Plasmodium falciparum mdr1 mutations and in vivo chloroquine resistance in Indonesia.

Mutations in the Pfmdr1 gene are reported to be associated with chloroquine resistance in some Plasmodium falciparum isolates. A polymerase chain reaction/restriction fragment length polymorphism method was used for the detection of Pfmdr1 mutations in chloroquine-resistant field isolates of P. falciparum collected in Irian Jaya. The frequency of Pfmdr1 mutations was significantly higher in chloroquine-resistant P. falciparum parasites than background frequencies observed in the same location. The 7G8 mutation was identified in some parasites although always in a mixed genotype status. Chloroquine-resistant P. falciparum specimens were characterized using the World Health Organization 28-day criteria, supplemented by demonstrating adequate chloroquine absorption and genetic analysis.

Mutations in the pfmdr1, dhfr and dhps genes of Plasmodium falciparum are associated with in-vivo drug resistance in West Papua, Indonesia.

This study (conducted in 1996-99) examines the association of mutations in pfmdr1, dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of Plasmodium falciparum with in-vivo drug resistance in West Papua, Indonesia. Initially, 85 patients infected with P. falciparum were treated with chloroquine, of whom 21 were cleared of parasites, 49 had parasitaemias classified as RI, RII or RIII resistance and 1 patient had recrudescent parasitaemia. Fansidar (pyrimethamine-sulfadoxine) was the second-line treatment and 18 patients were cleared of parasites and 31 had continuing infections classified as RI, RII or RIII resistance and 1 patient had recrudescent parasitaemia. The pfmdr1, dhfr and dhps genes were examined for mutations previously shown to be associated with resistance to these drugs. In this study, mutations in pfmdr1 were associated with chloroquine resistance and mutations in both dhfr and dhps were associated with Fansidar resistance in vivo. Interestingly, Gly-437 in dhps along with Arg-59/Asn-108 in dhfr were associated with RI, RII and RIII resistance whereas Glu-540 was highly associated with only RII and RIII Fansidar resistance. This finding supports the hypothesis that the molecular basis of RI, RII and RIII Fansidar resistance involves an accumulation of mutations in both dhfr and dhps. These results suggest that mutations in both dhfr and dhps genes are a good predictor of potential Fansidar treatment failure.

The T76 mutation in the pfcrt gene of Plasmodium falciparum and clinical chloroquine resistance phenotypes in Papua, Indonesia.

The T76 mutation in the pfcrt gene has been linked to chloroquine (CQ) resistance in Plasmodium falciparum. PCR-based analysis of pfcrt alleles was performed on pre-treatment samples from 107 individuals who had P. falciparum infections and lived in Papua, Indonesia. The results of a 28-day, in-vivo test revealed clinical resistance to CQ in 79 (74%) of the samples. The crude sensitivity of the pfcrt T76 assay for detecting the CQ-resistant infections in the samples was 96% and the crude specificity 52%. Discordance between pfcrt genotype and in-vivo phenotype was analysed either by genotyping of the merozoite surface protein-2 (to distinguish re-infection from recrudescence) or by amplification of the P. falciparum-specific small-subunit ribosomal RNA (ssrRNA) gene, using nested PCR (to detect any sub-patent but resistant parasites in infections misclassified as sensitive by the in-vivo test). When adjusting for the results of these analyses, the sensitivity and specificity of the pfcrt T76 assay for detecting the CQ-resistant infections became 93% and 82%, respectively. Overall, the present results indicate that the pfcrt T76 assay may be used to forecast therapeutic failure caused by CQ resistance. Validation requires exploration of the phenotype classifications based on the results of in-vivo tests, using genetic analyses that distinguish re-infection from recrudescence and detect microscopically subpatent parasitaemias.

The drug sensitivity and transmission dynamics of human malaria on Nias Island, North Sumatra, Indonesia.

Nias Island, off the north-western coast of Sumatra, Indonesia, was one of the first locations in which chloroquine-resistant Plasmodium vivax malaria was reported. This resistance is of particular concern because its ancient megalithic culture and the outstanding surfing conditions make the island a popular tourist destination. International travel to and from the island could rapidly spread chloroquine-resistant strains of P. vivax across the planet. The threat posed by such strains, locally and internationally, has led to the routine and periodic re-assessment of the efficacy of antimalarial drugs and transmission potential on the island. Active case detection identified malaria in 124 (17%) of 710 local residents whereas passive case detection, at the central health clinic, confirmed malaria in 77 (44%) of 173 cases of presumed 'clinical malaria'. Informed consenting volunteers who had malarial parasitaemias were treated, according to the Indonesian Ministry of Health's recommendations, with sulfadoxine-pyrimethamine (SP) on day 0 (for P. falciparum) or with chloroquine (CQ) on days 0, 1 and 2 (for P. vivax). Each volunteer was then monitored for clinical and parasite response until day 28. Recurrent parasitaemia by day 28 treatment was seen in 29 (83%) of the 35 P. falciparum cases given SP (14, 11 and four cases showing RI, RII and RIII resistance, respectively). Recurrent parasitaemia was also observed, between day 11 and day 21, in six (21%) of the 28 P. vivax cases given CQ. Although the results of quantitative analysis confirmed only low prevalences of CQ-resistant P. vivax malaria, the prevalence of SP resistance among the P. falciparum cases was among the highest seen in Indonesia. When the parasites present in the volunteers with P. falciparum infections were genotyped, mutations associated with pyrimethamine resistance were found at high frequency in the dhfr gene but there was no evidence of selection for sulfadoxine resistance in the dhps gene. Night-biting mosquitoes were surveyed by human landing collections and tested for sporozoite infection. Among the five species of human-biting anophelines collected, Anopheles sundaicus was dominant (68%) and the only species found to be infective--two (1.2%) of 167 females being found carrying P. vivax sporozoites. The risk of malarial infection for humans on Nias was considered high because of the abundance of asymptomatic carriers, the reduced effectiveness of the available antimalarial drugs, and the biting and infection 'rates' of the local An. sundaicus.