METHODS: A 28-day in vivo evaluation of the clinical and parasitological response to three-day course of AS + SP was carried out in two areas of high endemicity (Hodeidah and Al-Mahwit provinces, Tehama region) in Yemen according to standard WHO protocol 2009. Clinical and parasitological indices were monitored over a 28-day follow-up, and the outcome was PCR-corrected. The frequencies of mutations in the pfdhfr, pfdhps, and pfK13 genes were obtained by sequencing following amplification.
RESULTS: Eighty-six patients completed the study, with a cure rate of 96.5 % (94.2 % PCR-uncorrected). Whereas four (4.7 %) patients still showed parasitaemia on day 2 post-treatment, all were found negative for asexual malaria stages on days 3 and 7. The efficacy of gametocyte clearance was poor (14.5, 42.5 and 86.0 % on days 7, 14 and 28, respectively), with gametocytes persisting throughout the study in some patients. All the isolates sequenced had the pfk13 propeller domain wild-type allele, and mutations associated with SP failure were observed only for pfdhfr with the double mutation (S108N + N51I) found in 65.4 % of the isolates sequenced.
CONCLUSION: In Yemen, AS + SP therapy remains effective for the treatment of uncomplicated falciparum malaria. Mutations were not detected in pfk13 or pfdhps, though double mutations were observed for pfdhfr. The observed persistent gametocytaemia re-enforces calls to add a single dose primaquine to this ACT in order to minimizes the potential for transmission and enhance regional efforts to eliminate malaria.
METHODS: In order to investigate the efficacy of pre-clinical vaccine candidates in P. knowlesi-infected human cases, this study describes an in vitro invasion inhibition assay, using a P. knowlesi strain adapted to in vitro growth in human erythrocytes, PkA1-H.1. Recombinant proteins of P. knowlesi Duffy binding protein alpha (PkDBPα) and apical membrane antigen 1 (PkAMA1) were produced in Escherichia coli system and rabbit antibodies were generated from immune animals.
RESULTS: PkDBPα and PkAMA1 recombinant proteins were expressed as insoluble and produced as a functional refolded form for this study. Antibodies against PkDBPα and PkAMA1 specifically recognized recombinant proteins and native parasite proteins in schizont-stage parasites on the merozoite organelles. Single and combination of anti-PkDBPα and anti-PkAMA1 antibodies elicited strong growth inhibitory effects on the parasite in concentration-dependent manner. Meanwhile, IgG prevalence of PkDBPα and PkAMA1 were observed in 13.0 and 46.7% in human clinical patients, respectively.
CONCLUSION: These data provide support for the validation of in vitro growth inhibition assay using antibodies of DBPα and AMA1 in human-adapted P. knowlesi parasite PkA1-H.1 strain.
METHODS: Rosetting rates of clinical isolates pre- and post- brief (one hour) exposure to artesunate (AS, an ART derivative) were evaluated. The effects of AS-mediated rosetting on the post-AS-exposed parasite's replication and survival, as well as the extent of protection by AS-mediated rosetting on different parasite stages were investigated. The rosetting ligands, mechanisms, and gene mutations involved were studied.
FINDINGS: Brief AS exposure stimulated rosetting, with AS-resistant isolates forming more rosettes in a more rapid manner. AS-mediated rosetting enabled infected erythrocytes (IRBC) to withstand AS exposure for several hours and protected the IRBC from phagocytosis. When their rosetting ability was blocked experimentally, the post-AS exposure survival advantage by the AS-resistant parasites was abrogated. Deletions in two genes coding for PfEMP1 exon 2 (PF3D7_0200300 and PF3D7_0223300) were found to be associated with AS-mediated rosetting, and these mutations were significantly selected through time in the parasite population under study, along with the K13 mutations, a molecular marker of ART-resistance.
INTERPRETATION: Rapid ART parasite clearance is driven by the direct oxidative damages on IRBC by ART and the phagocytic destruction of the damaged IRBC. Rosetting serves as a rapid 'buying time' strategy that allows more parasites to complete schizont maturation, reinvasion and subsequent development into the intrinsically less ART-susceptible ring stage.
FUNDING: A*STAR, NMRC-OF-YIRG, HRC e-ASIA, Wellcome.