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    Prevalence of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) T76 mutation and in vivo chloroquine resistance in 1998 and 2001 in four study sites in Burkina Faso. TTF = total treatment failures; Accart = Accartville; Touss = Toussiana.

  • 1

    Djimde A, Doumbo OK, Steketee RW, Plowe CV, 2001. Application of a molecular marker for surveillance of chloroquine-resistant falciparum malaria. Lancet 358 :890–891.

    • Search Google Scholar
    • Export Citation
  • 2

    Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D, 2001. High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance gene pfmdr1. J Infect Dis 183 :1535–1538.

    • Search Google Scholar
    • Export Citation
  • 3

    Basco LK, Ringwald P, 2001. Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in Yaounde, Cameroon. J Infect Dis 183 :1828–1831.

    • Search Google Scholar
    • Export Citation
  • 4

    Chen N, Russell B, Staley J, Kotecka B, Nasveld P, Cheng Q, 2001. Sequence polymorphisms in pfcrt are strongly associated with chloroquine resistance in Plasmodium falciparum. J Infect Dis 183 :1543–1545.

    • Search Google Scholar
    • Export Citation
  • 5

    Pillai DR, Labbe AC, Vanisaveth V, Hongvangthong B, Pomphida S, Inkathone S, Zhong K, Kain KC, 2001. Plasmodium falciparum malaria in Laos: chloroquine treatment outcome and predictive value of molecular markers. J Infect Dis 183 :789–795.

    • Search Google Scholar
    • Export Citation
  • 6

    Tinto H, Zoungrana EB, Coulibaly SO, Ouédraogo JB, Traoré M, Guiguemdé TR, van Marck E, D’Alessandro U, 2002. Chloroquine and sulfadoxine-pyrimethamine efficacy for uncomplicated malaria treatment and haematological recovery in children in Bobo-Dioulasso, Burkina Faso, during a 3-year period (1998–2000). Trop Med Int Health 7 :925–930.

    • Search Google Scholar
    • Export Citation
  • 7

    World Health Organization, 2002. Monitoring Antimalarial Resistance. Geneva: World Health Organization. WHO/CDS/ CSR/EPH/ 2002.17.

  • 8

    Plowe CV, Djimdé A, Bouare M, Doumbo O, Wellems TE, 1995. Pyrimethamine and proguanil resistance-confering mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa. Am J Trop Med Hyg 52 :565–568.

    • Search Google Scholar
    • Export Citation
  • 9

    Tinto H, Ouédraogo JB, Erhart A, van Overmeir C, Dujardin C, van Marck E, Guiguemdé TR, D’Alessandro U, 2003. Relationship between the Pfcrt T76 and the Pfmdr-1 Y86 mutations and in vitro/in vivo chloroquine resistance in Burkina Faso, West Africa. Infect Genet Evol 3 :287–292.

    • Search Google Scholar
    • Export Citation
  • 10

    Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, Coulibaly D, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344 :257–263.

    • Search Google Scholar
    • Export Citation
  • 11

    Jelinek T, Aida AO, Peyerl-Hoffmann G, Jordan S, Mayor A, Heuschkel C, El Valy AO, von Sonnenburg F, Christophel EM, 2002. Diagnostic value of molecular markers in chloroquine-resistant falciparum malaria in southern Mauritania. Am J Trop Med Hyg 67 :449–453.

    • Search Google Scholar
    • Export Citation
  • 12

    Mboup S, Wypij D, Maguire JH, Wirth DF, 2002. In vitro chloroquine susceptibility and PCR analysis of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from Senegal. Am J Trop Med Hyg 66 :474–480.

    • Search Google Scholar
    • Export Citation
  • 13

    Rason MA, Ariey F, Rafidimanantsoa L, Andrianantenaina BH, Sahondra Harisoa JL, Randrianarivelojosia M, 2002. Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation detection tests. Parasite 9 :247–253.

