Prevalence of Plasmodium falciparum parasites

J Antimicrob Chemother doi:10.1093/jac/dky258

Prevalence of Plasmodium falciparum parasites resistant to sulfadoxine/pyrimethamine in the Democratic Republic of the Congo: emergence of highly resistant pfdhfr/pfdhps alleles Papy Nkoli Mandoko1, Florent Rouvier2, Lebon Matendo Kakina1, Destin Moke Mbongi3, Christine Latour2, Joris Losimba Likwela4,5, Dieudonne´ Ngoyi Mumba1, Stomy Karhemere Bi Shamamba1, Jean-Jacques Tamfum Muyembe1, Le´on Muepu Tshilolo3, Daniel Parzy2 and Ve´ronique Sinou6* 1

National Institute of Biomedical Research, Kinshasa, Democratic Republic of the Congo; 2Department of Biology, K-Plan, Grand Luminy Technopoˆle, Marseille, France; 3Centre de Formation et d’Appui Sanitaire (CEFA)/Centre Hospitalier Monkole, Kinshasa, Democratic Republic of the Congo; 4Department of Public Health, Faculty of Medicine and Pharmacy, University of Kisangani, Kisangani, Democratic Republic of the Congo; 5National Malaria Control Program, Kinshasa, Democratic Republic of the Congo; 6 UMR-MD3, University of Aix-Marseille, Faculty of Pharmacy, Marseille, France *Corresponding author. Aix-Marseille Universite´, Timone, 27 Boulevard Jean Moulin, 13005 Marseille, France. Tel: !33(0)4 91 32 46 06; Fax: !33(0)4 91 32 46 06; E-mail: [email protected]

Received 9 January 2018; returned 18 March 2018; revised 30 May 2018; accepted 6 June 2018 Background: In 2005, the Democratic Republic of the Congo (DRC) switched to artesunate/amodiaquine as the first-line antimalarial in response to increasing sulfadoxine/pyrimethamine resistance and adopted intermittent preventive treatment using sulfadoxine/pyrimethamine in pregnancy. Objectives: To determine the prevalence of molecular markers of sulfadoxine/pyrimethamine resistance in southwestern DRC 10 years after the new policy was instituted. Methods: From March 2014 to December 2015, blood samples were collected from symptomatic patients presenting to outpatient centres in urban and rural areas. A total of 2030 confirmed Plasmodium falciparum isolates were genotyped at codons associated with sulfadoxine/pyrimethamine resistance. Results: The prevalence of pfdhfr-N51I, C59R and S108N and pfdhps-A437G mutations was consistently high; the prevalence of the pfdhps-K540E mutation was low but increased since its first report in 2008 in the same region, reaching 17.6% by 2015. The pfdhps-A581G mutation increased from 4.5% in 2014 to 14.0% in 2015 at urban sites while in rural areas it remained low (4.0%). The mutations pfdhfr-I164L and pfdhps-A613S were detected for the first time in DRC. Also, 11 (0.8%) isolates revealed the presence of the newly described pfdhps-I431V mutation. Combining pfdhfr and pfdhps alleles, quintuple and sextuple mutations were observed, with the emergence of septuple (IRNI/IAGEGA)- and octuple (IRNI/VAGKGS)-mutant genotypes. Conclusions: Intermittent preventive treatment using sulfadoxine/pyrimethamine during pregnancy remains warranted in southwestern DRC. However, the expansion of pfdhps-K540E mutation and emergence of mutants that cause higher levels of sulfadoxine/pyrimethamine resistance is concerning and may present a challenge for future preventive interventions in the country.

Introduction Malaria is one of the main global health problems, with 3.2 billion people at risk of infection by the end of 2016. In 2016, there was a total of 216 million cases of malaria and an estimated 445 000 deaths, most of them in sub-Saharan Africa.1 The Democratic Republic of the Congo (DRC) has the highest number of deaths from malaria in Africa, as well as some of the highest reported malaria transmission rates in the world.1 Between 14 and 16 million

clinical cases occur annually,1 accounting for an estimated 47.1% of the overall mortality in children under 5 years old, 40%–45% of medical consultations and 30%–47% of hospitalizations.2 In addition, the disease is responsible for 41% of prenatal consultations and approximately half of hospitalizations during pregnancy leading to low birth weight, placental malaria, severe maternal anaemia and perinatal mortality.3 Antimalarial drug resistance in Plasmodium falciparum is the most pressing problem confronting malaria control in the DRC. In 2001, sulfadoxine/pyrimethamine

C The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. V

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Nkoli Mandoko et al.

