Skip to main content

Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda



Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods.


Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed.


Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC50 values for chloroquine were persistently low throughout the study period (17.4–24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower IC50s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10−8). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018.


This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.


Since the late 1940s, chloroquine was the mainstay for the treatment of Plasmodium falciparum infection. Heavy use of chloroquine, however, led to the emergence of P. falciparum parasites resistant to chloroquine in Southeast Asia and South America. The resistant parasites that first emerged in Southeast Asia spread to East Africa (Tanzania and Kenya) by 1980 [1, 2] and eventually across the malaria endemic regions of Africa [3]. Chloroquine was, therefore, withdrawn/discontinued for routine treatment of P. falciparum malaria in nearly all malaria endemic regions. However, with widespread discontinued use, numerous molecular-epidemiological studies showed that there was return of chloroquine susceptibility in P. falciparum field isolates [4]. This is supported by ex vivo [5,6,7,8,9,10,11,12,13,14,15,16] and in vivo drug-susceptibility studies [5, 17, 18]. Findings suggest that chloroquine might be re-used in the future as an option for the treatment and/or chemoprophylaxis on the condition that chloroquine sensitivity is maintained in the area. As the parasite is “skilled” in evading anti-malarial treatments, continuous surveillance on longitudinal persistence of chloroquine susceptibility by molecular and phenotypic analysis [8, 16, 18, 19] is needed.

In Uganda, first-line treatment for uncomplicated malaria was changed from chloroquine to chloroquine plus sulfadoxine/pyrimethamine in 2000, then again changed to artemether–lumefantrine in 2006 [20]. Several studies in different regions in Uganda reported high prevalence of ex vivo chloroquine resistant parasites (IC50s ≥ 100 nM) accompanied with high prevalence of lysine to threonine change at position 76 (K76T) in pfcrt [21,22,23,24]. However, a recent study showed recovery of chloroquine susceptibility in Eastern Uganda, Tororo; average IC50s decreased from 248 nM in 2010–2013 to 33 nM in the community and 57 nM in the hospital setting in 2016 [10]. Here, to investigate whether chloroquine sensitivity also recovered in other regions in Uganda and, if so, to examine the persistence of chloroquine sensitivity, ex vivo drug susceptibility studies for a 6-year period since 2013 in Gulu, Northern Uganda were performed. Results show that chloroquine susceptibility stably persisted during the study period with a significant decrease and eventual absence of the chloroquine resistant K76T alleles in pfcrt.


Study site

A comprehensive drug susceptibility assessment was conducted at St. Mary’s Hospital Lacor in Gulu, Northern Uganda (Fig. 1) from 2013 to 2018 [25, 26]: Oct–Nov 2013, May–Jun and Oct–Nov 2014, May–Jun and Oct 2015, Jun–Jul and Oct–Nov 2016, Jun 2017 and Jun 2018. Average temperature in the studied area is 24.6 °C and average annual rainfall is about 1507 mm with two rainy seasons; a smaller peak in April–May (average rainfall 150 mm) and a heavier peak in August–September (average rainfall 234 mm) [27]. Plasmodium falciparum is the most prevalent species and is mainly transmitted by Anopheles funestus and Anopheles gambiae as the major vectors.

Fig. 1

Study site, Gulu (red circle), Northern Uganda

Malaria control programmes in the studied region include vector control by long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), artemisinin-based combination therapy (ACT) together with improved diagnosis, management of severe malaria, and intermittent preventive treatment of malaria during pregnancy. These control measures were performed with funding from the Global Fund, USAID/PMI, DFID, World Vision and other partners [28]. Mass distribution of LLINs was first implemented in 2009–2010, which continued until 2013–2014. Through these extensive efforts, malaria burden was effectively reduced from 72% in 2009 to 29% in 2014 [27].


Initial screening was performed using RDT (SD BIOLINE Malaria Ag P.f/Pan test, Abbott, USA) for 1575 symptomatic patients who visited St. Mary’s Hospital Lacor. Inclusion criteria were: (a) patients that are P. falciparum positive by RDT and microscopy, (b) aged ≥ 6 months, and (c) with no history of taking anti-malarial drug(s) within 2 weeks before enrollment. Patients fulfilling the inclusion criteria were enrolled after obtaining written informed consent from the patients or parent/guardian(s). For children aged 7 to 17 years, separate assent was also obtained.

Ethical approval for this study was obtained from the Lacor Hospital Institutional Research and Ethics Committee (Ref; LHIREC 021/09/13), the Uganda National Council for Science and Technology (Ref; HS 1395), and Juntendo Research and Ethics Committee (Ref; 14-169).

Sample collection and ex vivo susceptibility assay for chloroquine and lumefantrine

Blood samples of approximately 100–500 µL (< 2 years old), and 1 mL (≥ 2 years old) were collected by peripheral venipuncture or finger prick and immediately transferred to the laboratory adjacent to the hospital. Thick and thin blood smears stained for 30 min with 2% Giemsa solution were used to determine parasitaemia.