    • Search Google Scholar
    • Export Citation
  • 14

    Ouedraogo JB, Dutheil Y, Tinto H, Traore B, Zampa H, Tall F, Coulibaly SO, Guiguemde TR, 1998. In vitro sensitivity of Plasmodium falciparum to halofantrine compared with chloroquine, quinine and mefloquine in the region of Bobo-Dioulasso, Burkina Faso (west Africa). Trop Med Int Health 3 :381–384.

    • Search Google Scholar
    • Export Citation
  • 15

    Diabaté A, Baldet T, Chandre F, Dabiré KR, Kengne P, Simard F, Guiguemdé TR, Guillet P, Hemingway J, Hougard JM, 2003. Kdr mutation, a genetic marker to assess events of introgression between the molecular M and S forms of Anopheles gambiae (Diptera, Culicidae) in the tropical savannah area of west Africa. J Med Entomol 40 :195–198.

    • Search Google Scholar
    • Export Citation
  • 16

    Abdel-Muhsin A, Mackinnon MJ, Ali E, Nassir EKA, Suleiman S, Ahmed S, Walliker D, Babiker HA, 2004. Evolution of drug-resistance genes in Plasmodium falciparum in an area of seasonal malaria transmission in eastern Sudan. J Infect Dis 189 :1239–1244.

    • Search Google Scholar
    • Export Citation
  • 17

    Talisuna AO, Langi P, Bakyaita N, Egwang T, Mutabingwa TK, Watkins W, van Marck E, D’Alessandro U, 2002. Intensity of malaria transmission, antimalarial-drug use and resistance in Uganda: what is the relationship between these three factors? Trans R Soc Trop Med Hyg 96 :310–317.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

USEFULNESS OF THE PLASMODIUM FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER T76 GENOTYPE FAILURE INDEX FOR THE ESTIMATION OF IN VIVO CHLOROQUINE RESISTANCE IN BURKINA FASO

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  • 1 Centre Muraz/Institut de Recherche en Sciences de la Santé, Bobo Dioulasso, Burkina Faso; Faculty of Medicine, University of Antwerp, Antwerp, Belgium; Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium

The prevalence of chloroquine (CQ) treatment failure and the genotype failure index was determined in four sentinel sites in Burkina Faso. In three sites, the genotype failure index varied between 1.7 and 3, a result confirming the relationship between the Plasmodium falciparum CQ resistance transporter (Pfcrt) T76 mutation and CQ resistance. In the remaining site, the genotype failure index was unusually low, 1.1, which was significantly different than that in the other sites (P < 0.00001). These findings are discussed. Often but not always, the prevalence of CQ resistance can be correctly estimated by the Pfcrt T76 genotype failure index.

The amount of chloroquine (CQ) resistance is usually estimated by carrying out World Health Organization (WHO) in vivo tests. However, this is a time-consuming task and results are difficult to interpret where re-infection is frequent.1 The identification of the relationship between the Plasmodium falciparum CQ resistance transporter (Pfcrt) T76 mutation and CQ resistance resulted in a series of field studies that investigated the usefulness of this molecular marker in estimating CQ resistance.25 Data from Mali suggest that its prevalence is 2–3-fold higher than in vivo CQ resistance.1 Two indexes, the genotype resistance index (GRI) and the genotype failure index (GFI), which correspond to the ratio between the age-adjusted frequency of the Pfcrt T76 mutation and the prevalence of parasitologic or clinical failure, respectively, were defined. They were proposed as useful tools to estimate, over large areas and without the need of carrying out expensive and time-consuming in vivo tests, CQ resistance, at least when it is less than 30–50%. However, the GRI and the GFI have not been applied or validated extensively. We therefore computed, based on the prevalence of the Pfcrt T76 mutation, the GFI in four sites in Bobo Dioulasso, Burkina Faso in two different years (1998 and 2001) and related it to the prevalence of CQ resistance estimated by in vivo 14 days test in 1998–1999 and 2000–2001.