was introduced as the first-line treatment drug for uncomplicated malaria, replacing chloroquine, which had been the first-line drug since the 1970s.4,5 However, resistance to sulfadoxine/pyrimethamine in the country had already emerged even before it was declared the first-line drug;5,6 thus, sulfadoxine/pyrimethamine was an interim solution that lasted 4 years before being replaced by artemisinin-based combination therapies.7 Despite the policy change, sulfadoxine/pyrimethamine is readily available in the Congolese market8,9 and its low cost has encouraged its continued use as monotherapy. Sulfadoxine/pyrimethamine is also recommended by the Congolese Malaria National Control Programme (MNCP) for use as intermittent preventive treatment (IPT) of malaria in pregnancy (SP-IPTp).10 In recent years, the concept of IPT has been extended to include infants (IPTi) and children (IPTc; now referred to as SMC, for seasonal malaria chemoprevention)11 and has been evaluated in several clinical trials in different settings with success.12,13 Since its recommendation by the WHO in 2012,14 SMC has been adopted as policy in several areas of the Sahel and subSahel with seasonal transmission,15 some of them located a little further north of the DRC. This prompted the MNCP to consider the possibility of introducing these interventions in Congolese settings to reduce the burden of malaria in these high-risk populations. Parasite susceptibility to sulfadoxine/pyrimethamine is influenced by mutations in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes that encode proteins involved in the folate biosynthesis pathway.16 In the pfdhfr gene, the mutations N51I, C59R, S108N and I164L are associated with pyrimethamine resistance.17,18 Similarly, five mutations in pfdhps, namely S436A, A437G, K540E, A581G and A613S/T have been implicated in resistance to sulfadoxine.19,20 The accumulation of mutations in pfdhfr and pfdhps genes leads to increasing levels of sulfadoxine/pyrimethamine resistance.21,22 The presence of the quintuple mutant that combines the pfdhfr triple N51I, C59R, S108N mutation with the pfdhps double A437G, K540E mutation is a significant predictor of clinical and parasitological sulfadoxine/pyrimethamine treatment failure23 and results in limited protective value of SP-IPTi.24 Accordingly, the WHO recommends that SP-IPTi should be implemented only when the prevalence of the K540E mutation (and thus the quintuple mutant) is ,50%.25Additional mutations in pfdhfr (I164L) and pfdhps (A581G, A613S/T) have emerged in Africa that, in combination with the pfdhfr/pfdhps quintuple mutant, confer higher resistance.26 In some parts of East Africa that reported .80% clinical failure, the sextuple A581G mutation has been associated with loss of protective efficacy of IPTi24 and IPTp.22–31 A recent metaanalysis concluded that IPTp efficacy was reduced when the prevalence of A581G was .10%.32 In West-Central Africa, an unusual pfdhps-I431V mutation has been seen to emerge in combination with A436S, A437G, A581G and A613S substitutions.33–35 To date, the I431V mutation has been reported from Nigeria33,35 and Cameroon,34 but recent observations support the view that pfdhps-I431V mutation is on the increase in West-Central Africa.35 Unlike in other African countries, studies investigating pfdhfr and pfdhps mutations have been limited in the DRC. Only five studies conducted on samples collected before or during the transition from sulfadoxine/pyrimethamine to artesunate/amodiaquine in 2005 have documented the prevalence of sulfadoxine/pyrimethamine resistance mutations.36 Hence, it may be of major interest to study the prevalence of these mutations to determine whether

sulfadoxine/pyrimethamine resistance has declined or increased in the country under the conditions of its restricted use. The present study, carried out in southwestern DRC, aimed to determine the prevalence of sulfadoxine/pyrimethamine resistance mutations in the general population 10 years after the policy change and SP-IPTp introduction.

Patients and methods Study sites and sample collection Samples analysed in this study were collected as part of a study designed to characterize the phenotypic and genotypic chemoresistance profile of P. falciparum to commonly used antimalarial drugs in the DRC using a fielddeployable laboratory (K-LMP LabTM; K-Plan, Villeurbanne, France). The study was conducted between March 2014 and December 2015 in the cityprovince of Kinshasa and in the Kongo Central province, southwestern DRC (Figure 1). Kinshasa is densely populated (1109 inhabitants/km2) whereas in Kongo Central the population density is only 100 inhabitants/km2. In 2014, the population of these provinces was estimated at 17 million inhabitants, of whom 5.5 million lived in Kongo Central.37 Malaria is hyperendemic with perennial transmission. In Kinshasa, malaria prevalence is around 11.9%, with lower prevalence in the city centre (0.5%) than in peri-urban areas (31.7%).38 The Kongo Central province recorded one of the highest prevalence rates for malaria in the DRC, at 47.1%.39 In Kinshasa, the study sites consisted of the urban-rural Kingasani (KGS) health zone (HZ) located in the southeastern area of Kinshasa and the urban Binza Ozone (BZO) HZ situated along the southern bank of the Congo River, directly opposite the city of Brazzaville, capital of the Republic of the Congo. The Kongo Central study sites consisted of the rural Kimpese (KPS) and Kisantu (KST) HZs, located, respectively, in the Cataractes and Lukaya Districts, both limited in the south by the northern border of Angola. In each HZ, participants were recruited from selected health facilities at two timepoints over the 2 year study period: KGS in March–April 2014 and February 2015 plus August–September 2015; BZO in August–September 2014 and September–November 2015; KPS in May–June 2014 and June– August 2015 and KST in November–December 2014 and November– December 2015. Febrile patients (age .2 years) were screened using a malaria rapid diagnostic test (SD Bioline Malaria Ag P.f/Pan, Standard Diagnostics, Inc., Gyeonggi-do, South Korea). Those that tested positive were invited to participate in the study. Briefly, 2–3 mL of blood was collected from each participant. From this sample, a thin blood smear was performed to determine the level of parasitaemia and species identification. Aliquots (250 lL) were removed and stored at #20 C. Parasite DNA extraction and analysis of pfdhfr and pfdhps genes was performed at AixMarseille University (Marseille, France).