At every visit from 2013 to 2018 (a total of nine times sampling period), ex vivo drug susceptibility studies were performed. Ex vivo susceptibility was evaluated for chloroquine and lumefantrine for the samples with parasitemia ≥ 0.05% as previously reported [29]. Parasite culture was incubated in the presence of chloroquine (25–1600 nM) or lumefantrine (1.25–80 nM) at 37 °C for 72 h in a gas atmosphere of 5% CO2, 5% O2 (AnaeroPack malaria culture system, Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan). Laboratory-maintained 3D7 clone was used for quality evaluation of pre-dosed drug plates. Parasite culture without antimalarials served as control. To evaluate parasite growth, thick smears were made from drug free culture after 72 h of incubation and the number of schizonts counted. If less than 5 schizonts were seen per field, the test samples included in that plate were not used for further analysis. Drug sensitivity was assessed using an enzyme-linked immunosorbent assay (ELISA) that quantifies parasite histidine-rich protein-2 (HRP-2) [30]. The effective concentration needed to inhibit P. falciparum growth by 50% (IC50) was determined by non-linear regression using an online ICEstimator software ( [31]. The quality of the ex vivo drug assay was evaluated based upon the level of fitness to the expected shape of the curve obtained by the inhibitory sigmoid Emax model [31].

pfcrt and pfmdr1 genotyping

Polymorphisms at amino acid positions 72–76 in the P. falciparum chloroquine resistance transporter gene (pfcrt) were determined by direct sequencing. In the P. falciparum multidrug resistance-1 (pfmdr1) gene, polymorphisms at codons 86, 184, 1034, 1042 and 1246 were determined by direct sequencing and/or restriction fragment length polymorphism (RFLP) analysis, as previously described [29, 32]. For direct sequencing, initial and nested PCR was done with PrimeSTAR Max DNA Polymerase (Takara Bio Inc., Japan) in 10 μL reaction mixture containing 1 μL of DNA template and 0.5 μM of each primer set. Excess primers and unincorporated nucleotides from the nested PCR product were enzymatically removed with ExoSAP-IT Kit (Amersham Biosciences, Buckinghamshire, UK) and direct sequence was performed (96 °C for 1 min, 25 cycles of 96 °C for 30 s, 50 °C for 30 s and 60 °C for 4 min, and a final cycle at 60 °C for 1 min) with a BigDye Terminator v1.1 cycle sequencing kit in the Applied Biosystems 3130/3130xL genetic analyzer (Life Technologies, Carlsbad, California, USA). Samples with overlapping peaks of at least 50% in height were considered harboring mixed genotypes.

Full sequencing of pfcrt

The full sequence of pfcrt was obtained either by whole-genome sequencing (n = 17) or target sequencing (n = 39). Whole genome sequence data was previously reported [26]. In brief, Acrodisc filters (Pall Corporation, New York, NY, USA) was used to reduce the extent of human DNA contamination from blood samples. Approximately 1–1.5 Gb of data per sample were obtained using Illumina instruments (Miseq and Hiseq 2000). Single-nucleotide polymorphisms were called at all genomic positions with > 80% frequency of > 10 reads support.

For target sequencing, the DNA fragment of a genomic region coding for pfcrt gene was amplified by PCR with primers (Pfcrt-F: 5′-TAC TTT CCC AAG TTG TAC TGC TTC TAA GCT-3′, Pfcrt-R: 5′-TTT ACC TAT TTA TCA AAA CAC CAA AAG GGA-3′), which covers the whole DNA sequence of pfcrt gene. PCR was performed with PrimeSTAR GXL DNA Polymerase (Takara Bio Inc., Japan) in 5 μL reaction mixture containing 1 μL of DNA solution and 0.25 μM of primer set. PCR conditions consisted of denaturation at 98 °C for 10 s, followed by 40 cycles of amplification (98 °C for 10 s, 60 °C for 15 s, and 68 °C for 5 min), with a final elongation period of 68 °C for 5 min. PCR products were diluted with 5 μL of pure water, electrophoresed in 2% agarose gel and stained with ethidium bromide. The PCR products were then purified with the ExoSAP-IT reagent (Affymetrix, USA). Libraries were prepared from the purified PCR products with Nextera XT DNA Library Prep Kit (Illumina, USA). The libraries were sequenced by MiSeq (Illumina) with the paired-end method and 250 bp of read length. The reads were also used to map the pfcrt gene sequence of P. falciparum 3D7 as a reference and assembled a single contiguous sequence by CLC Genomics Workbench (Qiagen). All sequences were deposited in the DNA Data Bank of Japan (DDBJ) with accession numbers LC498195–LC498250.

Statistical analysis

All statistical analyses were performed using R software (version 3.6.1). Data was analyzed using Kruskall Wallis test, Wilcoxon rank sum test, and Jonckheere-Terpstra test. p values < 0.05 were considered statistically significant.


Ex vivo drug susceptibility of chloroquine and lumefantrine

Of 1575 patients who visited St. Mary’s Hospital Lacor, 793 patients were enrolled based on P. falciparum-positive results by RDT (Fig. 2). The rest were excluded because of (a) absence of P. falciparum by microscopic examination (n = 535), (b) use of anti-malarial drug(s) within the last 2 weeks before enrollment (n = 198), or (c) other reasons (n = 49) (Fig. 2). The commonly used anti-malarial for pretreatment was artemether–lumefantrine (77%) (Table 1). Chloroquine use was confirmed in only 3 patients in 2013 and one patient in 2014.

