Skip to main content

Assessment of pfcrt 72-76 haplotypes eight years after chloroquine withdrawal in Kinshasa, Democratic Republic of Congo

Abstract

Background

In 2001, the World Health Organization (WHO) has recommended the use of artemisinin-based combination therapy (ACT) as the first-line treatment of uncomplicated malaria cases, as monotherapies had become ineffective in many parts of the world. As a result, the Democratic Republic of Congo (DRC) withdrew chloroquine (CQ) from its malaria treatment policy in 2002 and an artesunate (AS)-amodiaquine (AQ) combination became the ACT of choice in DRC in 2005. AQ-resistance (AQR) has been reported in several parts of the world and mutations in codons 72-76 of the Plasmodium falciparum chloroquine-resistance transporter (pfcrt) gene have been strongly correlated with resistance, especially mutations encoding the SVMNT haplotype. This haplotype was first identified in Southeast Asia and South America but was recently reported in two African countries neighbouring DRC. These facts raised two questions: the first about the evolution of CQ resistance (CQR) in DRC and the second about the presence of the SVMNT haplotype, which would compromise the use of AQ as a partner drug for ACT.

Methods

A total of 213 thick blood films were randomly collected in 2010 from a paediatric clinic in Kinshasa, DRC. Microscopy controls and real-time polymerase chain reaction (RT-PCR) were performed for Plasmodium species identification. Haplotypes of the pfcrt gene were determined by sequencing.

Results

The K76T mutation was detected in 145 out of 198 P. falciparum-positive samples (73.2%). In these 145 resistant strains, only the CVIET haplotype was detected.

Conclusions

This study is the first to assess the molecular markers of resistance to CQ and AQ after the introduction of ACT in DRC. The results suggest first that CQR is decreasing, as wild-type pfcrt haplotypes were found in only 26.8% of the samples and secondly that the SVMNT haplotype is not yet present in Kinshasa, suggesting that AQ remains valid as a partner drug for ACT in this region.

Background

In 2001, the World Health Organization (WHO) has recommended the use of artemisinin-based combination therapy (ACT) as the first-line treatment of uncomplicated malaria cases because monotherapies had become ineffective in many parts of the world due to the spread of Plasmodium falciparum strains resistant to almost all anti-malarial drugs in use [1].

Resistance to chloroquine (CQ), the drug that had been widely used for decades for malaria treatment [2], has been well characterized and it is now established that a mutation (K76T) occurring in the P. falciparum chloroquine-resistance transporter (pfcrt) gene is the key element [35]. Nevertheless many other mutations have been described in this gene [6].

Childs and colleagues were the first to show in vitro cross-resistance between amodiaquine (AQ) or its active metabolite and CQ [7]. Because of their pharmacological similarity, CQ and AQ were supposed to have the same molecular mechanism of resistance. Furthermore, Ochong et al., by analysing samples from a clinical trial held in southern Sudan, demonstrated a link between the K76T mutation and in vivo AQ resistance [8]. A few years later, other studies performed on the pfcrt gene revealed that some haplotypes defined by the aminoacids sequence at positions 72-76 of the CRT protein were related to a particular resistance profile and also to a specific geographical distribution. Sa et al. analysed reference strains and provided the first in vitro evidence that one of these haplotypes, the SVMNT (Ser-Val-Met-Asn-Thr), was strongly correlated with resistance to desethylamodiaquine (DEAQ), the active metabolite of AQ [9]. One year later, in vivo evidence of this link has been given by Beshir et al. who found that the presence of the SVMNT haplotype was sufficient to confer AQ resistance [10].

This haplotype was initially discovered in Asia and in South America [11, 12], but it has recently been found in two African countries: first in Tanzania in 2006 [13], then in 2010 in Angola [14]. Both countries border the Democratic Republic of Congo (DRC), where CQ, after being widely used, was officially abandoned in 2002 to be replaced first by sulphadoxine-pyrimethamine (SP) and then by an artesunate-amodiaquine (AS-AQ) combination in 2005 [15].

