Chloroquine-resistant Plasmodium falciparum infections among UK travellers returning with malaria after chloroquine prophylaxis

Abstract

Introduction

As the number of UK travellers visiting sub-Saharan Africa has risen over the past two decades, the proportion of imported malaria cases caused by Plasmodium falciparum has also increased.¹ Over the same period, resistance to the antimalarial drug chloroquine, used for both therapy and prophylaxis, has spread throughout the globe.² As a result, alternative therapies such as sulfadoxine/pyrimethamine and combination regimens containing artemisinin derivatives have been replacing chloroquine as first-line antimalarial therapy in many endemic regions, including sub-Saharan Africa. Recommended prophylaxis for travellers has also shifted away from chloroquine towards mefloquine, doxycycline or atovaquone/proguanil. Despite this shift, chloroquine remains available in UK pharmacies as a cheap over-the-counter preparation, and evidence from the Malaria Reference Laboratory database indicates that prophylactic use of chloroquine, usually in combination with proguanil, has continued. This may be partly because more effective recommended regimens such as atovaquone/proguanil, doxycycline and mefloquine are prescription-only medicines, or due to travellers' fear of side effects or issues of cost, depending upon the agent concerned.

Despite the widespread use of chloroquine prophylaxis, there have been few studies of chloroquine-resistant parasites among travellers with falciparum malaria. Studies in endemic areas have demonstrated a strong association between chloroquine treatment failure and carriage of mutant haplotypes of the pfcrt locus, which encodes CRT, a large transporter protein located in the parasite's digestive vacuole membrane.³ A study of 131 cases imported to France found that chloroquine/proguanil prophylaxis was strongly associated with the presence of one resistance-associated allele of pfcrt when compared with patients who had not used prophylaxis.⁴ We employed a novel multiplex real-time PCR that distinguishes three alleles of pfcrt to characterize parasite isolates from 66 patients presenting with falciparum malaria in the UK. We found that carriage of chloroquine-resistant parasites was significantly more common among those using chloroquine/proguanil for prophylaxis, compared with those using other prophylactic regimens.

Materials and methods

Patients and samples

Parasite DNA was obtained from 69 pre-treatment isolates of participants in a prospective study of disease severity⁵ using the QIAamp DNA Mini kit (Qiagen, UK). These were analysed by species-discriminating multiplex PCR, as described previously.⁶ Sixty-six of these were positive for P. falciparum monoinfection and were taken into the chloroquine-resistance analysis. Permission for the study was obtained from the Ethics Committee of University College London Hospitals.

Genotyping of the pfcrt locus

A multiplex, real-time PCR (qPCR) assay was developed using double-labelled probes with a different fluorophore, representing each of the three most common pfcrt alleles (N. Gadalla and C. J. Sutherland, unpublished data). Briefly, pfcrt DNA was amplified from each sample using previously described conditions and the amplification primers pfcrt F (TGG TAA ATG TGC TCA TGT GTT T) and pfcrt R (AGT TTC GGA TGT TAC AAA ACT ATA GT).⁷ Amplification was performed in a Corbett Rotorgene 3000 (Corbett, Sydney, Australia) in the presence of each of the three double-labelled probes, representing the wild-type and the two most common resistance-associated haplotypes at codons 72–76 of pfcrt. The probes were crt76CVMNK wild-type, 5′FAM-TGT GTA ATG AAT AAA ATT TTT GCT AA-BHQ1 (3D7 DNA from MR4 used as a positive control); crt76CVIET resistant, 5′JOE-TGT GTA ATTGAA ACA ATT TTT GCT AA-BHQ1 (Dd2 DNA from MR4 used as a positive control); and crt76SVMNT resistant, 5′ROX-AGT GTA ATG AAT ACA ATT TTT GCT AA-BHQ2 (7G8 DNA from MR4 used as a positive control).

Probes were synthesized by MWG, Germany. Validation and optimization of the method will be described in full elsewhere (N. Gadalla and C. J. Sutherland, unpublished data). Control parasite DNA was obtained directly from MR4 (www.mr4.org). Samples were considered positive for a particular genotype if a CT value of 35 cycles or fewer was obtained in at least two independent PCR experiments.

Results

Previous prophylaxis use among cases

Of the 66 isolates studied, 61 (92.4%) were from patients for whom information on malaria chemoprophylaxis use and travel itinerary was available. Of these 61 patients, 22 indicated that they had taken prophylaxis, 14 using chloroquine/proguanil, 3 using proguanil alone, 3 using mefloquine, 1 using doxycycline and 1 using pyrimethamine alone. Sixty patients had been exposed to P. falciparum while travelling in sub-Saharan Africa, with 22 countries visited in all. Nigeria (20 patients), Ghana (11 patients) and The Gambia (5 patients) were the most common destinations. One patient had been exposed in Cambodia.