Fig. 2

Flow chart of the study from screening to drug sensitivity assays, 2013–2018

Table 1 Commonly used anti-malarial drugs of patients attending St. Mary’s Hospital Lacor, Gulu (information obtained from excluded patients)

Among 793 blood samples obtained from enrolled patients, 203 were excluded because of very low parasitaemia (< 0.05%) or insufficient amount of blood, resulting in 590 samples used for ex vivo drug susceptibility assays. Also, ex vivo study for chloroquine in 2016 and lumefantrine in 2015 was not performed because of inadequate quality of pre-dosed drug plates. Thus, in total, 319 and 335 ex vivo drug-susceptibility assays for chloroquine and lumefantrine respectively were available for analyses. Background information of patients who participated in the study per year is shown in Table 2. Median age was 3.5 years (IQR 2.0–4.8) and hemoglobin level < 10 g/dL was observed in 27% of patients. Median parasitemia at enrollment were 0.2–3.5%, which significantly varied between studied years (p-value = 3.9 × 10−15, Kruskal–Wallis test). Beside parasitaemia at enrollment, no significant difference was observed in background factors among the studied years.

Table 2 Characteristics of participants evaluated for chloroquine and lumefantrine ex vivo susceptibility assay

Of 319 and 335 ex vivo drug-susceptibility assay for chloroquine and lumefantrine, respectively, 42 chloroquine and 48 lumefantrine assays were performed with O instead of AB blood-group serum due to the latter’s unavailability during the sampling period. These samples were excluded from further analysis. Thus, in summary, ex vivo drug study was successfully conducted for 239/277 samples (86.3%) for chloroquine and 168/287 for lumefantrine (58.5%) (Fig. 2). For chloroquine, only 1.3% (3/239) fulfilled the criteria for chloroquine resistance (IC50 > 100 nM) (Fig. 3a). From 2013 to 2018, the geometric means of the IC50s (17.4–24.9 nM) were much lower than the threshold for chloroquine resistance and was stable without any significant decrease or increase in trend throughout the study period (p-value = 0.32 Jonckheere-Terpstra test). The highest IC50 was 148.8 nM, observed in 2015 in a 9-year-old girl. For lumefantrine, IC50s displayed no specific trend over time ranging from 20.5 nM to 32.0 nM (p-value = 0.16, Jonckheere-Terpstra test). In all studied parasites, IC50 values were below the conservative cut-off of 50 nM for lumefantrine resistance [33], and lower than the 150 nM value [34] (Fig. 3b).

Fig. 3

Ex vivo sensitivity of P. falciparum to chloroquine and lumefantrine. Bold lines represent median IC50s. Faint horizontal lines represent the 25th and 75th interquartile range. Mean IC50 for chloroquine were 24.8 nM, 24.9 nM, 17.4 nM, 22.6 nM and 23.1 nM in 2013, 2014, 2015, 2017 and 2018, respectively; and for lumefantrine, 20.8 nM, 20.5 nM, 32.0 nM, 28.6 nM and 21.0 nM in 2013, 2014, 2016, 2017 and 2018, respectively. Cut-off sensitivity is based from literature

Allele prevalence and frequency of pfcrt and pfmdr1 polymorphisms

In pfcrt, the prevalence of chloroquine-resistant alleles (CVIET allele; amino acid position 72–76, mutation underlined) significantly decreased from 28.8% in 2013 to 1.1% in 2016 and finally could not be detected in 2017 (Fig. 4). Besides genome sequencing, the absence of minor alleles bearing CVIET was further confirmed by pfcrt target sequencing. In pfmdr1, chloroquine sensitive N86 allele was fixed or nearly fixed throughout the study period. Prevalence of the mutant allele at position 184 (Y184F) gradually increased from 2.4% in 2013 to 48.5% in 2018, albeit this trend was not significant (p-value = 0.13, Jonckheere-Terpstra trend test). Wild-type alleles were nearly fixed at other loci in pfmdr1.

Fig. 4

Temporal changes in pfcrt and pfmdr1 allele prevalence in all collected P. falciparum isolates

Association between ex vivo drug sensitivity and alleles in pfcrt and pfmdr1

In pfcrt, parasites carrying wild-type alleles showed significantly lower IC50s to chloroquine than those carrying mutant allele (geometric mean, 21.4 vs. 43.1 nM, p-value 3.9 × 10−8, Wilcoxon rank sum test) (Fig. 5). To see whether other polymorphism(s) besides those at position 72–76 played a role in the recovery of chloroquine susceptibility, entire sequences of pfcrt in 56 samples were analysed. IC50s were also successfully obtained in 44 samples (Table 3), in which 31 (71%) bore only wild-type alleles of the gene. The second (n = 5, 11%) most common haplotype, HP-4, corresponds to a prevalent mutant haplotype (CVMNT + A220S + Q271E + R371I) in Africa [3, 35]. Remaining eight parasites harbored minor haplotypes, all with wild type allele K76 and displayed IC50s to chloroquine of 18–35 nM. A previous study (16), has implicated C356R in the sensitivity recovery of chloroquine in the K76T-harbouring parasites, however this mutant allele was not found in the study area. These results further confirm the expansion of wild K76-harbouring parasites as the cause of chloroquine susceptibility reversal in the studied area, rather than additional mutation in the pfcrt gene.