This raised questions about the presence of the SVMNT haplotype in the DRC, which would jeopardize the use of AQ as a partner drug for ACT. These questions were addressed in this study.

Methods

Sample collection

A total of 213 thin and thick blood films (TBF) were randomly selected in April 2010 from the laboratory archives of the St Marc Paediatric Clinic of Masina (Kinshasa). All the smears were positive using microscopic recordings as selection criteria. The blood samples had been collected between January and March 2010 from children under five years who were suspected of malaria.

Malaria diagnosis

All blood smears underwent microscopic control performed by two experienced microscopists and the results were confirmed by real-time polymerase chain reaction (RT-PCR).

DNA extraction

The whole TBF was used for DNA extraction, leaving the thin smear for microscopic reading. Each TBF was first immersed in xylene for 10 s to avoid contamination between samples and to dissolve any residual oil from previous microscopic examinations. Then it was scratched out with a sterile scalpel as described previously by Cnops et al. [16]. The scraped material was transferred into a 1.5 ml sterile Eppendorf tube containing 100 μl of PBS and then used as a template for a semi-automated extraction using the Maxwell® 16 Cell LEV DNA purification Kit (Promega, USA). A DNA internal extraction and inhibition control was used to follow the extraction procedure (DIAcontrolDNA®, Diagenode, Belgium). DNA was stored at -20°C before processing.

Real-time PCR

The RT-PCR method for Plasmodium sp. identification was used as previously described [17]. Briefly, a four-primer PCR was used in two duplex reactions including a couple of forward primers (Pfal + Pviv and Pmal + Pova) with a universal reverse primer (plasmo2). The probes and primers were designed to detect genes of the small subunit 18S rRNA of Plasmodium species (Table 1). The mix consisted of: 12.5 μl of 2X Taqman Universal PCR Master Mix (Applied Biosystems, USA), 200 nM of all primers and probes except P. vivax at 100 nM (primers and probe), 2.5 μl of Internal Control primers and probes and the rest of water to make a total volume of 25 μl including 5 μl of DNA. PCR tests were run on a 7500 Fast Instrument (Applied Biosystems) and in the presence of positive controls (provided by the Parasitology Unit, Institute of Tropical Medicine, Antwerp and the Laboratory of Clinical Microbiology, University Hospital of Liège). PCR conditions were as follows: 2 min at 95°C, followed by 50 cycles of 15 s at 95°C and 60 s at 60°C.

Table 1 Primer and probe sequences

Pfcrt genotyping

The procedure previously described by Ménard et al. [18] was modified by adding an M13 fragment on the 5′ ends of the pfcrt primers (Table 1) to amplify a 154 bp fragment that included the pfcrt codons in positions 72-76 of all of the P. falciparum-positive samples. PCR tests were run on a Hybaid PX2 (ThermoFischer Scientific, USA) under the following conditions: 95°C for 10 min, followed by 45 cycles of 20 s at 95°C and 45 s at 60°C. Amplification was controlled on a microchip electrophoresis system using the MCE®-202 MultiNA (Shimadzu). Amplicons were purified on Sciclone G3 Automated Liquid Handling Workstation (Perkin Elmer, USA) using AgencourtCleanSEQ® kit (Agencourt Bioscience, USA).

Sequencing was performed on a 3130xl DNA sequencer (Applied Biosystems, USA).

Sequences were aligned using the GeneStudioTM® Professional software and were compared with the reference sequence of the CRT protein [GenBank: CAD50842.1] using the online BLASTx tool.

Ethical considerations

This study has received the ethical approbation of the Ministry of Public Health of the DRC and of the Institutional Committee of the Faculty of Medicine, University of Kinshasa.

Results

Malaria diagnosis confirmation

RT-PCR lead to the detection of 202/213 (93.4%) positive samples for P. falciparum including three mixed infections (P. falciparum + Plasmodium malariae).

