Between July, 2001, and December, 2010, 3202 hyperparasitaemic patients were treated. Most patients were younger than 15 years (appendix
). Most patients (2403) were given oral artesunate and divided doses of mefloquine. The remaining patients were given artesunate monotherapy (321 patients), artesunate in combination with either doxycycline (382 patients) or clindamycin (71 patients), or other combinations (25 patients). Patients who got blood transfusions within 24 hours of admission, or an incomplete course of or more than one parenteral dose of artesunate were excluded from the analysis. 2855 patients (1268 in Wang Pha, 975 in Mawker Thai, 467 in Maela, and 145 in Mae Khon Ken) remained eligible, of whom 1778 (62%) were men or boys. In 1759 of 2855 patients, artesunate monotherapy was given, or else the partner drug (mefloquine, doxycycline, or clindamycin) was given more than 48 h from the start of artesunate treatment. All patients survived, confirming the effectiveness of oral artesunate in uncomplicated hyperparasitaemia.18
Between 2001 and 2010, the median age of hyperparasitaemic patients increased slightly but significantly from 11 years to 15 years (p<0·0001). The proportion of patients developing severe malaria did not increase over time, nor did the ratio of severe to uncomplicated falciparum malaria cases, malaria mortality, nor the proportion of patients developing anaemia. We noted a significant increase in the proportion of patients with gametocytaemia on admission (p<0·0001; 7·5×10−6 test for trend).
Parasite clearance half-life lengthened from a geometric mean of 2·6 h (95% CI 2·5–2·7) in 2001, to 3·7 h (3·6–3·8) in 2010 (p<0·0001), compared with 5·5 h (5·2–5·9) in the 116 patients from western Cambodia between 2007 and 2010 (clearance data from three patients fitted poorly to the linear model [R2<0·8] and were thus excluded) (, ). We used a stringent threshold of a parasite clearance half-life of more than 6·2 h to categorise infections with slow or fast parasite clearance; the proportion of patients at our study centres with slow clearance rose from 0·6% in 2001, to 20% in 2010, compared with 42% of patients in western Cambodia in 2010 (). Parasite clearance rates in patients given artesunate monotherapy or given artesunate alone for more than 48 h (n=1759) before receiving partner drugs were similar to those in patients who were given combination treatments immediately, suggesting that increasing resistance to the partner drug cannot explain the temporal changes reported.
Parasite clearance half-life of Plasmodium falciparum on the Thailand–Myanmar border over 10 years
Change in parasite clearance half-life of Plasmodium falciparum 2001–10
No relation existed between parasite clearance half-life and age (p=0·439) or starting parasitaemia (p=0·598) in patients given artesunate monotherapy or given artesunate alone for more than 48 h before receiving partner drugs, nor in all patients (p=0·124 and p=0·569, respectively). The variable most strongly associated with increases in parasite clearance half-life was the date of patient admission (appendix
). This trend was strongly significant in all clinics sampled except Mae Khon Ken, Thailand (; appendix
). Only 7·4% of the variation in half-life could be explained by other admission variables. Location had a significant effect on parasite clearance; infections in northern locations (ie, Maela and Wang Pha) had slower clearance than those sampled in the south (ie, Mawker Thai and Mae Khon Ken) (appendix
Of 96 single nucleotide polymorphisms assessed, 93 gave robust genotype data. Of the 1583 infections genotyped, 1029 were single-clone infections (appendix
). From these, 148 unique 93-locus parasite genotypes were identified, each infecting between two and 13 patients (29 from 2001 to 2004, and 119 from 2007 to 2010) (appendix
). Patients harbouring parasites with the same 93-locus genotype had similar parasite clearance half-lives (), showing the important part played by parasite genotype. Between 2001 and 2004, parasite genotype had a significant effect on parasite clearance half-life (p=0·0166), even after adjusting for significant covariates (p=0·01). Two of the three patients with the longest parasite clearance half-lives recorded before 2004 had the same 93-locus genotype (). From 2007 to 2010, parasite genotype had a much stronger effect on parasite clearance half-life (p<0·0001) than it had between 2001 and 2004, and remained highly significant after adjusting for other covariates (F
=5·219, df=118, p<0·0001). Mean H2
increased from 0·30 (SD 0·14) in 2001–04, to 0·66 (0·04) in 2007–10. After adjustment for the effects of covariates, H2
rose from 0·32 (0·14) between 2001–04, to 0·58 (0·05) in 2007–10 (appendix
). The increase in H2
was tested by permutation and was significant both before (p<0·01) and after (p=0·04) adjustment for covariates (appendix
Effect of parasite genotype on parasite clearance rates
To investigate whether the increase in H2 was driven by alleles conferring slow parasite clearance, we removed 30% of the slowest clearing infections from the 2007–10 dataset, which caused H2 to decrease from 0·66 (SD 0·04) to 0·17 (0·07). Removal of 30% of the fastest clearing infections increased H2 to 0·70 (0·05), whereas random removal of 30% of infections did not alter H2 (mean H2 from 100 randomisations 0·67 (0·03); range 0·56–0·74). These in-silico experiments show that high H2 is driven by parasite genes that determine slow parasite clearance.
Admixture of parasites with the Cambodian phenotype could explain the parasite clearance distributions in 2001–02, and 2009–10, but not those between 2003 and 2008 (p<0·01). This finding suggests that the gradual increases in parasite clearance half-life are unlikely to be explained by a single-step process of importation or de-novo selection of a parasite population with the same characteristics as the resistant parasites prevalent in western Cambodia.
None of the 93-locus genotypes was common to patients from the Thailand–Myanmar border and western Cambodia. Pairwise allele sharing (ps) between the 25 slowest and 25 fastest clearing parasites from the Thailand–Myanmar border was compared with 30 slow-clearing parasites from western Cambodia. Cambodian parasites were more closely related (ps=0·453) to slow-clearing parasites than to fast-clearing parasites (ps=0·450) from the Thailand–Myanmar border. However, permutation tests show that this difference is not significant (p=0·137, 10 000 permutations).