Publications

Background Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates. Results In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype. Conclusions The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo .

Mass drug administration (MDA) with antimalarials has been shown to reduce prevalence and interrupt transmission in small populations, in populations with reliable access to antimalarial drugs, and in populations where sustained improvements in diagnosis and treatment are possible. Effective MDA eliminates drug-resistant parasites which has the long-term benefit of extending the useful therapeutic life of first-line therapies for all populations, not just the focal population where MDA was carried out. However, in order to plan elimination measures effectively, it is necessary to characterize the conditions under which failed MDA could exacerbate resistance. We use an individual-based stochastic model of Plasmodium falciparum transmission to evaluate this risk in large populations (>40K) where access to antimalarial treatments may not be uniformly high and where re-importation of drug-resistant parasites may be common. We find that drug-resistance evolution can be accelerated by MDA when all three of the following conditions are met: (1) strong genetic bottlenecking that falls short of elimination, (2) re-importation of resistant genotypes, and (3) continued selection pressure during routine case management post-MDA. Accelerated resistance levels are not immediate but follow the rebound of malaria cases post-MDA, if this is allowed to occur. Crucially, resistance is driven by the selection pressure during routine case management post-MDA and not the selection pressure exerted during the MDA itself. Second, we find that increasing treatment coverage post-MDA increases the probability of local elimination in low-transmission regions (PfPR < 2%) in scenarios with both low and high levels of drug-resistance importation. This emphasizes the importance of preparation and planning to ensure that MDA has a high probability of leading to elimination, and the necessity of supporting public health infrastructure to provide high coverage of diagnosis and treatment post-MDA.

Plasmodium falciparum, the protozoan parasite responsible for severe malaria infection, undergoes a complex life cycle. Infected red blood cells (iRBC) sequester in host cerebral microvessels, which underlies the pathology of cerebral malaria. Using immunohistochemistry on post mortem brain samples, we demonstrated positive staining for vascular endothelial growth factor (VEGF) on iRBC. Confocal microscopy of cultured iRBC revealed accumulation of VEGF within the parasitophorous vacuole, expression of host VEGF-receptor 1 and activated VEGF-receptor 2 on the surface of iRBC, but no accumulation of VEGF receptors within the iRBC. Addition of VEGF to parasite cultures had a trophic effect on parasite growth and also partially rescued growth of drug treated parasites. Both these effects were abrogated when parasites were grown in serum-free medium, suggesting a requirement for soluble VEGF receptor. We conclude that P. falciparum iRBC can bind host VEGF-R on the erythrocyte membrane and accumulate host VEGF within the parasitophorous vacuole, which may have a trophic effect on parasite growth.

In rural areas of South and Southeast Asia malaria is declining but febrile illnesses still account for substantial morbidity and mortality. Village health workers (VHWs) are often the first point of contact with the formal health system, and for patients with febrile illnesses they can provide early diagnosis and treatment of malaria. However, for the majority of febrile patients, VHWs lack the training, support and resources to provide further care. Consequently, treatable bacterial illnesses are missed, antibiotics are overused and poorly targeted, and patient attendance wanes along with declining malaria. This Open Letter announces the start of a new initiative, the Rural Febrile Illness (RFI) project, the first in a series of projects to be implemented as part of the South and Southeast Asian Community-based Trials Network (SEACTN) research programme. This multi-country, multi-site project will run in Bangladesh, Cambodia, Lao PDR, Thailand, and Myanmar. It will define the epidemiological baseline of febrile illness in nine remote and underserved areas of Asia where malaria endemicity is declining and access to health services is limited. The RFI project aims to determine the incidence, causes and outcomes of febrile illness; understand the opportunities, barriers and appetite for adjustment of the role of VHWs to include management of non-malarial febrile illnesses; and establish a network of community healthcare providers and facilities capable of implementing interventions designed to triage, diagnose and treat patients presenting with febrile illnesses within these communities in the future.

Theoretical models of the effect of population bottlenecks on genetic variation assume that individuals are removed at random from the population. We investigated this assumption in a naturally regulated, unstable population of Soay sheep ( Ovis aries ). During rapid population declines or ‘crashes’, individuals were not removed at random with respect to genotype: we found associations between individual survival and certain genotypes at five polymorphic protein or microsatellite DNA loci ( Ada, Got, Tf , MAF18 and OPACAP). Some loci appeared to show simple associations with survival whereas others had more complex interactions with crash year or age: all displayed different patterns of association between the sexes. Simple overdominance was not a general feature of our data; it seems likely that fluctuating selection, countervailing selection in different fitness components or frequency-dependent selection may explain the pattern and complexity of the associations shown at different loci. Our study cannot distinguish between selection acting at these loci or at other, closely linked loci. However, our empirical study implies that the molecular genetic outcome of population bottlenecks in natural populations does not always follow theoretical expectations based on the random removal of genotypes. Bottlenecks in which individuals are removed at random are distinct from bottlenecks in which there is scope for selection via non-random survival of individuals.

