47.

Response: This has been effected in the revised manuscript

Comment, Reviewer #1: Line 250: Table 2 in Column “Prevalence of mutation, %(n/N)” “N” is actually not shown, while it is stated in the column heading and in the footnote to the table. As I previously, suggested, N (the denominator) needs to be added for every number in this column consistent with the column’s heading. It would make it easier to comprehend the data and consistent with the format of data shown in the text.

Response: Thanks, this has been provided as guided in the revised manuscript

Comment, Reviewer #1: Line 114: “South east Asia” please change for Southeast or South East Asia.

Response: Thanks, this has been corrected in the revised manuscript.

Comment, Reviewer #1: Line 141-142 “…Confirmation of the presence of P. falciparum parasites in the stored(?) blood samples was done by two techniques namely malaria rapid diagnostic test (mRDT) and microscopy”. Were RDT and microscopy done before samples were frozen or after samples were thawed after storage. While RDTs are possible to do on frozen samples I am not sure if the quality slides could be produced after the blood is lysed upon thawing. If authors could clarify this statement.

Response: Sorry, we did not make it clear enough. Microscopy was performed in the field before the blood samples were frozen by the primary studies. In our current study, we conducted pfHRP-2 malaria rapid diagnosis (mRDT) to screen for frozen samples that had P. falciparum parasites. This has been re-written in the revised manuscript revised manuscript.

Comment, Reviewer #1: Line 205: “Correlation analysis was done using Fisher’s exact test to assess the relationship between mutations and year of sample collection” Suggest to change it to “.. using Fisher’s exact test to assess the differences in the prevalence of mutations in 2005 and 2013.”

Response: Thanks, this has been corrected in the revised manuscript.

Comment, Reviewer #1: Line 213 “…(4.2%, 6/142) K13 SNPs were detected (Table 2)”. Suggest to replace with “… SNPs were detected (Tables 1 and 2)” to reference the Tables in their order of appearance.

Response: Thanks for the comment, this has been corrected in the revised manuscript

Attachment

Submitted filename: RESPONSE TO REVIEWERs.doc

Click here for additional data file.^{(26.5KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0268095.r010

Decision Letter 4

Luzia Helena Carvalho

22 Apr 2022

Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in Uganda

PONE-D-21-33254R4

Dear Dr. Ocan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0268095.r011

Acceptance letter

Luzia Helena Carvalho

27 Apr 2022

PONE-D-21-33254R4

Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in Uganda

Dear Dr. Ocan:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. PCR cycling conditions for amplification of P. falciparum DNA fragments sandwiching pfcrt N326S, fd D193Y, arps10 V127M and mdr2 T484L genetic background mutations.

(DOC)

Click here for additional data file.^{(33KB, doc)}

S2 Table. Primer sets used during amplification of Plasmodium falciparum DNA.

(DOC)

Click here for additional data file.^{(47KB, doc)}

Attachment

Submitted filename: Response to Reviewers.doc

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Attachment

Submitted filename: RESPONSE TO THE REVIEWER_dec21.doc

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Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in Uganda

Moses Ocan

Fred Katabazi Ashaba

Savannah Mwesigwa

Kigozi Edgar

Moses R Kamya

Sam L Nsobya

Roles

Abstract

Introduction

Materials and methods

Ethics statement

Study design, site and population

DNA extraction and quantification

PCR amplification of P. falciparum K13 -propeller gene

Amplification of P. falciparum crt, fd, arps10 and pfmdr-2 genes

Sequence data analysis

Statistical analysis

Results

Prevalence of K13 single nucleotide polymorphisms in the P. falciparum parasites

Table 1. Single nucleotide polymorphisms identified in K13 gene in samples collected in Kampala and Jinja, Uganda in 2005 and 2013.

Table 2. Prevalence of single nucleotide polymorphisms in K13, Pfmdr-2 and Pfarps 10 genes in Plasmodium falciparum samples collected in Kampala and Jinja, Uganda in 2005 and 2013.

Prevalence of genetic background mutations, pfcrt N326S, fd D193Y, arps10 V127M and pfmdr-2 T484L in P. falciparum parasites in Uganda

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Author response to previous submission

Decision Letter 0

Luzia Helena Carvalho

Roles

Author response to Decision Letter 0

Decision Letter 1

Luzia Helena Carvalho

Roles

Author response to Decision Letter 1

Decision Letter 2

Luzia Helena Carvalho

Roles

Author response to Decision Letter 2

Decision Letter 3

Luzia Helena Carvalho

Roles

Author response to Decision Letter 3

Decision Letter 4

Luzia Helena Carvalho

Roles

Acceptance letter

Luzia Helena Carvalho

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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