To the Editor—Recent studies of Plasmodium falciparum [1–4] have shown that 1 unique allele of the pfcrt gene, which is located on chromosome 7, probably is responsible for chloroquine resistance in parasites in Asia and Africa. Recently, Wellems and Plowe [5] showed that 1 point mutation (pfcrt K76T) can be used to identify chloroquine-resistant strains. We used recent data to confirm this prediction and to propose practical ways of managing chloroquine in the islands in the Indian Ocean.
In 2000, a national network, Réseau d'Etude de la Résistance du Paludisme (RER), was established by the Ministry of Health and the Pasteur Institute of Madagascar to track the emergence of pockets of chloroquine-resistant P. falciparum strains. The RER uses in vivo and/or in vitro studies to produce useful and relevant data regarding the chemosensitivity of parasites from the whole Indian Ocean subregion. In Madagascar, chloroquine remains the first drug of choice because of previous experience with chloroquine chemosensitivity: no evidence of grade III resistance has ever been recorded in vivo, and in vitro tests confirm the low level of resistance [6]. Compared with other countries in which P. falciparum malaria is endemic, this situation is surprisingly favorable. On the other hand, since 1990, various studies have confirmed that resistant strains have emerged in the Comoro Islands and that their numbers are increasing, but these studies have not led to a change in treatment policy [7–9].
In association with the Madagascar national survey, a combined in vivo/in vitro study was performed in February 2001 by the RER in the Comoro Islands. We compared the nucleotide sequences of the pfcrt genes of 49 isolates from Grande Comore and 83 isolates from Madagascar. The Madagascar isolates could be divided into 3 distinct groups (table 1): 40 isolates were found throughout the island, 24 were chloroquine-resistant strains (IC50, ⩾100 nM), and 19 originated from malaria cases in the Mahajanga region of Madagascar, which is on the northwestern coast, nearest to the Comoro Islands, and is an area where human migration to and from the Comoro Islands is common.
Distribution of pfcrt K76T mutations among Plasmodium falciparum isolates from Madagascar and the Comoro Islands.
Distribution of pfcrt K76T mutations among Plasmodium falciparum isolates from Madagascar and the Comoro Islands.
The results collected by the RER confirm the results of previous studies [6–9] (table 2). We found that the therapeutic success rate was >80% for in vivo studies and that the IC50 values for chloroquine were low for isolates from Madagascar for in vitro studies. Conversely, therapy failed for >40% (30/73) of patients in the Comoro Islands in the in vivo tests, and 36% (9/25) of the P. falciparum isolates had an IC50 ⩾ 100 nM in the in vitro analysis of chloroquine chemosensitivity.
Therapeutic success rates for in vivo and vitro tests with chloroquine against Plasmodium falciparum isolates from Madagascar and the Comoro Islands.
Therapeutic success rates for in vivo and vitro tests with chloroquine against Plasmodium falciparum isolates from Madagascar and the Comoro Islands.
The polymorphism of the pfcrt gene was in complete accordance with the phenotypic data: no pfcrt K76T mutations were found in Madagascar. Even though we cannot be sure that the mutation is not present in Madagascar, this implies that this mutation is not involved in chloroquine resistance in this area; however, 62% (30/48) of the isolates tested from the Comoro Islands were found to harbor the pfcrt K76T mutation, which was associated with in vitro chloroquine resistance in the 21 isolates for which both in vivo and in vitro tests provided assessable results (9 of 9 isolates with IC50⩾100 nM vs. 7 of 13 isolates with IC50 < 100 nM; P < .05, Fisher's exact test). In summary, for public health policy, the presence of the pfcrt K76T mutation in the Indian Ocean subregion is associated with chloroquine resistance, whereas the absence of the pfcrt K76T mutation is associated with therapeutic success of first-line chloroquine use.
These are the first data on the pfcrt polymorphism in an area with chloroquine-sensitive strains. Our study suggests that chloroquine resistance has arisen in the Comoro Islands as a consequence of invasion by the new pfcrt K76T mutation from east Africa. In the past, Madagascar was protected from pandemic disease because of its isolation.
Madagascar must now try to prevent the invasion and spread of the pfcrt K76T mutation from areas with chloroquine-resistant strains. If nothing is done, the pfcrt K76T mutation soon will spread throughout Madagascar. We think that a localized genetic survey would be an appropriate first step in the public health policy toward incoming chloroquine-resistant strains. The low levels of investment required and the ease of the polymerase chain reaction test will create a balance between the burden of malaria and the economic capability of this developing country. We plan to carry out a detailed analysis of parasite gene flow between the different parasite populations of the islands, to identify pertinent sentinel sites. We hope that a survey network, such as the RER, can be used to protect Madagascar from chloroquine-resistant strains. This procedure then could be used to protect the few other remaining areas around the world in which chloroquine-sensitive strains are endemic.