This is part of a larger study that investigated CQ and sulfadoxine-pyrimethamine efficacy in children between 6 months and 15 years of age. This study was reviewed and approved by institutional Ethical Board at Centre Muraz. Informed consent for participation in the study was obtained from the children’s parents or guardians. In the following analysis, only CQ efficacy has been considered. Details of the study methodology have been reported elsewhere.6 Outcomes were defined according to the WHO modified classification for monitoring antimalarial drug resistance.7 Total treatment failure (TTF) was defined as the sum of early treatment failure (ETF), late clinical failure (LCF), and late parasitologic failure (LPF).

Blood samples for the molecular analysis were collected on no. 3 filter paper (Whatman, Maidstone, United Kingdom) at day 0 before treatment. The DNA was extracted using the Chelex-100 method.8 Detection of the Pfcrt T76 mutation was done by using a polymerase chain reaction, followed by-sequence-specific restriction enzyme digestion (http://www.medschool.umaryland.edu/CVD/plowe.html). The GFI was computed by dividing the frequency of the Pfcrt T76 mutation by the prevalence of TTF.

Between 1998 and 2001, a total of 1,068 patients were enrolled in the four sites. The mean age of the children included in the study was similar in the four study sites and varied between 51.7 months in Lena in 1998–1999 to 52 months in Toussiana in 1998 and 2001. Outcome was known for 884 (83%) patients who completed the follow-up. The TTF increased from 20.4% (69 of 337) in 1998–1999 to 28.7% (157 of 547) in 2000–2001 (odds ratio [OR] = 0.6; 95% confidence interval [CI] = 0.4–0.9, P = 0.0006), and was composed of mostly LCF or LPF. Such an increase occurred in all sites, although the difference between the two periods was statistically significant (OR = 0.5, 95% CI = 0.2–0.9, P = 0.03) at only one site (Lena).

Seven hundred sixteen pre-treatment blood samples (340 in 1998 and 376 in 2001) were randomly selected and analyzed for polymorphism in the Pfcrt 76 locus. In all sites, the prevalence of Pfcrt T76 mutation increased between 1998 and 2001 (Figure 1), but the difference between the two years was statistically significant only in Accartville (OR = 0.5, 95% CI = 0.3–1, P = 0.03). In Bama, the prevalence of the mutation was low (22% in 1998 and 33% in 2001) and significantly different from the other sites (P < 0.00001 for both years). In three sites (Accartville, Lena, and Toussiana) the prevalence of the Pfcrt T76 mutation was always higher than the TTF (Figure 1) and the GFI ranged between 1.7 and 3. However, in both years in Bama, the prevalence of the Pfcrt T76 mutation was almost identical to that of the TTF with a GFI of 1.1.

The GFI has been proposed, on the basis of data from Mali showing its stability at sites with different levels of transmission, levels of immunity, and ethnic composition, as a simple and reliable tool to monitor CQ resistance in places where this is less than 30–50%.1 We found similar results in most but not all our study sites. The GFI in Accartville, Lena, and Toussiana ranged between 1.7 and 3, and was constant over the study period. However, in Bama we observed a low prevalence of the Pfcrt T76 mutation, a result that was different from those reported by other studies in west Africa.912 Similar finding have been reported in a study carried out in Madagascar where only 3 of the 43 isolates were resistant to CQ in vitro and none carried the mutation.13 The results from Bama are unexpected because the prevalence of CQ resistance is approximately 20–30%, which is similar to that reported in previous studies,6,14 and also similar to the prevalence of the Pfcrt T76 mutation, which resulted in a GFI of 1.1 for both years studied. An overestimation of CQ resistance can be ruled out because the slides were regularly subjected to quality control in all four sites. The mean age of patients was also comparable between the four study sites, thus ruling out a possible role of the acquired immunity in explaining the difference observed. A possible explanation of different GFIs at various sites is that CQ resistance has a multigenic basis in which the Pfcrt T76 mutation is necessary but not sufficient.15

Another specific and selective factor in Bama is the much higher frequency of Anopheles gambiae M (Mopti) compared with the other sites where both forms M and S (Savanna) occur.16 High transmission has been associated with a faster spread of CQ resistance, once this has emerged.15 In Uganda, CQ resistance seems to spread faster, regardless of drug pressure in low and high transmission areas.17 In Bama, where malaria transmission is high, this does not seem to happen. The high frequency of An. gambiae M could be involved in this phenomenon, although it is not clear how.