Genotyping of the pfcrt locus

All 66 isolates were successfully tested for pfcrt genotype at codons 72–76 in at least two PCR assays, with full agreement. Most isolates (65%) harboured only parasites with the common African chloroquine-resistant haplotype CVIET, reflecting the geographical origin of the infections (Table 1). Three isolates comprised a mixture of genotypes encoding CVMNK and CVIET at codons 72–76 of pfcrt. The southeast Asian/South American chloroquine-resistant haplotype SVMNT was observed in one isolate, acquired in Cambodia. As our probes were designed to identify these three alleles only, we cannot rule out the possibility that other less common haplotypes were also present in some infections.

Prophylaxis use and carriage of chloroquine-resistant genotypes

Thirteen of the fourteen patients (92.9%) presenting with P. falciparum malaria after taking chloroquine/proguanil chemoprophylaxis harboured parasites with the CVIET pfcrt haplotype, which is associated with resistance to chloroquine. In contrast, 30 of 47 patients (63.8%) who did not take prophylaxis or who took prophylaxis other than chloroquine/proguanil harboured the CVIET or SVMNT haplotype (OR 0.136; 95% CI 0.003–1.084; two-sided Fisher's exact test P = 0.047). Considering only the 22 patients with recorded prophylaxis use, 3 of the 8 patients (37.5%) who used prophylaxis other than chloroquine/proguanil presented with chloroquine-resistant haplotypes (OR 0.046; 95% CI 0.0009–0.733; P = 0.011). Optimal compliance was reported by 1 of the 8 patients taking prophylaxis other than chloroquine (12.5%) and by 6 of the 14 patients taking chloroquine/proguanil (42.9%). Chloroquine-resistant genotypes were found in 27 of 39 cases (69.2%) who had not used prophylaxis.

Cases of malaria among travellers on prophylaxis

Compliance with malaria prophylaxis was further examined among an anonymized sample of 145 patients treated at the Hospital for Tropical Diseases for P. falciparum malaria between August 2000 and July 2005. (As identifiers were removed for this analysis, we cannot say how many of these were among the 66 patients providing parasite DNA samples for resistance genotyping.) All 145 patients reported some use of a recognized prophylactic regimen: 76 had used chloroquine/proguanil, 29 used proguanil alone and 19, 18 and 3 patients used doxycycline, mefloquine and atovaquone/proguanil, respectively. Thirty of the 76 patients (39.5%) using chloroquine/proguanil reported good adherence to the regimen, defined as having missed three doses or fewer. In contrast, among the 69 patients using other regimens, only 10 (14.5%) reported good adherence (OR 3.85, 95% CI 1.61–9.69; P = 0.0008).

Discussion

We have found evidence that chloroquine-resistant pfcrt genotypes are common among imported P. falciparum malaria cases in the UK, as has been shown for imported cases in France.⁴ Further, resistant genotypes are significantly more likely to occur among those who have been taking chloroquine/proguanil chemoprophylaxis than among other travellers using different prophylactic drugs or taking no chemoprophylaxis at all. Thirteen of 14 patients using chloroquine/proguanil for prophylaxis carried chloroquine-resistant pfcrt genotypes. Eight of these 14, including the single patient among them carrying wild-type parasites, reported suboptimal compliance with their prophylactic regimen. We conclude that chloroquine/proguanil does not provide adequate protection against falciparum malaria for travellers to Africa, and the same is likely to be true for malaria-endemic areas of Asia.

Among 145 malaria patients treated in the Hospital for Tropical Diseases who reported some use of prophylaxis prior to becoming ill, those using chloroquine/proguanil were significantly more likely to have missed fewer than three doses during the period of protection. This suggests that full compliance with chloroquine/proguanil does not offer the level of protection against P. falciparum malaria achieved by full compliance with other regimens.

The therapeutic and preventative performance of antimalarials may differ. For example, the suboptimal efficacy of sulfadoxine/pyrimethamine as a therapeutic drug for malaria in sub-Saharan Africa contrasts with its successful use as intermittent preventive treatment for pregnant women⁸ and infants.⁹ The widespread failure of chloroquine as treatment for falciparum malaria throughout the tropics and subtropics is well documented.¹⁰ We have presented evidence that chloroquine is also failing to provide adequate malaria prophylaxis for travellers. The guidelines issued by the UK Advisory Committee for Malaria Prevention in UK Travellers (available at www.malaria-reference.co.uk), which do not recommend chloroquine prophylaxis in sub-Saharan Africa, should be rigorously adhered to.

Acknowledgements

We thank the patients who consented to participate in this study. We are grateful to the staff of the Department of Clinical Parasitology, Hospital for Tropical Diseases, to Dr Richard Jennings and to Elizabeth King, Immunology Unit, LSHTM, for assistance with sample collection. This study was supported by the Special Trustees, Hospital for Tropical Diseases. C. J. S. is supported by the Health Protection Agency, UK. N. G. is supported by an International Atomic Energy Agency Fellowship.

Transparency declarations

None to declare.

References