DNA extraction and sequencing DNA was extracted from 200 lL of collected blood using the QIAamp DNA Mini Kit (QIAGEN, Germany). Once a sample was confirmed as P. falciparum positive by real-time quantitative PCR (qPCR),40 it was subjected to pfdhfr (codons 16, 51, 59, 108 and 164) and pfdhps (codons 431, 436, 437, 540, 581 and 613) mutational analysis. The target regions were amplified using the Red Diamond Taq polymerase kit (Eurogentec, Seraing, Belgium). Primer sequences and PCR reaction conditions are indicated in Table 1. Aliquots of amplified products were subjected to sequencing analysis by Eurofins Genomics (Germany). Sequence alignments were performed with CodonCode Aligner software (CodonCode Corporation, Centerville, USA).

Statistical analysis Data were analysed with XLSTAT version 2014.4.08 (SPSS Inc., Chicago, IL, USA). Changes in mutation and genotype frequency over the study period


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Highly resistant pfdhfr/pfdhps in the DRC

Figure 1. Map of the DRC with study areas. Table 1. Primer sequences and PCR reaction conditions used for the detection of pfdhfr (A16V, N51C, C59R, S108N and I164L) and pfdhps (I431V, S436A/F, A437G, K540E, A581G, A613S/F) mutations Gene and primer pfdhfr DHFR-F DHFR-R pfdhps DHPS-F DHPS-R

Sequence (50 to 30 )

Position

Product size (bp)

PCR conditions

ACG-TTT-TCG-ATA-TTT-ATG-C TCA-CAT-TCA-TAT-GTA-CTA-TTT-ATT-C

20–38 557–581

562

94 C % 5 min; 40%[94 C % 20 s, 52 C % 20 s, 72 C % 40 s]; 72 C for 5 min

GTT-GAA-CCT-AAA-CGT-GCT-GT TTC-ATC-ATG-TAA-TTT-TTG-TTG-TG

1225–1244 1874–1896

672

94 C % 5 min; 40%[94 C % 20 s, 54 C % 20 s, 72 C % 40 s]; 72 C for 5 min

for each study site were evaluated using v2 and Fisher’s two-tailed tests. A P value of ,0.05 was considered statistically significant. Mixed alleles were categorized as mutants.

Ethics Ethics approval was obtained from the Ethics Committee of the Centre de Formation et d’Appui Sanitaire/Centre Hospitalier Monkole (N/Re´f: 01/CEFAMONKOLE/CEL/2013). Blood samples were collected after written informed consent, in French or in the main language used in Kinshasa and Kongo Central, was obtained from each patient or their parent/guardian in cases of minor-aged patients. Participants found to be positive for malaria were treated according to national guidelines.

Results Characteristics of study participants A total of 2172 people aged 2–96 years old, including 577 (26.6%) from KGS, 472 (21.7%) from BZO, 592 (27.3%) from KPS and 531 (24.4%) from KST, participated in the study (Figure 2). There were marked differences in the study subject demographic and parasitological characteristics between locations (Table 2). The median age [P , 0.001 by the Wilcoxon rank-sum (Mann–Whitney) test] and fever (temperature 37.5 C) at health facility presentation

(v2 " 84.678, P , 0.0001) were lower at KGS compared with BZO, KPS and KST, and the geometric mean parasitaemia was lower in BZO compared with the other three study sites [P , 0.0001 by the Wilcoxon rank-sum (Mann–Whitney) test]. Only a few patients reported prior history of sulfadoxine/pyrimethamine (50/2115; 2.4%) usage prior to enrolment into the study. Overall, 2030 (93.5%) samples were confirmed as P. falciparum positive by qPCR. Out of these, 1714 (84.4%) and 1435 (70.7%) were successfully sequenced for pfdhfr and pfdhps genes, respectively.