A major limitation of this study is that we observed a GFI close to 1 only in one site, although the study was conducted in two different years (1998 and 2001). In the other three sites, estimating the amount of CQ resistance by dividing the prevalence of the Pfcrt T76 mutation by 2 or 3 seems to confirm the usefulness of the GFI.1 It would be important to determine whether this is still the case in areas of extremely high transmission, such as Bama.

Figure 1.
Figure 1.

Prevalence of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) T76 mutation and in vivo chloroquine resistance in 1998 and 2001 in four study sites in Burkina Faso. TTF = total treatment failures; Accart = Accartville; Touss = Toussiana.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 1; 10.4269/ajtmh.2005.73.171

*

Address correspondence to Umberto D’Alessandro, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium. E-mail: udalessandro@itg.be

Authors’ addresses: Halidou Tinto, Boroma Sanou, Jean Bosco Ouédraogo, and Tinga Robert Guiguemdé, Centre Muraz/Institut de Recherche en Sciences de la Santé, BP 153 Bobo Dioulasso, Burkina Faso, Telephone: 226-20-97-01-02; Fax: 226-20-97-28-24, E-mail: tintoh@hotmail.com. Jean-Claude Dujardin, Chantal van Overmeir, Annette Erhart, and Umberto D’Alessandro, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium, Telephone: 32-3-247-6354, Fax: 32-3-247-6362, E-mail: udalessandro@itg.be. Eric van Marck, Faculty of Medicine, University of Antwerp, Universiteitsplein 1 B-2610, Antwerp, Belgium, Telephone: 32-3-820-2540, Fax: 32-3-820-2501, E-mail: Eric.Van.Marck@uza.be.

Acknowledgments: We thank the parents of the children included in this study for their participation. We also thank the health staff of the health centers where the study was conducted for their collaboration.

Financial support: We thank the French Co-operation for its support of the field study and the Belgium Co-operation for support of the laboratory work through a training grant to Halidou Tinto. This work was also partially support by Société de Pathologie Exotique through a prize awarded to Halidou Tinto.

REFERENCES

  • 1

    Djimde A, Doumbo OK, Steketee RW, Plowe CV, 2001. Application of a molecular marker for surveillance of chloroquine-resistant falciparum malaria. Lancet 358 :890–891.

    • Search Google Scholar
    • Export Citation
  • 2

    Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D, 2001. High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance gene pfmdr1. J Infect Dis 183 :1535–1538.

    • Search Google Scholar
    • Export Citation
  • 3

    Basco LK, Ringwald P, 2001. Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in Yaounde, Cameroon. J Infect Dis 183 :1828–1831.

    • Search Google Scholar
    • Export Citation
  • 4

    Chen N, Russell B, Staley J, Kotecka B, Nasveld P, Cheng Q, 2001. Sequence polymorphisms in pfcrt are strongly associated with chloroquine resistance in Plasmodium falciparum. J Infect Dis 183 :1543–1545.

    • Search Google Scholar
    • Export Citation
  • 5

    Pillai DR, Labbe AC, Vanisaveth V, Hongvangthong B, Pomphida S, Inkathone S, Zhong K, Kain KC, 2001. Plasmodium falciparum malaria in Laos: chloroquine treatment outcome and predictive value of molecular markers. J Infect Dis 183 :789–795.

