Timing of infection and prior immunization with respiratory syncytial virus (RSV) in RSV-enhanced allergic inflammation

Respiratory syncytial virus (RSV) infection has been shown to be a risk factor for the development of allergy in humans and mice. The allergy-enhancing properties of RSV may be dependent on atopic background and an individual's history of RSV infection. We examined the influence of the timing of infection and prior inoculation with RSV in a mouse model of allergic asthma. Mice were sensitized to and challenged with ovalbumin (OVA) and were inoculated with RSV either before or during the sensitization or challenge period. One group of mice was inoculated with RSV both before sensitization to OVA and during challenge with OVA. Increased pulmonary expression of interleukin (IL)-4, IL-5, and IL-13 mRNA and aggravated alveolitis and hypertrophy of mucus-producing cells were observed only when OVA-sensitized mice were inoculated with RSV shortly before or during challenge with OVA. Despite protection against viral replication, prior inoculation with RSV did not abrogate RSV-enhanced, OVA-induced expression of T helper 2 (Th2) cytokines in the lung. In conclusion, inoculation with RSV enhances allergic disease only when the immune system has already been Th2-primed by the allergen (i.e., OVA). This RSV-enhanced allergy is not completely abrogated by prior inoculation with RSV

Inferred relatedness and heritability in malaria parasites

Malaria parasites vary in phenotypic traits of biomedical or biological interest such as growth rate, virulence, sex ratio and drug resistance, and there is considerable interest in identifying the genes that underlie this variation. An important first step is to determine trait heritability (H2). We evaluate two approaches to measuring H2 in natural parasite populations using relatedness inferred from genetic marker data. We collected single-clone Plasmodium falciparum infections from 185 patients from the Thailand–Burma border, monitored parasite clearance following treatment with artemisinin combination therapy (ACT), measured resistance to six antimalarial drugs and genotyped parasites using 335 microsatellites. We found strong relatedness structure. There were 27 groups of two to eight clonally identical (CI) parasites, and 74 per cent of parasites showed significant relatedness to one or more other parasites. Initially, we used matrices of allele sharing and variance components (VC) methods to estimate H2. Inhibitory concentrations (IC50) for six drugs showed significant H2 (0.24 to 0.79, p = 0.06 to 2.85 × 10−9), demonstrating that this study design has adequate power. However, a phenotype of current interest—parasite clearance following ACT—showed no detectable heritability (H2 = 0–0.09, ns) in this population. The existence of CI parasites allows the use of a simple ANOVA approach for quantifying H2, analogous to that used in human twin studies. This gave similar results to the VC method and requires considerably less genotyping information. We conclude (i) that H2 can be effectively measured in malaria parasite populations using minimal genotype data, allowing rational design of genome-wide association studies; and (ii) while drug response (IC50) shows significant H2, parasite clearance following ACT was not heritable in the population studied

Considerations on the use of nucleic acid-based amplification for malaria parasite detection

<p>Abstract</p> <p>Background</p> <p>Nucleic acid amplification provides the most sensitive and accurate method to detect and identify pathogens. This is primarily useful for epidemiological investigations of malaria because the infections, often with two or more <it>Plasmodium </it>species present simultaneously, are frequently associated with microscopically sub-patent parasite levels and cryptic mixed infections. Numerous distinct equally adequate amplification-based protocols have been described, but it is unclear which to select for epidemiological surveys. Few comparative studies are available, and none that addresses the issue of inter-laboratory variability.</p> <p>Methods</p> <p>Blood samples were collected from patients attending malaria clinics on the Thai-Myanmar border. Frozen aliquots from 413 samples were tested independently in two laboratories by nested PCR assay. Dried blood spots on filter papers from the same patients were also tested by the nested PCR assay in one laboratory and by a multiplex PCR assay in another. The aim was to determine which protocol best detected parasites below the sensitivity level of microscopic examination.</p> <p>Results</p> <p>As expected PCR-based assays detected a substantial number of infected samples, or mixed infections, missed by microscopy (27 and 42 for the most sensitive assay, respectively). The protocol that was most effective at detecting these, in particular mixed infections, was a nested PCR assay with individual secondary reactions for each of the species initiated with a template directly purified from the blood sample. However, a lesser sensitivity in detection was observed when the same protocol was conducted in another laboratory, and this significantly altered the data obtained on the parasite species distribution.</p> <p>Conclusions</p> <p>The sensitivity of a given PCR assay varies between laboratories. Although, the variations are relatively minor, they primarily diminish the ability to detect low-level and mixed infections and are sufficient to obviate the main rationale to use PCR assays rather than microscopy or rapid diagnostic tests. The optimal approach to standardise methodologies is to provide PCR template standards. These will help researchers in different settings to ensure that the nucleic acid amplification protocols they wish to use provide the requisite level of sensitivity, and will permit comparison between sites.</p

