To our knowledge, this study is the first to examine changes in the distribution of pfcrt haplotype frequencies in Sri Lanka over an extended period of decreasing malaria prevalence. Although the sample size is small, the data indicates a significant trend in haplotype frequencies. HRM analysis was a sensitive and high-throughput method for detecting haplotypes that was less labor-intensive and more cost-efficient than cloning and direct sequencing of samples.
We found a significant change in haplotype frequencies between 1996 and 1998, suggesting that there was strong selection pressure in favor of the SVMNT haplotype and against the wild type. The replacement of wild-type parasites in the population with the SVMNT mutants corresponds to a period of continuous chloroquine use as first treatment and decreasing malaria incidence in the district of Anadhurapura. The total number of malaria cases in the district declined from 7,995 in 1996 to 5,528 cases in 1998 and continued to drop even more rapidly over the next 10 years, corresponding with nationwide trends. By 2004, the nationwide prevalence of P. falciparum in Sri Lanka was 520, and by 2006, only 27 cases were reported. The sole prevalence of the SVMNT parasites between 2004 and 2006 may indicate a fixation of the haplotype on the island.
The change in frequency might be explained by SVMNT parasites' relative fitness over both the wild-type CVMNK and the mutant CVIET parasites under the standard treatment regimen at the time. Before May 2008, chloroquine (CQ) was the standard first-line treatment of malaria cases in Sri Lanka. Furthermore, a Sri Lankan study conducted several years ago suggested that recrudescent CQ-resistant malaria strains had a lower detectability rate and a higher gametocyte rate that may have conferred an additional survival advantage over CQ-sensitive strains.6
Recent studies also suggest that CVIET mutants may incur a higher fitness cost than SVMNT mutants, which was indicated by the recovery of CQ-sensitive strains after the cessation of drug pressure in regions where the CVIET haplotype was prevalent but not in regions of high SVMNT prevalence.11
This finding may be informative in planning future treatment policies, because artemisinin-based combination therapy (ACT) has replaced CQ as first-line therapy for P. falciparum
-infected patients in Sri Lanka as of 2008.3
Furthermore, several recent studies indicate that the SVMNT mutations may be sufficient to confer resistance to amodiaquine.11,12
Although the current ACT regimen used in Sri Lanka is artemether-lumefantrine, the high prevalence of the SVMNT haplotype found in Sri Lanka cautions against the widespread adoption of amodiaquine-based ACTs in the country.
The highly prevalent SVMNT type strain in Sri Lanka may be related to parasites in India, where the SVMNT haplotype has also reached near fixation.14
There is evidence the Indian strains are related to strains from Papua New Guinea.14
In contrast, the CVIET-type parasites have generally been traced to a common origin at the Thai–Cambodian border, which subsequently spread throughout Southeast Asia as well as Africa.10
Recent research suggests importation of cases may play a significant role in hindering elimination efforts on islands.15
As the malaria elimination program progresses, future tracking of pfcrt
haplotypes, along with other data, will be informative in determining the frequency and origin of imported cases.