Pfcrt haplotypes

Pfcrt genotyping was successfully performed for 198 P. falciparum samples from the 202 positive samples detected by RT-PCR. Among them, 145 K76T mutants (73.2%) were detected and they were all harbouring the CVIET haplotype. The wild strains had the CVMNK haplotype. No other haplotypes were detected.

Discussion

Nearly ten years after the withdrawal of CQ in the first-line treatment of malaria in the DRC, CQR strains remain above the warning threshold established by WHO. However the prevalence of pfcrt 76 T mutants continues to decline as shown in previous studies performed in DR Congo where a slow reversion in sensitivity has been recorded. Indeed, the percentage of Pf resistant strains went from 100% in 2000 [19], to 93% in 2002 [20], 83.8% in 2008 [21] and 73.2% in the present study. Finally, over the last ten years, the prevalence of resistant strains has decreased by 26.8%. In other countries however, the decrease was much more significant like in Malawi where CQR fell from 85% to 13% in 8 years [22] after replacing CQ by SP or in Gabon with a drop of 55% (100% to 45%) in 6 years [23]. This fact could be explained by the large area covered by DRC that is more difficult to control, and by the relatively high cost of ACT compared to former monotherapies, resulting in a slower anti-malarial policy shift. Until 2007, CQ was still the most widely used anti-malarial drug in the DRC [24] although it was officially removed since 2002 and firstly replaced by SP. Aside from the efficiency with which new drug policies were implemented, malaria transmission intensity in the region and the use of many other anti-malarial drugs in addition to the national policy have been suggested to explain the difference in trend [25].

The CVIET haplotype was the only resistant haplotype identified in the present study. Indeed, it is the most prevalent haplotype among the five discovered to date in Africa and it seems that it has migrated from Southeast Asia to Africa via India [26, 27]. Some studies have described this haplotype as a necessary but insufficient condition to confer AQ resistance [28, 29]. Beshir et al. suggested that if the CVIET haplotype is highly prevalent, AQ can still be effective and further mutations on the P. falciparum multidrug resistance gene (pfmdr1) are required for the development of clinically significant AQ resistance [10].

The only existing results on 72-76 pfcrt haplotypes that are available for the DRC refer to samples collected in 2000. The study identified 100% of resistant strains and these were distributed as follows: 14/27 (51.8%) SVIET, 12/27 (44.4%) CVIET and 1/27 (3.7%) of CVMNT [18]. Studies on a larger scale should be more representative of the high prevalence of the SVIET haplotype, which appears to be rare elsewhere in the world. In fact, it was found before only once, in a study performed in Papua New Guinea [30].

Our results showed that the SVMNT haplotype is not yet present in Kinshasa suggesting that AQ remains valid as a partner drug for ACT. However, continuous monitoring is necessary because, initially absent from Africa, the SVMNT haplotype suddenly appeared in Tanzania in 2004, where it was not present the year before [13]. This fact shows how the emergence of a new haplotype can be spontaneous. This haplotype has been also found recently in Angola by Gama et al. who supposed that this haplotype may have been imported from Brazil [14]. Frosch et al. showed that Angola and Tanzania were among African countries where AQ was more extensively used than in other countries during the last decade [31]. This fact could have contributed to the selection of SVMNT resistant strains, as suggested by Sa and by others, who stipulated that AQ had a prominent role in the selection of parasites carrying the SVMNT haplotype [10, 13, 32, 33]. In fact, SVMNT haplotype is predominant in countries where AQ was early and widely used [32].

Malaria is a disease of great geographical diversity and, as DRC is a very large country, the results of this study concern only the region of Kinshasa and further studies are needed.

Conclusion

This study confirms the slow reverse in sensitivity to CQ in Kinshasa. This is the first to assess resistance to AQ through pfcrt haplotypes after CQ withdrawal, especially based on TBFs. AQ remains effective as a partner molecule for ACT in Kinshasa but monitoring of P. falciparum genomic resistance is mandatory.