Fig. 5

Association between allele prevalence and mean IC50 to chloroquine and lumefantrine. N is the number of samples available for comparison. Allele prevalence was compared between wild-type and mutant alleles using Wilcoxon rank sum test, and comparisons with p values of < 0.05 are significant. p values < 0.0001 are indicated by *

Table 3 Chloroquine susceptibility in pfcrt haplotypes obtained from sequencing the entire pfcrt gene

Association between ex vivo drug sensitivity and respective pfmdr1 alleles, however, could not be properly evaluated because of substantial deviation in allele frequencies except for position 184. At this position, no significant difference in chloroquine IC50s was observed between wild and mutant alleles (25.3 nM vs. 22.0 nM, p-value = 0.192, Wilcoxon rank sum test) (Fig. 5). Analysis of susceptibility to lumefantrine indicated that parasites carrying pfcrt CVIET exhibited significantly lower IC50s than those with CVMNK (13.4 vs. 28.0 nM, p-value = 8.1 × 10−5, Wilcoxon rank sum test) (Fig. 5). No significant difference in IC50s for lumefantrine was found between parasites with pfmdr1 Y184 (25.0 nM) and Y184F (30.0 nM).


In Uganda, chloroquine was officially withdrawn and replaced with artemether–lumefantrine in 2006. The present analysis revealed that chloroquine susceptibility has returned and is stably maintained for at least 6-years in Gulu, Northern Uganda. This is the first report in Uganda to demonstrate stable and persistent recovery of chloroquine sensitivity using both phenotypic and genotypic approaches.

The prevalence of K76 allele in pfcrt rapidly increased from 67% in 2013 to complete fixation in 2017. This is most probably due to K76-harbouring parasites outcompeting the K76T-harbouring parasites because of fitness advantage in the absence of chloroquine selection pressure, as previously observed in Malawi [36, 37]. Indeed, recent transfection studies also show that K76T confers a substantial fitness cost to parasites [35, 38]. This fitness cost can be partly explained by a functional impairment in haemoglobin digestion and subsequent reduction in the supply of amino acids in K76T harbouring parasites [39]. In another possible scenario, a back mutation from T to K at position 76 in pfcrt may potentially induce chloroquine sensitivity. As an example, the chloroquine-susceptible 106/1 clone had mutant alleles at positions 74 and 75 but showed wild-type K allele at position 76 (CVIEK) [40]. The only difference in the pfcrt haplotype at position 72–76 in this clone and the widely prevalent chloroquine-resistant haplotype (CVIET) is at amino acid position 76. In the present analysis, however, no such haplotype (CVIEK) was found, negating this possibility. Also, no evidence of additional mutation in pfcrt such as C350R which has been reported to be associated with the restoration of chloroquine susceptibility [16] was obtained. Taken together, these results strongly suggest that neither back mutation nor additional mutations in pfcrt was associated with the observed recovery of chloroquine sensitivity in the study area.

It is of note that recovery of chloroquine sensitivity after its withdrawal occurred much earlier in Gulu than in other regions in Uganda [9, 22,23,24, 41]. In 2013, as much as 65% of parasites displayed ex vivo chloroquine resistance [24] and 60–80% carried K76T allele in Tororo, Eastern Uganda [42]. In contrast, present results revealed that prevalence of ex vivo chloroquine-resistance and K76T alleles were already 6% and 29%, respectively, in 2013, indicating a faster recovery or re-emergence of chloroquine sensitive strains in the region. Despite the government’s move to change the national treatment policy, chloroquine might be used as self-treatment and/or prophylaxis. Such chloroquine use potentially creates various levels of chloroquine selecting pressure in the region, which would be one of the important factors that affects the speed of recovery of susceptible parasites [43]. However, countrywide anti-malarial survey reported no considerable difference of chloroquine use between Tororo and this study area [44,45,46], suggesting that this could not be a main factor for the observed findings.

Piperaquine use in Tororo might explain the observed difference to some extent. Dihydroartemisinin-piperaquine has been used as a second line treatment for uncomplicated malaria in Uganda. In Tororo, this regimen was widely used in different drug trials for malaria treatment [47, 48] and chemoprevention [24, 49,50,51,52]. Previous studies indicated that dihydroartemisinin-piperaquine treatment selected the N86Y allele in pfmdr1 in Uganda [48, 52,53,54], albeit one study questioned this association [55]. Approximately one-third of parasites possessed the N86Y in 2010–2013 in Tororo [9], which was much higher than that found in the studied area (2%). Since N86Y is associated with chloroquine resistance, it might be plausible that the N86Y mutation selected by piperaquine played a role in the slower recovery of chloroquine sensitivity in Tororo.

In contrast to chloroquine, average lumefantrine IC50 values in the present analysis (21–29 nM) were considerably higher than those in Eastern Uganda (3.0–5.4 nM) [9, 24]. It has been suggested that decrease in lumefantrine susceptibility are associated with wild type alleles in pfcrt and pfmdr1 [48, 56,57,58,59]. In this study higher IC50s to lumefantrine were observed in K76 sequences than those with K76T. The higher prevalence of K76 allele in our study area than that in Eastern Uganda [9, 23, 42] may partly explain the observed lumefantrine susceptibility.