Prevalence of pfdhfr and pfdhps mutations The prevalence of pfdhfr mutant alleles N51I and S108N was high in all sampling, totalling more than 97% and 99%, respectively, while the prevalence of the mutation C59R was slightly lower, ranging from 86.0% to 90.8% (Figure 3a). The rare I164L mutant allele was observed in only one isolate (0.1%; 1/1715) collected from KGS. No mutation was observed at codon 16; this codon was excluded from any further analysis. The most prevalent pfdhps mutations affected codons 437 and 436, and were present in 97.6% (1416/1451) and 13.3% (192/1441) of isolates, respectively. At HZ level, KPS and KST in the Kongo Central province exhibited the highest prevalence of the S436A mutation at 21.5% and 15.9%, respectively, compared with KGS (6.1%) and BZO (8.7%) 3 of 12

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2172 participants enrolled from 03/2014 to 12/2015

577 from Kingasani (Kinshasa)

472 from Binza Ozone (Kinshasa)

592 from Kimpese (Kongo Central)

531 from Kisantu (Kongo Central)

Screening for P. falciparum

530 real-time PCR positive




Screening for pfdhfr/pfdhps mutations

pfdhfr 407 successful complete sequence 0 partially sequenced

Reasons for failure * Insufficient quality of DNA band on gel * Insufficient quality of sequences


pfdhps 326 successful complete sequence 9 partially sequenced






Figure 2. Patient enrolment.

Table 2. Demographic and parasitological characteristics of the study population in the DRC Study site Parameters

KGS (n " 577)

Median age, years (IQR) Sex, female, % (n/N) Geometric mean parasitaemia, % (95% CI) Febrile at presentation (37.5 C), % (n/N) Antimalarial treatment before presentation, % (n/N) artemisinin combination therapy, % (n) quinine, % (n) sulfadoxine/pyrimethamine, % (n) other, % (n)

9.0 (4.0–20.0) 55.4 (318/574) 0.73 (0.28–1.18) 69.4 (390/562) 23.0 (128/557) 1.8 (10) 16.9 (94) 2.1 (12) 2.1 (12)

BZO (n " 472)

KPS (n " 592)

KST (n " 531)

15.0 (6.0–27.3) 56.8 (266/468) 0.47 (0.11–0.83) 85.7 (403/470) 8.8 (40/452) 0.7 (3) 6.0 (27) 0.2 (1) 2.0 (9)

13.0 (6.0–26.0) 50.9 (299/587) 0.75 (0.33–1.16) 83.1 (483/581) 20.4 (118/579) 5.7 (33) 7.1 (41) 4.7 (27) 2.9 (17)

11.5 (5.0–22.0) 56.8 (301/530) 0.77 (0.49–1.05) 91.5 (483/528) 4.9 (26/527) 0.8 (4) 2.1 (11) 1.9 (10) 0.2 (1)


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(a) KGS

100 90

BZO

80

KPS KST

Prevalence (%)

70 60 50 40 30 20 10 0 N51I

C59R

S108N

I164L

Prevalence (%)

(b) 100

KGS

90

BZO

80

KPS KST

70 60 50 40 30 20 10 0 I431V

S436A

A437G

K540E

A581G

A613S

Figure 3. Prevalence of mutations in pfdhfr (a) and pfdhps (b) genes over the 2 year period by study site.

(Figure 3b). The prevalence of K540E mutation was comparable among study sites with the highest observed in BZO (17.3%) and the lowest in KST (12.8%). The A581G mutation was most prevalent in Kinshasa, both in KGS (6.7%) and BZO (9.9%); it was observed at 3.4% in KPS and 4.4% in KST. The mutations pfdhps-I431V and A613S were detected in KGS (1.8% and 1.5%, respectively), in BZO (1.0% for both) and in KPS (0.5% for both), while being absent in KST.

Temporal distribution of pfdhfr and pfdhps mutations Between 2014 and 2015, samples collected from KPS and KST displayed steady prevalence at all codons except for codon 540 in KPS where the mutant allele increased from 12.4% in 2014 to 18.0% in 2015 (P " 0.27) (Figure 4). Continued monitoring of this allele is needed to evaluate whether this is a significant trend over time. By contrast, BZO and KGS showed temporal changes in allele prevalence. At BZO, samples collected in 2014 had lower prevalence of mutation in all pfdhps codons compared with samples collected in 2015. The prevalence of pfdhps mutations at codons 436, 437, 540 and 581 was shown to be significantly different (P " 0.03, 0.03, 0.04 and 0.03, respectively) by v2 analysis. At KGS, the prevalence of mutation at codon 581 significantly increased from 2.5% in 2014 to 13.4% in 2015 (P , 0.0001). An increase in the prevalence of mutation at codon 540 was also observed, although this was not statistically significant (P " 0.37). The mutations at codons 431 and 613 were not found in 2014 at this site; however, they

were present in 2015 in 4.7% (P " 0.003) and 3.9% (P " 0.008) of isolates, respectively.