Abbreviations

ACT:

Artemisinin-based combination therapy

AQ:

Amodiaquine

AS:

Artesunate

CQ:

Chloroquine

DNA:

Deoxyribonucleic acid

DRC:

Democratic Republic of Congo

RT-PCR:

Real-time polymerase chain reaction

SP:

Sulphadoxine-pyrimethamine

TBF:

Thick blood film

WHO:

World health organization.

References

  1. World Health Organization: Antimalarial drug combination therapy. Report of a WHO technical consultation. 2001, Geneva: WHO

    Google Scholar 

  2. Wellems TE: Plasmodium chloroquine resistance and the search for a replacement antimalarial drug. Science. 2002, 298: 124-126. 10.1126/science.1078167.

    Article  CAS  PubMed  Google Scholar 

  3. Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naude B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE: Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell. 2000, 6: 861-871. 10.1016/S1097-2765(05)00077-8.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Sidhu AB, Verdier-Pinard D, Fidock DA: Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science. 2002, 298: 210-213. 10.1126/science.1074045.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su X-Z, Noruma T, Fidock DA, Wellems TE, Plowe CV: A molecular marker for chloroquine resistant falciparum malaria. N Engl J Med. 2001, 344: 257-263. 10.1056/NEJM200101253440403.

    Article  CAS  PubMed  Google Scholar 

  6. Chen N, Kyle DE, Pasay C, Fowler EV, Baker J, Peters JM, Cheng Q: pfcrt Allelic types with two novel amino acid mutations in chloroquine resistant Plasmodium falciparum isolates from the Philippines. Antimicrob Agents Chemother. 2003, 47: 3500-3505. 10.1128/AAC.47.11.3500-3505.2003.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Childs GE, Boudreau EF, Milhous WK, Wimonwattratee T, Pooyindee N, Pang L, Davidson DE: A comparison of the in vitro activities of amodiaquine and desethylamodiaquine against isolates of Plasmodium falciparum. Am J Trop Med Hyg. 1989, 40: 7-11.

    CAS  PubMed  Google Scholar 

  8. Ochong EO, van den Broek IV, Keus K, Nzila A: Short report: association between chloroquine and amodiaquine resistance and allelic variation in the Plasmodium falciparum multiple drug resistance 1 gene and the chloroquine resistance transporter gene in isolates from the upper Nile in southern Sudan. Am J Trop Med Hyg. 2003, 69: 184-187.

    CAS  PubMed  Google Scholar 

  9. Sa JM, Twu O, Hayton K, Reyes S, Fay MP, Ringwald P, Wellems TE: Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine. Proc Natl Acad Sci U S A. 2009, 106: 18883-18889. 10.1073/pnas.0911317106.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Beshir K, Sutherland CJ, Merinopoulos I, Durrani N, Leslie T, Rowland M, Hallett RL: Amodiaquine resistance in Plasmodium falciparum malaria in Afghanistan is associated with the pfcrt SVMNT allele at codons 72 to 76. Antimicrob Agents Hemother. 2010, 54: 3714-3716. 10.1128/AAC.00358-10.

    Article  CAS  Google Scholar 

  11. Wootton JC, Feng X, Ferdig MT, Cooper RA, Mu J, Baruch DI, Magill AJ, Su XZ: Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum. Nature. 2002, 418: 320-323. 10.1038/nature00813.

    Article  CAS  PubMed  Google Scholar 

  12. Mehlotra RK, Fujioka H, Roepe PD, Janneh O, Ursos LMB, Jacobs-Lorena V, McNamara DT, Bockarie MJ, Kazura JW, Kyle DE, Fidock DA, Zimmerman PA: Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with Pfcrt polymorphism in Papua New Guinea and South America. Proc Natl Acad Sci U S A. 2001, 98: 12689-12694. 10.1073/pnas.221440898.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Alifrangis M, Dalgaard MB, Lusingu JP, Vestergaard LS, Staalsoe T, Jensen AT, Enevold A, Ronn AM, Khalil IF, Warhurst DC, Lemnge MM, Theander TG, Bygbjerg IC: Occurrence of the Southeast Asian/South American SVMNT haplotype of the chloroquine-resistance transporter gene in Plasmodium falciparum in Tanzania. J Infect Dis. 2006, 193: 1738-1741. 10.1086/504269.