    • Search Google Scholar
    • Export Citation
  • 6

    Tinto H, Zoungrana EB, Coulibaly SO, Ouédraogo JB, Traoré M, Guiguemdé TR, van Marck E, D’Alessandro U, 2002. Chloroquine and sulfadoxine-pyrimethamine efficacy for uncomplicated malaria treatment and haematological recovery in children in Bobo-Dioulasso, Burkina Faso, during a 3-year period (1998–2000). Trop Med Int Health 7 :925–930.

    • Search Google Scholar
    • Export Citation
  • 7

    World Health Organization, 2002. Monitoring Antimalarial Resistance. Geneva: World Health Organization. WHO/CDS/ CSR/EPH/ 2002.17.

  • 8

    Plowe CV, Djimdé A, Bouare M, Doumbo O, Wellems TE, 1995. Pyrimethamine and proguanil resistance-confering mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa. Am J Trop Med Hyg 52 :565–568.

    • Search Google Scholar
    • Export Citation
  • 9

    Tinto H, Ouédraogo JB, Erhart A, van Overmeir C, Dujardin C, van Marck E, Guiguemdé TR, D’Alessandro U, 2003. Relationship between the Pfcrt T76 and the Pfmdr-1 Y86 mutations and in vitro/in vivo chloroquine resistance in Burkina Faso, West Africa. Infect Genet Evol 3 :287–292.

    • Search Google Scholar
    • Export Citation
  • 10

    Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, Coulibaly D, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344 :257–263.

    • Search Google Scholar
    • Export Citation
  • 11

    Jelinek T, Aida AO, Peyerl-Hoffmann G, Jordan S, Mayor A, Heuschkel C, El Valy AO, von Sonnenburg F, Christophel EM, 2002. Diagnostic value of molecular markers in chloroquine-resistant falciparum malaria in southern Mauritania. Am J Trop Med Hyg 67 :449–453.

    • Search Google Scholar
    • Export Citation
  • 12

    Mboup S, Wypij D, Maguire JH, Wirth DF, 2002. In vitro chloroquine susceptibility and PCR analysis of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from Senegal. Am J Trop Med Hyg 66 :474–480.

    • Search Google Scholar
    • Export Citation
  • 13

    Rason MA, Ariey F, Rafidimanantsoa L, Andrianantenaina BH, Sahondra Harisoa JL, Randrianarivelojosia M, 2002. Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation detection tests. Parasite 9 :247–253.

    • Search Google Scholar
    • Export Citation
  • 14

    Ouedraogo JB, Dutheil Y, Tinto H, Traore B, Zampa H, Tall F, Coulibaly SO, Guiguemde TR, 1998. In vitro sensitivity of Plasmodium falciparum to halofantrine compared with chloroquine, quinine and mefloquine in the region of Bobo-Dioulasso, Burkina Faso (west Africa). Trop Med Int Health 3 :381–384.

    • Search Google Scholar
    • Export Citation
  • 15

    Diabaté A, Baldet T, Chandre F, Dabiré KR, Kengne P, Simard F, Guiguemdé TR, Guillet P, Hemingway J, Hougard JM, 2003. Kdr mutation, a genetic marker to assess events of introgression between the molecular M and S forms of Anopheles gambiae (Diptera, Culicidae) in the tropical savannah area of west Africa. J Med Entomol 40 :195–198.

    • Search Google Scholar
    • Export Citation
  • 16

    Abdel-Muhsin A, Mackinnon MJ, Ali E, Nassir EKA, Suleiman S, Ahmed S, Walliker D, Babiker HA, 2004. Evolution of drug-resistance genes in Plasmodium falciparum in an area of seasonal malaria transmission in eastern Sudan. J Infect Dis 189 :1239–1244.

    • Search Google Scholar
    • Export Citation
  • 17

    Talisuna AO, Langi P, Bakyaita N, Egwang T, Mutabingwa TK, Watkins W, van Marck E, D’Alessandro U, 2002. Intensity of malaria transmission, antimalarial-drug use and resistance in Uganda: what is the relationship between these three factors? Trans R Soc Trop Med Hyg 96 :310–317.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Umberto D’Alessandro, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium.
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