Prevalence of pfdhfr and pfdhps genotypes Table 3 shows the prevalence of genotypes in pfdhfr and pfdhps genes. The WT pfdhfr (NCSI) and pfdhps (ISAKAA) were detected at very low levels (0.5% and 1.7%, respectively). The triple IRN mutant pfdhfr was predominantly high across all sites with an overall prevalence of 87.3%. In the case of the pfdhps gene, the ISGKAA genotype was the most common (70.1%), followed by IA/FGKAA, which occurred in 11.4% of samples. Prevalence of double-mutant (ISGEAA) and triple-mutant (ISGEGA) genotypes was 9.6% and 4.0%, respectively. The I431V mutant occurred in diverse forms with a total prevalence of 0.8% (0.1% VSGKGA, 0.6% VAGKGS and 0.1% VSGEGS). It should be noted that the VSGEGS genotype was observed in one sample containing mixed alleles (codons 431, 540, 581 and 613), leading to the possibility that this genotype did not actually occur within a single genome in this sample. As expected, combining the two loci revealed that more than 71% of the parasite isolates were of the pfdhfr/pfdhps IRNI/ ISGKAA and IRNI/IAGKAA genotypes (Table 4). The quintuple mutant, IRNI/ISGEAA, was present in 9.7% of the samples and the sextuple mutant, IRNI/ISGEGA, was present in 3.6% of the samples. Although in low proportions (,1%), more highly mutated septuple and octuple mutants were observed. The septuple and octuple mutants identified were due to the combination of the triple IRNI pfdhfr-mutant genotype with the quadruple (IAGEGA) and quintuple (VAGKGS, VSGEGS) mutants of pfdhps, respectively.

Temporal distribution of pfdhfr and pfdhps genotype Table S1 (available as Supplementary data at JAC Online) shows the temporal distribution of pfdhfr and pfdhps genotypes. There was a decline in the prevalence of the pfdhfr and pfdhps WT genotypes from 0.9% and 2.7% in 2014 to 0.1% and 0.6% in 2015, respectively. The IRNI genotype remained relatively stable over time at all four sites; in contrast, temporal changes were seen in pfdhpsmutant genotypes across sites. An increase in prevalence of the pfdhps double-mutant ISGEAA genotype was observed at BZO and KPS from 6.3% and 8.6% in 2014 to 13.2% and 14.6% in 2015, respectively. Additionally, prevalence of the triple-mutant ISGEGA genotype was found to increase in KGS and BZO from 2.5% and 4.6% to 7.0% and 9.6%, respectively by 2015 (Figure 5). Table S2 shows the temporal distribution of genotypes in combined pfdhfr and pfdhps genes. Because the pfdhfr triple mutation was above 85% in the 2014–15 period, the prevalence of pfdhfr/ pfdhps quintuple IRNI/ISGEAA- and sextuple IRNI/ISGEGAmutated genotypes tracked the pfdhps double and triple mutation prevalence. Thus, the prevalence of the quintuple INRI/ISGEAA mutant was found to go from 7.4% and 8.9% in samples collected in 2014 to 12.9% and 14.9% in samples collected in 2015 in BZO (P " 0.24) and KPS (P " 0.14), respectively (Figure 6). The proportion of isolates with sextuple mutations (IRNI/ISGEGA) increased markedly between 2014 and 2015 in KGS from 1.9% to 7.3% (P " 0.03) and in BZO from 4.6% to 9.1% (P " 0.29). By contrast, the sextuple IRNI/ISGEGA genotype was found at a low prevalence in KPS and KST in 2014 and in 2015 (1.6% and 1.0% in KPS, and 1.4% and 4.0% in KPS, respectively) (Figure 6). 5 of 12

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KGS

90

80

80

70

70

60 50 40 30

50 40

**

70

70

60

60

pfdhps

L 64

I1

08

S1

N5

S4

I4

31

L 64

08

I1

S1

C5

1I

0 36 A4 A 37 G K5 40 A5 E 81 G A6 13 S

0 V

10 N

10 9R

20

N

I4 31 S4 V 36 A4 A 37 G K5 40 A5 E 81 G A6 13 S

30

20

1I

8N 64 L

40

C5

30

2014 2015

50

9R

Prevalrnce (%)

80

40

KST

100 90

50

pfdhps

pfdhfr

I4 31 S4 V 36 A4 A 37 G K5 40 A5 E 81 G A6 13 S

2014 2015

80

N5

I1

pfdhfr

90

pfdhfr

S1 0

1I N5

I4

pfdhps KPS

9R

31 S4 V 36 A4 A 37 G K5 40 A5 E 81 G A6 13 S

L 64

I1

S1 0

C5

N5

8N

0 9R

0 1I

10

100

*

20

10

pfdhfr

*

*

C5

***

2014 2015

60

30 **

*

BZO

100

90

20

Prevalence (%)

2014 2015

Prevalence (%)

Prevalence (%)

100

pfdhps 2

Figure 4. Distribution and prevalence of pfdhfr and pfdhps mutations at the four study sites. P value calculated by v test: *P , 0.05, **P , 0.01 and ***P , 0.0001.