    Article  CAS  PubMed  Google Scholar 

  14. Gama BE, de Carvalho GA P, LutucutaKosi FJ, de Oliveira NK A, Fortes F, Rosenthal PJ, Daniel Ribeiro CT, da Cruz MD F: Plasmodium falciparum isolates from Angola show the SVMNT haplotype in the pfcrt gene. Malar J. 2010, 9: 174-10.1186/1475-2875-9-174.

    Article  PubMed Central  PubMed  Google Scholar 

  15. Kazadi WM, Vong S, Makina BN, Mantshumba JC, Kabuya W, Kebela B, Ngimbi NP: Assessing the efficacy of chloroquine and sulfadoxine-pyrimethamine for treatment of uncomplicated Plasmodium falciparum malaria in the Democratic Republic of Congo. Trop Med Int Health. 2003, 8: 868-875. 10.1046/j.1365-3156.2003.01098.x.

    Article  CAS  PubMed  Google Scholar 

  16. Cnops L, Van Esbroeck M, Bottieau E, Jacobs J: Giemsa-stained thick blood films as a source of DNA for Plasmodium species-specific real-time PCR. Malar J. 2010, 9: 370-10.1186/1475-2875-9-370.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Cnops L, Jacobs J, Van Esbroeck M: Validation of a four-primer real-time PCR as a diagnostic tool for single and mixed Plasmodium infections. Clin Microbiol Infect. 2011, 17: 1101-1107. 10.1111/j.1469-0691.2010.03344.x.

    Article  CAS  PubMed  Google Scholar 

  18. Menard S, Morlais I, Tahar R, Sayang C, IssamouMayengue P, Iriart X, Benoit-Vical F, Lemen B, Magnaval J-F, Awono-Ambene P, Basco LK, Berry A: Molecular monitoring of Plasmodium falciparum drug susceptibility at the time of the introduction of artemisinin-based combination therapy in Yaoundé, Cameroon: implications for the future. Malar J. 2012, 11: 113-10.1186/1475-2875-11-113.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Severini C, Menegon M, Sannella AR, Paglia MG, Narciso P, Matteelli A, Gulletta M, Caramello P, Canta F, Xayavong MV, Moura IN, Pieniazek NJ, Taramelli D, Majori G: Prevalence of pfcrt point mutations and level of chloroquine resistance in Plasmodium falciparum isolates from Africa. Infect Genet Evol. 2006, 6: 262-268. 10.1016/j.meegid.2005.07.002.

    Article  CAS  PubMed  Google Scholar 

  20. Wilson PE, Kazadi W, Kamwendo DD, Mwapasa V, Purfield A, Meshnick SR: Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay. Acta Trop. 2005, 93: 97-106. 10.1016/j.actatropica.2004.09.010.

    Article  CAS  PubMed  Google Scholar 

  21. Mobula L, Lilley B, Tshefu AK, Rosenthal PJ: Resistance-mediating Polymorphisms in Plasmodium falciparum infections in Kinshasa, Democratic Republic of the Congo. Am J Trop Med Hyg. 2009, 80: 555-558.

    PubMed  Google Scholar 

  22. Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimdé AA, Kouriba B, Taylor TE, Plowe CV: Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine in Malawi. J Infect Dis. 2003, 187: 1870-1875. 10.1086/375419.

    Article  PubMed  Google Scholar 

  23. Schwenke A, Brandts C, Philipps J, Winkler S, Wernsdorfer WH, Kremsner PG: Declining chloroquine resistance of Plasmodium falciparum in Lambarene, Gabon from 1992 to 1998. Wien Klin Wochenschr. 2001, 113: 63-64.