Molecular epidemiological analysis displayed a large increase in the proportion of parasites carrying the Y184F mutation in pfmdr1. This occurred at the same time when substantial reduction on the pfcrt K76T mutation was observed, particularly between 2014 and 2015. One possible explanation would be an increase in lumefantrine usage results in the selection of these alleles in this area. Indeed, previous in vivo studies have demonstrated that artemether–lumefantrine treatment selected for these alleles (pfcrt K76 and Y184F) in Africa including Uganda [24, 60, 61]. In the present study, IC50s for lumefantrine were significantly higher in pfcrt K76 harboring parasites than those harboring K76T; while, Y184F allele did not show significantly high IC50s for lumefantrine, consistent with previous transfection study that revealed no association of Y184F mutation to in vitro lumefantrine susceptibility [59]. Thus, in vivo selection for Y184F allele after artemether–lumefantrine treatment may be because of mechanisms other than susceptibility to lumefantrine.


The study shows the stable persistence of chloroquine-susceptibility with the fixation of pfcrt K76 in Northern Uganda. This observation implies the possibility of future clinical trials for potential re-use of chloroquine as an option for malaria treatment or prevention. Such trial was performed in Malawi where longtime stable chloroquine susceptibility has been evidenced and has revealed that weekly chemoprophylaxis with chloroquine showed 78% lower risk of clinical malaria than intermittent sulfadoxine-pyrimethamine [62]. Similar trial in Uganda would provide an insight into potential re-introduction of chloroquine. However, further evidence of long-time persistence of return of chloroquine susceptibility is warranted in other endemic areas in Uganda before the implementation of clinical trials.

Availability of data and materials

The primary datasets used and analyzed during the current study are available from the corresponding author on reasonable request.



Artemisinin-based combination therapy


DNA data bank of Japan


Department for international development (UK)


Deoxyribonucleic acid


Enzyme-linked immunosorbent assay


Histidine-rich protein-2

IC50 :

50% growth inhibitory concentration


Indoor residual spray


Long-lasting insecticidal mosquito net

pfcrt :

Plasmodium falciparum chloroquine resistance transporter gene

pfmdr1 :

Plasmodium falciparum multidrug resistance-1


Polymerase chain reaction


President’s malaria initiative


Rapid diagnostic test


United States Agency for International Development


  1. 1.

    Campbell CC, Chin W, Collins WE, Teutsch SM, Moss DM. Chloroquine-resistant Plasmodium falciparum from East Africa: cultivation and drug sensitivity of the Tanzanian I/CDC strain from an American tourist. Lancet. 1979;2:1151–4.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2.

    Fogh S, Jepsen S, Effersoe P. Chloroquine-resistant Plasmodium falciparum malaria in Kenya. Trans R Soc Trop Med Hyg. 1979;73:228–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Mita T, Tanabe K, Kita K. Spread and evolution of Plasmodium falciparum drug resistance. Parasitol Int. 2009;58:201–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4.

    Ocan M, Akena D, Nsobya S, Kamya MR, Senono R, Kinengyere AA, et al. Persistence of chloroquine resistance alleles in malaria endemic countries: a systematic review of burden and risk factors. Malar J. 2019;18:76.

    PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Mita T, Kaneko A, Lum JK, Bwijo B, Takechi N, Zungu IL, et al. Recovery of chloroquine sensitivity and low prevalence of the Plasmodium falciparum chloroquine resistance transporter gene mutation K76T following the discontinuance of chloroquine use in Malawi. Am J Trop Med Hyg. 2003;68:413–5.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Akala HM, Eyase FL, Cheruiyot AC, Omondi AA, Ogutu BR, Waters NC, et al. Antimalarial drug sensitivity profile of western Kenya Plasmodium falciparum field isolates determined by a SYBR Green I in vitro assay and molecular analysis. Am J Trop Med Hyg. 2011;85:34–41.

    PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Eyase FL, Akala HM, Ingasia L, Cheruiyot A, Omondi A, Okudo C, et al. The role of Pfmdr1 and Pfcrt in changing chloroquine, amodiaquine, mefloquine and lumefantrine susceptibility in western-Kenya P falciparum samples during 2008–2011. PLoS ONE. 2013;8:e64299.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Lucchi NW, Komino F, Okoth SA, Goldman I, Onyona P, Wiegand RE, et al. In vitro and molecular surveillance for antimalarial drug resistance in Plasmodium falciparum parasites in Western Kenya reveals sustained artemisinin sensitivity and increased chloroquine sensitivity. Antimicrob Agents Chemother. 2015;59:7540–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Rasmussen SA, Ceja FG, Conrad MD, Tumwebaze PK, Byaruhanga O, Katairo T, et al. Changing antimalarial drug sensitivities in Uganda. Antimicrob Agents Chemother. 2017;61:e01516–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Fall B, Pascual A, Sarr FD, Wurtz N, Richard V, Baret E, et al. Plasmodium falciparum susceptibility to anti-malarial drugs in Dakar, Senegal, in 2010: an ex vivo and drug resistance molecular markers study. Malar J. 2013;12:107.

    PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Issaka M, Salissou A, Arzika I, Guillebaud J, Maazou A, Specht S, et al. Ex vivo responses of Plasmodium falciparum clinical isolates to conventional and new antimalarial drugs in Niger. Antimicrob Agents Chemother. 2013;57:3415–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Quashie NB, Duah NO, Abuaku B, Quaye L, Ayanful-Torgby R, Akwoviah GA, et al. A SYBR Green 1-based in vitro test of susceptibility of Ghanaian Plasmodium falciparum clinical isolates to a panel of anti-malarial drugs. Malar J. 2013;12:450.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  13. 13.