Discussion In 2005, the DRC switched to artemisinin-based combination therapies as first-line treatment for uncomplicated malaria in response to increasing sulfadoxine/pyrimethamine resistance and at the same time adopted SP-IPTp.7 This study analyses the polymorphisms and prevalence of mutations in pfdhfr and pfdhps genes in isolates collected from the general population 10 years after the policy change in the southwestern region of DRC. To our knowledge, this is the first report that includes a large number (2030) of samples and where all mutations of pfdhfr and pfdhps genes predictive of sulfadoxine/pyrimethamine therapeutic failure were screened. Our results demonstrate that pfdhfr polymorphisms associated with sulfadoxine/pyrimethamine resistance persist at high frequency in southwestern DRC. In this area, the proportion of N51I

and S108N mutations in P. falciparum isolates was already near saturation in 2008,41 and did not vary in 2014–15. Likewise, during the same period, more than 89% of P. falciparum isolates carried pfdhfr mutant allele C59R, a proportion that was around 80% in isolates collected in 2008 in Kinshasa.41 Thus, our study revealed an intense level of 87.7% of the pfdhfr triple mutant IRN responsible for pyrimethamine resistance. This is higher than the 60.8%–65.1% pfdhfr triple-mutant prevalence reported in other Western provinces in 2004,42 when sulfadoxine/pyrimethamine was being used as the first-line drug. Similarly, the pfdhps-A437G mutation has continued to increase after 2008, reaching 97.7% over the 2014–15 period. Over the same period, the prevalence of pfdhps-K540E was 14.7%, resulting in a prevalence of the quintuple pfdhfr/pfdhps mutant (IRN/GE) of 9.3%. The quintuple mutant rate found in this study validates low sulfadoxine/pyrimethamine resistance in the southwestern part of DRC, indicating that SP-IPTp is still expected to be


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Table 3. Prevalence of pfdhfr and pfdhps genotypes over the 2 year period in the study area Percentage (no.) of isolates Haplotype pfdhfr NCSI (WT) ICSI NCNI ICNI NRNI IRNI IRNL pfdhps ISAKAA (WT) IA/FAKAA ISGKAA IA/FGKAA ISGKGA ISGEAA IAGKGA IAGEAA ISGEGA VSGKGA IAGEGA VAGKGS VSGEGS

No. of mutations 0 1 1 2 2 3 4 0 1 1 2 2 2 3 3 3 3 4 5 5

KGS

BZO

KPS

KST

all sites

n " 407 0.5 (2) 0.2 (1) 0.2 (1) 10.3 (42) 1.7 (7) 86.7 (353) 0.2 (1) n " 326 1.5 (5) 0.6 (2) 77.9 (254) 3.7 (12) 0.6 (2) 9.2 (30) — 0.3 (1) 4.3 (14) 0.3 (1) — 1.5 (5) —

n " 393 0.8 (3) — — 12.0 (47) 1.0 (4) 86.3 (339) — n " 311 3.5 (11) 0.3 (1) 70.4 (219) 6.1 (19) 1.3 (4) 9.3 (29) 0.3 (1) 0.3 (1) 6.7(21) — 0.6 (2) 1.0 (3) —

n " 464 0.6 (3) 0.2 (1) 0.2 (1) 8.2 (38) 0.4 (2) 90.3 (419) — n " 408 — 1.0 (4) 64.0 (261) 18.6 (76) 1.0 (4) 11.5 (47) — 1.5 (6) 2.0 (8) — — 0.2 (1) 0.2 (1)

n " 450 0.2 (1) — — 13.8 (62) 0.4 (2) 85.6 (385) — n " 390 2.0(8) 0.3 (1) 69.7 (272) 14.6 (57) 0.5 (2) 7.9 (31) — 1.0 (4) 3.8 (15) — — — —

n " 1714 0.5 (9) 0.1 (2) 0.1 (2) 11.0 (189) 0.9 (15) 87.3 (1496) 0.1 (1) n " 1435 1.7 (24) 0.6 (8) 70.1 (1006) 11.4 (164) 0.8 (12) 9.6 (137) 0.1 (1) 0.8 (12) 4.0 (58) 0.1 (1) 0.1 (2) 0.6 (9) 0.1 (1)

Mutated codons are shown in bold. A dash indicates the absence of this genotype.