A Randomised Controlled Trial of Artemether-Lumefantrine Versus Artesunate for Uncomplicated Plasmodium falciparum Treatment in Pregnancy

Rose McGready and colleagues show that an artemether-lumefantrine regimen is well tolerated and safe in pregnant Karen women with uncomplicated falciparum malaria, but efficacy is inferior to artesunate, probably because of low drug concentrations in later pregnancy

Changes in the Treatment Responses to Artesunate-Mefloquine on the Northwestern Border of Thailand during 13 Years of Continuous Deployment

Background: Artemisinin combination treatments (ACT) are recommended as first line treatment for falciparum malaria throughout the malaria affected world. We reviewed the efficacy of a 3-day regimen of mefloquine and artesunate regimen (MAS ), over a 13 year period of continuous deployment as first-line treatment in camps for displaced persons and in clinics for migrant population along the Thai-Myanmar border. Methods and Findings: 3,264 patients were enrolled in prospective treatment trials between 1995 and 2007 and treated with MAS. The proportion of patients with parasitaemia persisting on day-2 increased significantly from 4.5% before 2001 to 21.9% since 2002 (p&lt;0.001). Delayed parasite clearance was associated with increased risk of developing gametocytaemia (AOR = 2.29; 95% CI, 2.00-2.69, p = 0.002). Gametocytaemia on admission and carriage also increased over the years (p = 0.001, test for trend, for both). MAS efficacy has declined slightly but significantly (Hazards ratio 1.13; 95% CI, 1.07-1.19, p&lt;0.001), although efficacy in 2007 remained well within acceptable limits: 96.5% (95% CI, 91.0-98.7). The in vitro susceptibility of P. falciparum to artesunate increased significantly until 2002, but thereafter declined to levels close to those of 13 years ago (geometric mean in 2007: 4.2 nM/l; 95% CI, 3.2-5.5). The proportion of infections caused by parasites with increased pfmdr1 copy number rose from 30% (12/ 40) in 1996 to 53% (24/45) in 2006 (p = 0.012, test for trend). Conclusion: Artesunate-mefloquine remains a highly efficacious antimalarial treatment in this area despite 13 years of widespread intense deployment, but there is evidence of a modest increase in resistance. Of particular concern is the slowing of parasitological response to artesunate and the associated increase in gametocyte carriage. © 2009 Carrara et al

Defining Plasmodium falciparum Treatment in South West Asia: A Randomized Trial Comparing Artesunate or Primaquine Combined with Chloroquine or SP

INTRODUCTION: Antimalarial resistance has led to a global policy of artemisinin-based combination therapy. Despite growing resistance chloroquine (CQ) remained until recently the official first-line treatment for falciparum malaria in Pakistan, with sulfadoxine-pyrimethamine (SP) second-line. Co-treatment with the gametocytocidal primaquine (PQ) is recommended for transmission control in South Asia. The relative effect of artesunate (AS) or primaquine, as partner drugs, on clinical outcomes and gametocyte carriage in this setting were unknown. METHODS: A single-blinded, randomized trial among Afghan refugees in Pakistan compared six treatment arms: CQ; CQ+(single-dose)PQ; CQ+(3 d)AS; SP; SP+(single-dose)PQ, and SP+(3 d)AS. The objectives were to compare treatment failure rates and effect on gametocyte carriage, of CQ or SP monotherapy against the respective combinations (PQ or AS). Outcomes included trophozoite and gametocyte clearance (read by light microscopy), and clinical and parasitological failure. FINDINGS: A total of 308 (87%) patients completed the trial. Failure rates by day 28 were: CQ 55/68 (81%); CQ+AS 19/67 (28%), SP 4/41 (9.8%), SP+AS 1/41 (2.4%). The addition of PQ to CQ or SP did not affect failure rates (CQ+PQ 49/67 (73%) failed; SP+PQ 5/33 (16%) failed). AS was superior to PQ at clearing gametocytes; gametocytes were seen on d7 in 85% of CQ, 40% of CQ+PQ, 21% of CQ+AS, 91% of SP, 76% of SP+PQ and 23% of SP+AS treated patients. PQ was more effective at clearing older gametocyte infections whereas AS was more effective at preventing emergence of mature gametocytes, except in cases that recrudesced. CONCLUSIONS: CQ is no longer appropriate by itself or in combination. These findings influenced the replacement of CQ with SP+AS for first-line treatment of uncomplicated falciparum malaria in the WHO Eastern Mediterranean Region. The threat of SP resistance remains as SP monotherapy is still common. Three day AS was superior to single-dose PQ for reducing gametocyte carriage. TRIAL REGISTRATION: ClinicalTrials.gov NCT00959517

Adaptive Copy Number Evolution in Malaria Parasites

Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2–11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation