Jennifer M. Peters

Artemisinin-Induced Dormancy in Plasmodium falciparum: Duration, Recovery Rates, and Implications in Treatment Failure

BACKGROUND Despite the remarkable activity of artemisinin and its derivatives, monotherapy with these agents has been associated with high rates of recrudescence. The temporary arrest of the growth of ring-stage parasites (dormancy) after exposure to artemisinin drugs provides a plausible explanation for this phenomenon. METHODS Ring-stage parasites of several Plasmodium falciparum lines were exposed to different doses of dihydroartemisinin (DHA) alone or in combination with mefloquine. For each regime, the proportion of recovering parasites was determined daily for 20 days. RESULTS Parasite development was abruptly arrested after 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 dependent, with recovery rates ranging from 0.044% to 1.313%. Repeated treatment with DHA or with DHA in combination with mefloquine led to a delay in recovery and an approximately 10-fold reduction in total recovery. Strains with different genetic backgrounds appeared to vary in their capacity to recover. CONCLUSIONS These results imply that artemisinin-induced arrest of growth occurs readily in laboratory-treated parasites and may be a key factor in P. falciparum malaria treatment failure.

Role of pfmdr1 amplification and expression in induction of resistance to artemisinin derivatives in Plasmodium falciparum.

ABSTRACT 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.

High diversity and rapid changeover of expressed var genes during the acute phase of Plasmodium falciparum infections in human volunteers

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 laboratory-induced 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.

pfcrt Allelic Types with Two Novel Amino Acid Mutations in Chloroquine-Resistant Plasmodium falciparum Isolates from the Philippines

ABSTRACT 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 μM) 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.

Transcription and Expression of Plasmodium falciparum Histidine-Rich Proteins in Different Stages and Strains: Implications for Rapid Diagnostic Tests

Background Although 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. Methods Total 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. Results Transcription 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. Conclusions The 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.

Genetic diversity of the DBLα region in Plasmodium falciparum var genes among Asia-Pacific isolates

In Plasmodium falciparum a highly polymorphic multi-copy gene family, var, encodes the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1), which has an important role in cytoadherence and immune evasion. Using previously described universal PCR primers for the first Duffy binding-like domain (DBLalpha) of var we analysed the DBLalpha repertoires of Dd2 (originally from Thailand) and eight isolates from the Solomon Islands (n=4), Philippines (n=2), Papua New Guinea (n=1) and Africa (n=1). We found 15-32 unique DBLalpha sequence types among these isolates and estimated detectable DBLalpha repertoire sizes ranging from 33-38 to 52-57 copies per genome. Our data suggest that var gene repertoires generally consist of 40-50 copies per genome. Eighteen DBLalpha sequences appeared in more than one Asia-Pacific isolate with the number of sequences shared between any two isolates ranging from 0 to 6 (mean=2.0 +/-1.6). At the amino acid level DBLalpha sequence similarity within isolates ranged from 45.2 +/- 7.1 to 50.2 +/- 6.9%, and was not significantly different from the DBLalpha amino acid sequence similarity among isolates (P>0.1). Comparisons with published sequences also revealed little overlap among DBLalpha sequences from different regions. High DBLalpha sequence diversity and minimal overlap among these isolates suggest that the global var gene repertoire is immense, and may potentially be selected for by the hosts protective immune response to the var gene products, PfEMP1.

Mutations in Cytochrome b Resulting in Atovaquone Resistance Are Associated with Loss of Fitness in Plasmodium falciparum

ABSTRACT Drug resistance in malarial parasites has become a major obstacle in the control of the disease. Strategies are urgently needed to control the development of resistance and to possibly reverse existing resistance. One key element required to reverse malaria drug resistance is for the parasites to “pay” a biological “cost” or suffer a loss of fitness when acquiring resistance to antimalarial drugs. Such a situation would be a disadvantage to the resistant parasites in the absence of drug pressure. We compared here the relative fitness of atovaquone-resistant Plasmodium falciparum K1 clones with single and double base mutations in their cytochrome b genes to their parent clones during erythrocytic stages in the absence of drug pressure. We found that the double amino acid mutation (M133I and G280D) is associated with a 5 to 9% loss of fitness and that the single amino acid change of M133I did not result in any detectable loss of fitness. Molecular modeling of the interaction of P. falciparum cytochrome b with ubiquinone led to the prediction that a loss of fitness of the malaria parasites would result from the G280D mutation due to its close proximity to the putative ubiquinone-binding site. This appears to have resulted in a weakening of the cytochrome b-ubiquinone complex, thereby causing the electron transport chain to become less efficient. Our results suggest that the prevalence of resistant parasites may decrease after the drug usage is discontinued.

Switching rates of Plasmodium falciparum var genes: faster than we thought?

Plasmodium falciparum undergoes antigenic variation by switching the expressed erythrocyte membrane protein (PfEMP)1. This family of proteins plays an important role in the development of chronic, recrudescent P. falciparum malaria, acquired immunity and severe malaria. However, little is known about the switching mechanism or switching rates in the human host. Here, we estimate the switch rate of var genes, using recently published data describing the var gene transcripts detected in blood taken from human volunteers during acute P. falciparum infections and a mathematical model of the in-host dynamics. The overall switch rate of PfEMP1 predicted during the initial stage of infection ( approximately 18% switching parasites per generation) is much higher than previously reported. The implications of the predicted switching rates are discussed.

Agricultural Intensification Exacerbates Spillover Effects on Soil Biogeochemistry in Adjacent Forest Remnants

Land-use intensification is a central element in proposed strategies to address global food security. One rationale for accepting the negative consequences of land-use intensification for farmland biodiversity is that it could ‘spare’ further expansion of agriculture into remaining natural habitats. However, in many regions of the world the only natural habitats that can be spared are fragments within landscapes dominated by agriculture. Therefore, land-sparing arguments hinge on land-use intensification having low spillover effects into adjacent protected areas, otherwise net conservation gains will diminish with increasing intensification. We test, for the first time, whether the degree of spillover from farmland into adjacent natural habitats scales in magnitude with increasing land-use intensity. We identified a continuous land-use intensity gradient across pastoral farming systems in New Zealand (based on 13 components of farmer input and soil biogeochemistry variables), and measured cumulative off-site spillover effects of fertilisers and livestock on soil biogeochemistry in 21 adjacent forest remnants. Ten of 11 measured soil properties differed significantly between remnants and intact-forest reference sites, for both fenced and unfenced remnants, at both edge and interior. For seven variables, the magnitude of effects scaled significantly with magnitude of surrounding land-use intensity, through complex interactions with fencing and edge effects. In particular, total C, total N, δ15N, total P and heavy-metal contaminants of phosphate fertilizers (Cd and U) increased significantly within remnants in response to increasing land-use intensity, and these effects were exacerbated in unfenced relative to fenced remnants. This suggests movement of livestock into surrounding natural habitats is a significant component of agricultural spillover, but pervasive changes in soil biogeochemistry still occur through nutrient spillover channels alone, even in fenced remnants set aside for conservation. These results have important implications for the viability of land-sparing as a strategy for balancing landscape-level conservation and production goals in agricultural landscapes.

Physical Linkage to Drug Resistance Genes Results in Conservation of var Genes among West Pacific Plasmodium falciparum Isolates

The multicopy var gene family encoding the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 is highly diverse, with little overlap between different P. falciparum isolates. We report 5 var genes (varS1-varS5) that are shared at relatively high frequency among 63 genetically diverse P. falciparum isolates collected from 5 islands in the West Pacific region. The varS1, varS2, and varS3 genes were localized to the internal region on chromosome 4, approximately 200 kb from pfdhfr-ts, whereas varS4 and varS5 were mapped to an internal region of chromosome 7, within 100 kb of pfcrt. The presence of varS2 and varS3 were significantly correlated with the pyrimethamine-resistant pfdhfr genotype, whereas varS4 was strongly correlated with the chloroquine-resistant pfcrt genotype. Thus, the conservation of these var genes is the result of their physical linkage with drug-resistant genes in combination with the antimalarial drug pressure in the region.