efficacious. This is in line with other studies carried out in Central Africa,43–45 and elsewhere in western DRC,42 in contrast to eastern DRC, where this quintuple mutant was observed at high prevalence, with K540E mutation detected at .50% in the locality of Rutshuru, Nord Kivu province.7 One explanation for this is its proximity to the neighbouring countries in East Africa, Rwanda and Uganda, which have a very high prevalence of sulfadoxine/pyrimethamineresistant parasites and sustained sulfadoxine/pyrimethamine drug pressure.45–47 These countries reported prevalences of K540E mutation (as a marker for the quintuple-mutant genotype) higher than the 50% cut-off,26 and have been classified as unsuitable for SP-IPTi by the WHO.25 Thus, given that the quintuple-mutant genotype is less likely to be found in our study area during actual treatment with sulfadoxine/pyrimethamine, SP-IPTi could be beneficial to our studied population. However, when compared with the data obtained by Mobula et al.,41 our results show an upward trend of the prevalence of K540E mutation from 9.5% in 2008 to 12.1% in 2014 and 17.5% in 2015, indicating that the prevalence of the pfdhfr/pfdhps quintuple mutant may increase further in the coming years. This more recent increase of K540E mutation occurred in a period of much lower drug pressure, as sulfadoxine/pyrimethamine is no longer used for case management but only for IPTp as recommended by the authorities. However, sulfadoxine/pyrimethamine continues to predominate in the antimalarial drug market both in rural and urban areas.8,9 In 2013, it was reported to constitute 31.1% of all antimalarials purchased in Kinshasa,8 suggesting it is still widely used for uncomplicated malaria treatment. In addition,

it is likely that the use of another antifolate such as co-trimoxazole (trimethoprim/sulfamethoxazole) for prophylaxis against opportunistic infections among HIV-infected people may be contributing to the sustained selection pressure. In certain parts of East Africa, there are reports that even in the presence of very high penetration of the pfdhfr/pfdhps quintuplemutant genotype (including K540E mutation), sulfadoxine/pyrimethamine may continue to be beneficial for IPTp in reducing adverse outcomes of malaria in pregnancy.48–50 Settings where the sextuple A581G mutation is present are of even greater concern, with studies showing no effect of sulfadoxine/pyrimethamine on malaria infection and parasite densities,30,31,50,51 as well as increased risk of severe malaria for the offspring.52 Moreover, one earlier study from northern Tanzania suggested that sulfadoxine/pyrimethamine may cause harm in such settings.28 In the DRC, the A581G mutation was first reported in 2002 at 30% prevalence in Rutshuru, eastern DRC, which is located in an area of widespread drug resistance, near Rwanda.26 This site reported about 60% clinical failure and a loss of the effectiveness of SP-IPTp at reducing low birthweight.53 In our results, the A581G mutants were found in 5.8% of all isolates typed. When delineated by study area, the prevalence of 581G was low (4.5%) in urban and rural sites in 2014 but rapidly increased over 1 year in urban areas reaching 14.0% by 2015, while it remained at levels around 4.0% in the rural sites. The differences in levels of resistance markers between urban and rural areas, which both represent high malaria endemicity, leads to speculation that other local factors are contributing



Table 4. Prevalence of pfdhfr/pfdhps genotypes over the 2 year period Percentage (no.) of isolates pfdhfr/pfdhps genotype NCSI/ISAKAA NCSI/ISGKAA ICNI/ISAKAA NRNI/ISAKAA NCNI/ISGKAA ICNI/ISGKAA NRNI/IAAKAA NRNI/ISGKAA IRNI/ISAKAA ICNI/IAGKAA ICNI/ISGKGA ICNI/ISGEAA IRNI/IAAKAA IRNI/ISGKAA ICNI/IAGEAA ICNI/ISGEGA IRNI/IA/FGKAA IRNI/ISGKGA IRNI/ISGEAA IRNI/IAGKGA IRNI/IAGEAA IRNI/ISGEGA IRNI/VSGKGA IRNI/IAGEGA IRNI/VAGKGS IRNI/VSGEGS

No. of mutations

KGS (n " 282)

BZO (n " 307)

KPS (n " 386)

KST (n " 367)

all sites (n " 1342)

0 1 2 2 2 3 3 3 3 4 4 4 4 4 5 5 5 5 5 6 6 6 6 7 8 8

0.7 (2) — 0.4 (1) — — 7.4 (21) — 1.8 (5) 0.4 (1) 0.4 (1) — 1.1 (3) 0.4 (1) 68.1 (192) — — 3.9 (11) 0.4 (1) 8.5 (24) — 0.4 (1) 4.3 (12) 0.4 (1) — 1.8 (5) —

1.0 (3) — 0.7 (2) 0.3 (1) — 9.4 (29) — 1.0 (3) 1.6 (5) 0.3 (1) 0.3 (1) 0.3 (1) 0.3 (1) 59.3 (182) — 0.3 (1) 5.9 (18) 1.0 (3) 9.8 (30) 0.3 (1) — 6.5 (20) — 0.7 (2) 1 (3) —

— 0.3 (1) — — 0.3 (1) 6.0 (23) 0.3 (1) 0.3 (1) — 0.8 (3) — 0.3 (1) 0.5 (2) 57.3 (221) 0.3 (1) 0.5 (2) 17.9 (69) 1.0 (4) 11.9 (46) — 0.8 (3) 1.3 (5) — — 0.3 (1) 0.3 (1)