For many countries, as Professor Greenwood points out, the answer to his question is a resounding ‘yes’.1 Perhaps the more pertinent question is, with so many more interventions now available, ‘how?’. The two most important new tools that have been added to our malaria control arsenal since the 1950s are artemisinin combination therapies and insecticide-treated bed nets. These are most likely to be effective when used together, in combination with indoor residual spraying where there are suitable vectors. The problem we are faced with is a lack of experience of using such an elimination strategy on a large scale, leading to a lack of data, particularly on the best way to roll out ACTs. In the past, mass screening and treatment, mass drug administration and large-scale replacement of first-line therapies have all been attempted, with varying degrees of success. In the context of newly arisen artemisinin resistance and the diminishing effectiveness of the pyrethroid insecticides, particular care must be taken to preserve the effectiveness of these compounds for as long as possible, whilst achieving maximum impact in a wide variety of epidemiological settings.

Primaquine is the only drug providing radical cure of Plasmodium vivax malaria. It is not recommended for breastfeeding women as it causes hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals, and breast milk excretion and thus infant exposure are not known.Healthy G6PD-normal breastfeeding women with previous P. vivax infection and their healthy G6PD-normal infants between 28 days and 2 years old were enrolled. Mothers took primaquine 0.5 mg/kg/day for 14 days. Primaquine and carboxyprimaquine concentrations were measured in maternal venous plasma, capillary plasma, and breast milk samples and infant capillary plasma samples taken on days 0, 3, 7, and 13.In 20 mother-infant pairs, primaquine concentrations were below measurement thresholds in all but 1 infant capillary plasma sample (that contained primaquine 2.6 ng/mL), and carboxyprimaquine was likewise unmeasurable in the majority of infant samples (maximum value 25.8 ng/mL). The estimated primaquine dose received by infants, based on measured breast milk levels, was 2.98 µg/kg/day (ie, ~0.6% of a hypothetical infant daily dose of 0.5 mg/kg). There was no evidence of drug-related hemolysis in the infants. Maternal levels were comparable to levels in nonlactating patients, and adverse events in mothers were mild.The concentrations of primaquine in breast milk are very low and therefore very unlikely to cause adverse effects in the breastfeeding infant. Primaquine should not be withheld from mothers breastfeeding infants or young children. More information is needed in neonates.NCT01780753.

Plasmodium-infected erythrocytes lose their normal deformability and become susceptible to splenic filtration. In animal models, this is one mechanism of antimalarial defense. To assess the effect of acute falciparum malaria on splenic filtration, we measured the clearance of heated 51Cr-labeled autologous erythrocytes in 25 patients with acute falciparum malaria and in 10 uninfected controls. Two groups of patients could be distinguished. Sixteen patients had splenomegaly, markedly accelerated clearance of the labeled erythrocytes (clearance half-time, 8.4 +/- 4.4 minutes [mean +/- SD] vs. 62.5 +/- 36.5 minutes in controls; P less than 0.001), and a lower mean hematocrit than did the patients without splenomegaly (P less than 0.001). In the nine patients without splenomegaly, clearance was normal. After institution of antimalarial chemotherapy, however, the clearance in this group accelerated to supernormal rates similar to those in the patients with splenomegaly, but without the development of detectable splenomegaly. Clearance was not significantly altered by treatment in the group with splenomegaly. Six weeks later, normal clearance rates were reestablished in most patients in both groups. We conclude that splenic clearance of labeled erythrocytes is enhanced in patients with malaria if splenomegaly is present and is enhanced only after treatment if splenomegaly is absent. Whether this enhanced splenic function applies to parasite-infected erythrocytes in patients with malaria and has any clinical benefit will require further studies.

Summary 1. We use structured demographic accounting to decompose the contribution to the variance in relative population growth between 1971 and 1997 in female red deer on Rum, Scotland, in terms of different fitness components and age categories. 2. During the first 10 years (1971–80) the variance in relative population growth was small (0·00246) as the population grew from 58 to 148 having been culled previously. After this the relative variance in population growth increased to 0·00575 between 1981 and 1989, and increased again to 0·00848 between 1990 and 1997. 3. In the first phase of population growth (1971–80) changes in the birth rate contributed most to the variance in relative population growth (30%), then adult winter survival (23%), with calf summer and calf winter survival contributing less than 10%. Birth rate tended to covary strongly with adult winter survival, and to a lesser extent with calf winter survival. 4. The contribution of birth rate to the variance in relative population growth diminished to less than 5% in both the periods 1981–89 and 1990–97. In contrast, the contribution of adult winter survival increased to 36% in 1980s and 40% in 1990s. At the same time, the covariation of adult winter survival with calf winter survival increased successively to reach 40% in 1990–97. 5. Changes in population age structure were small and contributed little to the variance in relative population growth (≈ 1% over the entire study). In the 1970s the 5–7‐year‐old group contributed most (23%) to the variance in relative population growth, though this covaried with 8–12‐year‐olds. In the 1980s the 8–12‐year‐olds contributed most (27%) and now the covariation was with older animals (> 12‐year‐olds). This pattern persisted in the 1990s, though there was covariation with 5–7‐year‐olds too. 6. Birth rate, calf winter survival and adult winter survival were all density‐dependent. The strength of density dependence within each vital rate did not differ between the 1970s, 1980s and 1990s; however, there were differences in the means of these vital rates relative to population size between the three periods. 7. The implications of temporal changes in the most important vital rates, the ‘key factors’, contributing to the variance in relative population growth rate are discussed.

Rickettsial diseases have not been described previously from Laos, but in a prospective study, acute rickettsial infection was identified as the cause of fever in 115 (27%) of 427 adults with negative blood cultures admitted to Mahosot Hospital in Vientiane, Laos. The organisms identified by serologic analysis were Orientia tsutsugamushi (14.8%), Rickettsia typhi (9.6%), and spotted fever group rickettsia (2.6% [8 R. helvetica, 1 R. felis, 1 R. conorii subsp. indica, and 1 Rickettsia "AT1"]). Patients with murine typhus had a lower frequency of peripheral lymphadenopathy than those with scrub typhus (3% vs. 46%, p<0.001). Rickettsioses are an underrecognized cause of undifferentiated febrile illnesses among adults in Laos. This finding has implications for the local empiric treatment of fever.

The antagonistic pleiotropy (AP) theory of ageing predicts genetically based trade-offs between investment in reproduction in early life and survival and performance in later life. Laboratory-based research has shown that such genetic trade-offs exist, but little is currently known about their prevalence in natural populations. We used random regression ‘animal model’ techniques to test the genetic basis of trade-offs between early-life fecundity (ELF) and maternal performance in late life in a wild population of red deer ( Cervus elaphus ) on the Isle of Rum, Scotland. Significant genetic variation for both ageing rates in a key maternal performance measure (offspring birth weight) and ELF was present in this population. We found some evidence for a negative genetic covariance between the rate of ageing in offspring birth weight and ELF, and also for a negative environmental covariance. Our results suggest rare support for the AP theory of ageing from a wild population.

<ns3:p>Chagas disease is a neglected disease caused by <ns3:italic>Trypanosoma cruzi</ns3:italic> that currently affects about 7 million people in the Americas and the American diaspora. Only two drugs are available to treat Chagas disease, both are associated with a high incidence of adverse events, particularly in adults, and they are also complex to administer. New treatments are needed urgently, but there is no established method of evaluating new drugs. We describe the study protocol for a pilot phase 2 descriptive observational study of parasite dynamics within the host, followed by a randomised evaluation of the pharmacokinetic-pharmacodynamic properties of subcurative doses of anti-chagasic drugs in patients with chronic Chagas disease. This study is divided into three stages. The general objective of the study is to characterise the dynamics of the parasite within the host at steady state and the pharmacokinetic-pharmacodynamic relationships for each of the three drugs studied. This is based on characterising the structure and parameters of a pharmacodynamic model of parasite turnover within the host. Each stage of the study targets specific key parameters in the pharmacodynamic model of parasite dynamics (both the blood stage and the tissue stage) for individual research participants. The primary outcome measure for all three stages is blood-stage parasite density, measured by quantitative PCR. Upon completion, research participants will receive definitive treatment for Chagas disease in accordance with national guidelines. Registration ISRCTN (ISRCTN26467068; 01/07/2021).</ns3:p>

No abstract is provided for this article.