    Salissou A, Zamanka H, Biyghe Binze B, Riviere T, Tichit M, Ibrahim ML, et al. Low prevalence of pfcrt resistance alleles among patients with uncomplicated falciparum malaria in Niger six years after chloroquine withdrawal. Malar Res Treat. 2014;2014:614190.

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Tinto H, Bonkian LN, Nana LA, Yerbanga I, Lingani M, Kazienga A, et al. Ex vivo anti-malarial drugs sensitivity profile of Plasmodium falciparum field isolates from Burkina Faso five years after the national policy change. Malar J. 2014;13:207.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  15. 15.

    Mbaye A, Gaye A, Dieye B, Ndiaye YD, Bei AK, Affara M, et al. Ex vivo susceptibility and genotyping of Plasmodium falciparum isolates from Pikine, Senegal. Malar J. 2017;16:250.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  16. 16.

    Pelleau S, Moss EL, Dhingra SK, Volney B, Casteras J, Gabryszewski SJ, et al. Adaptive evolution of malaria parasites in French Guiana: reversal of chloroquine resistance by acquisition of a mutation in pfcrt. Proc Natl Acad Sci USA. 2015;112:11672–7.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Laufer MK, Thesing PC, Eddington ND, Masonga R, Dzinjalamala FK, Takala SL, et al. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med. 2006;355:1959–66.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Laufer MK, Thesing PC, Dzinjalamala FK, Nyirenda OM, Masonga R, Laurens MB, et al. A longitudinal trial comparing chloroquine as monotherapy or in combination with artesunate, azithromycin or atovaquone-proguanil to treat malaria. PLoS ONE. 2012;7:e42284.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Frosch AE, Laufer MK, Mathanga DP, Takala-Harrison S, Skarbinski J, Claassen CW, et al. Return of widespread chloroquine-sensitive Plasmodium falciparum to Malawi. J Infect Dis. 2014;210:1110–4.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Nanyunja M, Nabyonga Orem J, Kato F, Kaggwa M, Katureebe C, Saweka J. Malaria treatment policy change and implementation: the case of Uganda. Malar Res Treat. 2011;2011:683167.

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Nsobya SL, Kiggundu M, Nanyunja S, Joloba M, Greenhouse B, Rosenthal PJ. In vitro sensitivities of Plasmodium falciparum to different antimalarial drugs in Uganda. Antimicrob Agents Chemother. 2010;54:1200–6.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Kamugisha E, Bujila I, Lahdo M, Pello-Esso S, Minde M, Kongola G, et al. Large differences in prevalence of Pfcrt and Pfmdr1 mutations between Mwanza, Tanzania and Iganga, Uganda-a reflection of differences in policies regarding withdrawal of chloroquine? Acta Trop. 2012;121:148–51.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Mbogo GW, Nankoberanyi S, Tukwasibwe S, Baliraine FN, Nsobya SL, Conrad MD, et al. Temporal changes in prevalence of molecular markers mediating antimalarial drug resistance in a high malaria transmission setting in Uganda. Am J Trop Med Hyg. 2014;91:54–61.

    PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Tumwebaze P, Conrad MD, Walakira A, LeClair N, Byaruhanga O, Nakazibwe C, et al. Impact of antimalarial treatment and chemoprevention on the drug sensitivity of malaria parasites isolated from ugandan children. Antimicrob Agents Chemother. 2015;59:3018–30.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Balikagala B, Mita T, Ikeda M, Sakurai M, Yatsushiro S, Takahashi N, et al. Absence of in vivo selection for K13 mutations after artemether–lumefantrine treatment in Uganda. Malar J. 2017;16:23.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. 26.

    Ikeda M, Kaneko M, Tachibana SI, Balikagala B, Sakurai-Yatsushiro M, Yatsushiro S, et al. Artemisinin-resistant Plasmodium falciparum with high survival rates, Uganda, 2014–2016. Emerg Infect Dis. 2018;24:718–26.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Simple O, Mindra A, Obai G, Ovuga E, Odongo-Aginya EI. Influence of climatic factors on malaria epidemic in Gulu District, Northern Uganda: a 10-year retrospective study. Malar Res Treat. 2018;2018:5482136.

    PubMed  PubMed Central  Google Scholar 

  28. 28.

    Uganda Bureau of Statistics (UBOS) and ICF International. Uganda Malaria Indicator Survey 2014–15. 2015. Accessed 15 Jan 2020.

  29. 29.

    Sekihara M, Tachibana SI, Yamauchi M, Yatsushiro S, Tiwara S, Fukuda N, et al. Lack of significant recovery of chloroquine sensitivity in Plasmodium falciparum parasites following discontinuance of chloroquine use in Papua New Guinea. Malar J. 2018;17:434.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Noedl H, Wernsdorfer WH, Miller RS, Wongsrichanalai C. Histidine-rich protein II: a novel approach to malaria drug sensitivity testing. Antimicrob Agents Chemother. 2002;46:1658–64.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Le Nagard H, Vincent C, Mentre F, Le Bras J. Online analysis of in vitro resistance to antimalarial drugs through nonlinear regression. Comput Methods Programs Biomed. 2011;104:10–8.

    PubMed  Article  PubMed Central  Google Scholar 

  32. 32.

    Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC. The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol. 2000;108:13–23.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33.

    Kaddouri H, Djimde A, Dama S, Kodio A, Tekete M, Hubert V, et al. Baseline in vitro efficacy of ACT component drugs on Plasmodium falciparum clinical isolates from Mali. Int J Parasitol. 2008;38:791–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Basco LK, Bickii J, Ringwald P. In vitro activity of lumefantrine (benflumetol) against clinical isolates of Plasmodium falciparum in Yaounde, Cameroon. Antimicrob Agents Chemother. 1998;42:2347–51.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Dhingra SK, Gabryszewski SJ, Small-Saunders JL, Yeo T, Henrich PP, Mok S, et al. Global spread of mutant PfCRT and its pleiotropic impact on Plasmodium falciparum multidrug resistance and fitness. mBio. 2019;10:e02731-18.

    PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Mita T, Kaneko A, Lum JK, Zungu IL, Tsukahara T, Eto H, et al. Expansion of wild type allele rather than back mutation in pfcrt explains the recent recovery of chloroquine sensitivity of Plasmodium falciparum in Malawi. Mol Biochem Parasitol. 2004;135:159–63.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. 37.

    Laufer MK, Takala-Harrison S, Dzinjalamala FK, Stine OC, Taylor TE, Plowe CV. Return of chloroquine-susceptible falciparum malaria in Malawi was a reexpansion of diverse susceptible parasites. J Infect Dis. 2010;202:801–8.

    PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Petersen I, Gabryszewski SJ, Johnston GL, Dhingra SK, Ecker A, Lewis RE, et al. Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter. Mol Microbiol. 2015;97:381–95.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    Lewis IA, Wacker M, Olszewski KL, Cobbold SA, Baska KS, Tan A, et al. Metabolic QTL analysis links chloroquine resistance in Plasmodium falciparum to impaired hemoglobin catabolism. PLoS Genet. 2014;10:e1004085.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  40. 40.

    Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, et al. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell. 2000;6:861–71.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Asua V, Vinden J, Conrad MD, Legac J, Kigozi SP, Kamya MR, et al. Changing molecular markers of antimalarial drug sensitivity across Uganda. Antimicrob Agents Chemother. 2019;63:e01818-18.

    PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Tumwebaze P, Tukwasibwe S, Taylor A, Conrad M, Ruhamyankaka E, Asua V, et al. Changing antimalarial drug resistance patterns identified by surveillance at three sites in Uganda. J Infect Dis. 2017;215:631–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Rosenthal PJ. The interplay between drug resistance and fitness in malaria parasites. Mol Microbiol. 2013;89:1025–38.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Sears D, Kigozi R, Mpimbaza A, Kakeeto S, Sserwanga A, Staedke SG, et al. Anti-malarial prescription practices among outpatients with laboratory-confirmed malaria in the setting of a health facility-based sentinel site surveillance system in Uganda. Malar J. 2013;12:252.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    ACTwatch Group and PACE.ACTwatch Study Reference Document: Uganda Outlet Survey 2013. Washington DC: PSI; 2014. Accessed 15 Jan 2020.

  46. 46.

    Sserwanga A, Sears D, Kapella BK, Kigozi R, Rubahika D, Staedke SG, et al. Anti-malarial prescription practices among children admitted to six public hospitals in Uganda from 2011 to 2013. Malar J. 2015;14:331.

    PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    The Four Artemisinin-Based Combinations (4ABC) Study Group. A head-to-head comparison of four artemisinin-based combinations for treating uncomplicated malaria in African children: a randomized trial. PLoS Med. 2011;8:e1001119.

    Article  CAS  Google Scholar 

  48. 48.

    Conrad MD, LeClair N, Arinaitwe E, Wanzira H, Kakuru A, Bigira V, et al. Comparative impacts over 5 years of artemisinin-based combination therapies on Plasmodium falciparum polymorphisms that modulate drug sensitivity in Ugandan children. J Infect Dis. 2014;210:344–53.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Bigira V, Kapisi J, Clark TD, Kinara S, Mwangwa F, Muhindo MK, et al. Protective efficacy and safety of three antimalarial regimens for the prevention of malaria in young Ugandan children: a randomized controlled trial. PLoS Med. 2014;11:e1001689.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  50. 50.

    Nankabirwa JI, Wandera B, Amuge P, Kiwanuka N, Dorsey G, Rosenthal PJ, et al. Impact of intermittent preventive treatment with dihydroartemisinin-piperaquine on malaria in Ugandan schoolchildren: a randomized, placebo-controlled trial. Clin Infect Dis. 2014;58:1404–12.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Kakuru A, Jagannathan P, Muhindo MK, Natureeba P, Awori P, Nakalembe M, et al. Dihydroartemisinin–piperaquine for the prevention of malaria in pregnancy. N Engl J Med. 2016;374:928–39.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Nankabirwa JI, Conrad MD, Legac J, Tukwasibwe S, Tumwebaze P, Wandera B, et al. Intermittent preventive treatment with dihydroartemisinin–piperaquine in Ugandan schoolchildren selects for Plasmodium falciparum transporter polymorphisms that modify drug sensitivity. Antimicrob Agents Chemother. 2016;60:5649–54.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Taylor AR, Flegg JA, Holmes CC, Guerin PJ, Sibley CH, Conrad MD, et al. Artemether–lumefantrine and dihydroartemisinin–piperaquine exert inverse selective pressure on Plasmodium falciparum drug sensitivity-associated haplotypes in Uganda. Open Forum Infect Dis. 2017;4:ofw229.