0.3 (1) — 0.5 (2) — — 10.1 (37) — 0.5 (2) 0.8 (3) 1.1 (4) — 1.4 (5) 0.3 (1) 60.8 (223) — 0.3 (1) 10.6 (39) 0.5 (2) 8.2 (30) — 1.6 (6) 3.0 (11) — — — —

0.4 (6) 0.1 (1) 0.4 (5) 0.1 (1) 0.1 (1) 8.2 (110) 0.1 (1) 0.8 (11) 0.7 (9) 0.7 (9) 0.1 (1) 0.7 (10) 0.4 (5) 61.0 (818) 0.1 (1) 0.3 (4) 10.2 (137) 0.7 (10) 9.7 (130) 0.1 (1) 0.7 (10) 3.6 (48) 0.1 (1) 0.1 (2) 0.7 (9) 0.1 (1)

Mutated codons are shown in bold. A dash indicates the absence of this genotype.

to local expansion of mutant parasites. Such factors may be that the urban sites in the city-province of Kinshasa have a higher density of drug shops than the rural sites and there is easier access to healthcare and services;54 thus, resulting in greater drug pressure and higher resistance levels. Another explanation may be the importation of sulfadoxine/pyrimethamine-resistant parasites to Kinshasa from Brazzaville, the capital of the neighbouring Republic of Congo. A prevalence of 33% pfdhfr/pfdhps quintuple (IRNI/ IGEKAA) and 25% sextuple (IRNI/IGEGAA) mutation was recorded in 2012–13 among pregnant women in Brazzaville.55 The data presented here show that sulfadoxine/pyrimethamine use in urban settings was not just associated with the expansion of K540E and A581G mutations but also with the emergence of the highly resistant pfdhfr-I164L and pfdhps-A613S alleles. In our study, the A613S mutant allele was always found in association with A437G, A581G and the uncommon I431V mutations. While the effect of this pfdhps-I431V mutation on antifolate resistance has not yet been established, it is possible that it may interfere with the efficient binding of sulfadoxine conferring increased sulfadoxine/pyrimethamine resistance.35 Our study is not without limitations. Considering the size (2 345 000 km2) of the DRC and the different patterns and drivers of malaria in the country, the results of this study are not

generalizable to all Congolese settings. Sulfadoxine/pyrimethamine resistance appears more pronounced in the east than the west of the country. Thus, for a proper, evidence-based implementation of IPT programmes and an understanding of regions where the strategy may be compromised, countrywide monitoring of the mutations is essential.

Conclusions This study provides an update on the prevalence of mutations related to resistance to sulfadoxine/pyrimethamine treatment in the southwestern region of the DRC. SP-IPTp remains warranted in this setting. However, the emergence of mutants and related genotypes that cause higher levels of sulfadoxine/pyrimethamine resistance is concerning and calls for continued surveillance to monitor the protective efficacy of sulfadoxine/pyrimethamine and the prevalence of resistance-mediating polymorphisms.

Acknowledgements We thank all the study participants for agreeing to take part in this study. Many thanks also to the local authorities for support, to the health staff and to the laboratory technicians of the different health centres for their


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Figure 5. Distribution and prevalence of pfdhfr and pfdhps genotypes at the four sites. WT, wild-type (NCSI, for pfdhfr; ISAKAA, for pfdhps). KGS, Kingasani; BZO, Binza Ozone; KPS, Kimpese; KST, Kisantu. cooperation in sample collection. The authors thank also Deborah Sene´ (University of Aix-Marseille, France) and Christophe Gaillardot (University of Aix-Marseille, France) for their technical assistance during the laboratory analyses. Expertise France (France) is thanked for its external support. P. N. M., D. M. M. and L. M. K. are grateful to the 5% Initiative (indirect contribution of France to the Global Fund to fight AIDS, Tuberculosis and Malaria) for personal support through the grant number 12INI214.

Funding This work was supported by the 5% Initiative (indirect contribution of France to the Global Fund to fight AIDS, Tuberculosis and Malaria) under the grant number 12INI214.

Transparency declarations None to declare.

Author contributions V. S., D. P., J. L. L. and L. M. T. conceived the study, and participated in its design and coordination. P. N. M. supervised sample collection in the field and participated in writing the manuscript. V. S. interpreted the data and wrote the manuscript. D. M. M. and L. M. K. carried out sample collection. F. R. carried out molecular assays and performed statistical analysis. C. L. participated in molecular assays and data analysis. D. P., D. N. M., S. K. B. S and J-J. T. M. reviewed the final manuscript. All authors read and approved the final version of the manuscript.



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Figure 6. Distribution and prevalence of pfdhfr/pfdhps genotypes at the four sites. Represented are WT (NCSI/ISAKAA), pfdhfr triple mutant (N51I, C59R, S108N) and pfdhps alleles at codons 431, 436, 437, 540, 581 and 613.

Supplementary data Tables S1 and S2 appear as Supplementary data at JAC Online.

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