    CAS  PubMed  Google Scholar 

  24. MeasureDHS, ICF Macro: [http://www.measuredhs.com] EDS RDC 2007

  25. Gharbi M, Flegg JA, Hubert V, Kendjo E, Metcalf JE, Bertaux L, Guérin PJ, Le Bras J, Members of the French National Reference Centre for Imported Malaria Study: Longitudinal study assessing the return of chloroquine susceptibility of Plasmodium falciparum in isolates from travellers returning from West and Central Africa, 2000-2011. Malar J. 2013, 12: 35-10.1186/1475-2875-12-35.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Awasthi G, Prasad GBKS, Das A: Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol. 2011, 41: 705-709. 10.1016/j.ijpara.2011.03.002.

    Article  PubMed  Google Scholar 

  27. Anderson TJ, Nair S, Qin H, Singlam S, Brockman A, Paiphun L, Nosten F: Are transporter genes other than the chloroquine resistance locus (pfcrt) and multidrug resistance gene (pfmdr) associated with antimalarial drug resistance?. Antimicrob Agents Chemother. 2005, 49: 2180-2188. 10.1128/AAC.49.6.2180-2188.2005.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Holmgren GJ, Hamrin J, Svard J, Mårtensson A, Gil JP, Bjorkman A: Selection of pfmdr1 mutations after amodiaquine monotherapy and amodiaquine plus artemisinin combination therapy in East Africa. Infect Genet Evol. 2007, 7: 562-569. 10.1016/j.meegid.2007.03.005.

    Article  CAS  PubMed  Google Scholar 

  29. Nsobya SL, Dokomajilar C, Joloba M, Dorsey G, Rosenthal PJ: Resistance-mediating Plasmodium falciparum pfcrt and pfmdr1 alleles after treatment with artesunate-amodiaquine in Uganda. Antimicrob Agents Chemother. 2007, 51: 3023-3025. 10.1128/AAC.00012-07.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Nagesha SH, Casey GJ, Rieckmann KH, Fryauff DJ, Laksana BS, Reeder JC, Maguire JD, Baird JK: New haplotypes of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene among chloroquine-resistant parasite isolates. Am J Trop Med Hyg. 2003, 68: 398-402.

    CAS  PubMed  Google Scholar 

  31. Frosch AEP, Venkatesan M, Laufer MK: Patterns of chloroquine use and resistance in sub-Saharan Africa: a systematic review of household survey and molecular data. Malar J. 2011, 10: 11-10.1186/1475-2875-10-11.

    Article  Google Scholar 

  32. Sa JM, Twu O: Protecting the malaria drug arsenal: halting the rise and spread of amodiaquine resistance by monitoring the PfCRT SVMNT type. Malar J. 2010, 9: 374-10.1186/1475-2875-9-374.

    Article  PubMed Central  PubMed  Google Scholar 

  33. Folarin OA, Bustamante C, Gbotosho GO, Sowunmi A, Zalis MG, Oduola AMJ, Happi CT: In vitro amodiaquine resistance and its association with mutations in pfcrt and pfmdr1 genes of Plasmodium falciparum isolates from Nigeria. Acta Trop. 2011, 120: 224-230. 10.1016/j.actatropica.2011.08.013.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank the Molecular Biology Unit of the University Hospital of Liège, Belgium.

This work was supported by the Coopération Universitaire pour le Développement (CUD)/Belgium.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dieudonné Makaba Mvumbi.

Additional information

Competing interests

The authors declare that they have no competing interest

Authors’ contributions

MMD collected samples, carried out the DNA extraction, performed RT-PCR analysis and drafted the manuscript. RB and RS supervised genotyping analysis.ML, KL and SN conceived and designed the study protocol. BL participated in collecting samples. PM provided materials. MPH, KN and PDM supervised the study. All authors read and approved the manuscript.

Rights and permissions

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Cite this article

Mvumbi, D.M., Boreux, R., Sacheli, R. et al. Assessment of pfcrt 72-76 haplotypes eight years after chloroquine withdrawal in Kinshasa, Democratic Republic of Congo. Malar J 12, 459 (2013). https://doi.org/10.1186/1475-2875-12-459

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/1475-2875-12-459

Keywords