    PubMed  Article  PubMed Central  Google Scholar 

  54. 54.

    Conrad MD, Mota D, Foster M, Tukwasibwe S, Legac J, Tumwebaze P, et al. Impact of intermittent preventive treatment during pregnancy on Plasmodium falciparum drug resistance-mediating polymorphisms in Uganda. J Infect Dis. 2017;216:1008–17.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Yeka A, Wallender E, Mulebeke R, Kibuuka A, Kigozi R, Bosco A, et al. Comparative efficacy of artemether–lumefantrine and dihydroartemisinin–piperaquine for the treatment of uncomplicated malaria in Ugandan children. J Infect Dis. 2019;219:1112–20.

    PubMed  Article  PubMed Central  Google Scholar 

  56. 56.

    Sisowath C, Petersen I, Veiga MI, Martensson A, Premji Z, Bjorkman A, et al. In vivo selection of Plasmodium falciparum parasites carrying the chloroquine-susceptible pfcrt K76 allele after treatment with artemether–lumefantrine in Africa. J Infect Dis. 2009;199:750–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Venkatesan M, Gadalla NB, Stepniewska K, Dahal P, Nsanzabana C, Moriera C, et al. Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether–lumefantrine and artesunate–amodiaquine. Am J Trop Med Hyg. 2014;91:833–43.

    PubMed  PubMed Central  Article  Google Scholar 

  58. 58.

    Baraka V, Tinto H, Valea I, Fitzhenry R, Delgado-Ratto C, Mbonye MK, et al. In vivo selection of Plasmodium falciparum Pfcrt and Pfmdr1 variants by artemether–lumefantrine and dihydroartemisinin–piperaquine in Burkina Faso. Antimicrob Agents Chemother. 2015;59:734–7.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  59. 59.

    Veiga MI, Dhingra SK, Henrich PP, Straimer J, Gnadig N, Uhlemann AC, et al. Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies. Nat Commun. 2016;7:11553.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  60. 60.

    Dokomajilar C, Nsobya SL, Greenhouse B, Rosenthal PJ, Dorsey G. Selection of Plasmodium falciparum pfmdr1 alleles following therapy with artemether–lumefantrine in an area of Uganda where malaria is highly endemic. Antimicrob Agents Chemother. 2006;50:1893–5.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  61. 61.

    Baliraine FN, Rosenthal PJ. Prolonged selection of pfmdr1 polymorphisms after treatment of falciparum malaria with artemether–lumefantrine in Uganda. J Infect Dis. 2011;204:1120–4.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. 62.

    Divala TH, Mungwira RG, Mawindo PM, Nyirenda OM, Kanjala M, Ndaferankhande M, et al. Chloroquine as weekly chemoprophylaxis or intermittent treatment to prevent malaria in pregnancy in Malawi: a randomised controlled trial. Lancet Infect Dis. 2018;18:1097–107.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references


We specially thank study participants, participants’ guardians for consent to participate in the study as well as study teams from St. Mary’s Hospital Lacor and the Department of Tropical Medicine and Parasitology Juntendo University for sample collection. We also thank Hiroaki Nakanishi and Kazuyuki Saito for technical assistance.


This study was financially supported by Grants-in-aid for scientific research [26460515, 26305015, 17H04074], Health and Labour Sciences Research Grants [H26-Iryokiki-Ippan-004] from Ministry of Health, Labour and Welfare (MEXT) of Japan, AMED under Grant Number JP15km0908001 and by the Global Health Innovative Technology (GHIT) Fund (G2015-210) awarded to Professor Toshihiro Mita. Further funding was from Strategic Promotion of International Cooperation to Accelerate Innovation in Africa (MEXT) (16jm0410004j0005) and the GHIT Fund (G2013-105) awarded to Prof Toshihiro Horii as well as from Juntendo University Collaborative Project Research Grant (PRO 2019-21) awarded to Dr. Betty Balikagala.

Author information




BB, TH and TMita designed and coordinated the study; BB, MSY, SIT, MI, MY, OTK, EHN, MS, NF, SY, WO, PSO, DAA, MAA, NMQP, TT, EIO, EK, MO, TH and TMita performed the field study; BB, MSY, SIT, MI, MY, NF, NT, MH and TMori performed the laboratory work; BB and TMita analysed and interpreted the data; BB, NMQP and TMita wrote the manuscript. All authors contributed significantly to this work. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Toshihiro Mita.

Ethics declarations

Ethics approval and consent to participate

Ethical approval for the study was obtained from Lacor Hospital Institutional Research and Ethics Committee (Ref; LHIREC 021/09/13), the Uganda National Council for Science and Technology (Ref; HS 1395), and Juntendo Research and Ethics Committee (Ref; 14-169).

Consent for publication

Prior to participation, all study subjects consented to the publication of study results in medical literature in an anonymized manner.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Balikagala, B., Sakurai-Yatsushiro, M., Tachibana, S. et al. Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda. Malar J 19, 76 (2020).

Download citation


  • Chloroquine sensitivity
  • Persistent recovery
  • Plasmodium falciparum
  • Uganda