Background Despite the remarkable activity of artemisinin and its derivatives their monotherapy has been associated with high rates of recrudescence. The temporary growth arrest of ring stage parasites (dormancy) following exposure to artemisinin drugs provides a plausible explanation for this phenomenon. Methods Ring stage parasites of several P. falciparum lines were exposed to different doses of dihydroartemisinin (DHA) alone or in combination with mefloquine (MQ). For each regime the proportion of parasites recovering was determined daily for 20 days. Results Parasite development was abruptly arrested following a single exposure to DHA, with some parasites being dormant for up to 20 days. Approximately 50% of dormant parasites recovered to resume growth within the first 9 days. The overall proportion of parasites recovering was dose dependant with recovery rates ranging from 0.044% to 1.313%. Repeated treatment with DHA, or DHA in combination with MQ, led to a delay in recovery and a ~10 fold reduction in total recovery. Strains with different genetic backgrounds appear to vary in their capacity to recover. Conclusions These results imply that artemisinin-induced growth arrest occurs readily in laboratory treated parasites, and may be a key factor in treatment failure of P. falciparum malaria.

Mutations in the pfcrt and pfmdr1 genes have been associated with chloroquine resistance in Plasmodium falciparum. Ten and five mutations, respectively, have been identified in these genes from chloroquine-resistant parasites worldwide. Mutation patterns in pfcrt revealed that chloroquine resistance evolved independently in southeast Asia, South America, and Papua New Guinea. However, the evolution of chloroquine resistance in the rest of the Pacific region is unclear. In this study, we examined sequence polymorphisms in these genes in isolates from Morong, Philippines, and compared them to known chloroquine resistance sequences. Two novel mutations, A144T and L160Y, were identified outside of the 10 known mutations in pfcrt in Morong isolates. These novel mutations were identified only in parasites with K76T and N326D but without the common A220S mutation found in most chloroquine-resistant isolates. This represents a unique chloroquine resistance allelic type (K76T/A144T/L160Y/N326D) not previously found elsewhere in the world. One Morong isolate also had an additional C72S mutation, whereas only one isolate possessed an allelic type typical of chloroquine resistance in Asia. Parasites with the novel pfcrt allelic types were resistant to chloroquine in vitro and were unresponsive to verapamil (0.9 M) chemosensitization, similar to chloroquine-resistant parasites from South America and Papua New Guinea. These results suggest that chloroquine resistance evolved independently in the Philippines and represents a second chloroquine resistance founder event in the South Pacific.

Artemisinin and its derivatives are the most rapidly acting and efficacious antimalarial drugs currently available. Although resistance to these drugs has not been documented, there is growing concern about the potential for resistance to develop. In this paper we report the selection of parasite resistance to artelinic acid (AL) and artemisinin (QHS) in vitro and the molecular changes that occurred during the selection. Exposure of three Plasmodium falciparum lines (W2, D6, and TM91C235) to AL resulted in decreases in parasite susceptibilities to AL and QHS, as well as to mefloquine, quinine, halofantrine, and lumefantrine. The changes in parasite susceptibility were accompanied by increases in the copy number, mRNA expression, and protein expression of the pfmdr1 gene in the resistant progenies of W2 and TM91C235 parasites but not in those of D6 parasites. No changes were detected in the coding sequences of the pfmdr1, pfcrt, pfatp6, pftctp, and pfubcth genes or in the expression levels of pfatp6 and pftctp. Our data demonstrate that P. falciparum lines have the capacity to develop resistance to artemisinin derivatives in vitro and that this resistance is achieved by multiple mechanisms, to include amplification and increased expression of pfmdr1, a mechanism that also confers resistance to mefloquine. This observation is of practical importance, because artemisinin drugs are often used in combination with mefloquine for the treatment of malaria.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins expressed on the surface of P. falciparum-infected erythrocytes undergo antigenic variation by switching the gene expressed within a repertoire of approximately 50 var genes per haploid genome. The switching of PfEMP1 plays an important role in the survival and pathogenesis of the parasite. To understand how a parasite switches its var gene expression in human infections, we investigated the composition and change of var gene transcripts during the acute phase of well-defined laboratoryinduced P. falciparum infections in naïve human hosts. Multiple var transcripts, with the same dominant transcript, were identified in samples collected after three to four asexual-parasite cycles in two volunteers infected with cloned 3D7 P. falciparum via mosquito bites. A major change in composition and frequency of var gene transcripts was observed between the culture used to infect the mosquitoes and the parasites recovered from the infected volunteers. A further change was seen when infected blood from a mosquito-infected volunteer was either passaged to other volunteers or cultured in vitro. The diversity of var transcripts did not increase with time. The results suggest that the switch of var gene expression is reinitiated after mosquito transmission and that var genes may rapidly switch from the first gene expressed after liver stage, but subsequent switching occurs at a much lower rate.

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Plasmodium falciparum erythrocyte membrane protein 1, which is encoded by the var multigene family, is expressed on the surface of P. falciparum-infected erythrocytes and has been implicated in many of the complications associated with falciparum malaria. Transcriptional switching of var is commonly investigated using in vitro cultured parasites, because parasite material from patients is limited. We investigated the affect of short-term in vitro cultivation on var gene transcription in patient samples. A significant reduction in the overall abundance of var transcripts was observed during the first approximately 10 days of culture. The rate of down-regulation was not constant among all var genes; genes with an upsA, -D, and -E 5' flanking region had a significantly faster rate than genes with an upsB or -C flanking region. These results have significant implications for the investigation of associations between var transcription and clinical manifestations using parasites that have been enriched by in vitro culture.

BackgroundAlthough rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates.MethodsTotal RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle.ResultsTranscription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed.ConclusionsThe levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.

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