Biomedical subjects
Richard Carter
Publications and source records attributed to Richard Carter.
Real-time quantitative PCR for analysis of genetically mixed infections of malaria parasites: technique validation and applications.
A technique that can distinguish and quantify genetically different malaria parasite clones in a mixed infection reliably and with speed and accuracy would be very useful for researchers. Many current methods of genotyping and quantification fall down on a number of aspects relating to their ease of use, sensitivity, cost, reproducibility and, not least, accuracy. Here we report the development and validation of a method that offers several advantages in terms of cost, speed and accuracy over conventional PCR or antibody-based methods. Using real-time quantitative PCR (RTQ-PCR) with allele-specific primers, we have accurately quantified the relative proportions of clones present in laboratory prepared ring-stage mixtures of two genetically distinct clones of the rodent malaria parasite Plasmodium chabaudi chabaudi. Accurate and reproducible measurement of the amount of genomic DNA representing each clone in a mixture was achieved over 100-fold range, corresponding to 0.074% parasitised erythrocytes at the lower end. To demonstrate the potential utility of this method, we include an example of the type of application it could be used for. In this case, we studied the growth rate dynamics of mixed-clone infections of P. chabaudi using an avirulent/virulent clone combination (AS (PYR) and AJ) or two clones with similar growth rate profiles (AQ and AJ). The modification of the technique described here should enable researchers to quickly extract accurate and reliable data from in-depth studies covering broad areas of interest, such as analyses of clone-specific responses to drugs, vaccines or other selection pressures in malaria or other parasite species that also contain highly polymorphic DNA sequences.
The paroxysm of Plasmodium vivax malaria.
The paroxysms of Plasmodium vivax malaria are antiparasite responses that, although distressing to the human host, almost never impart serious acute pathology. Using plasma and blood cells from P. vivax patients, the cellular and noncellular mediators of these events have been studied ex vivo. The host response during a P. vivax paroxysm was found to involve T cells, monocytes and neutrophils, and the activity, among others, of the pyrogenic cytokines tumor necrosis factor alpha and interleukin 2 in addition to granulocyte macrophage-colony stimulating factor. However, interferon gamma activity, associated with serious acute pathogenesis in other studies on malaria, was absent. Induction of the cytokines active during a P. vivax paroxysm depends upon the presence of parasite products, which are released into the plasma before the paroxysm. Chemical identification of these natural parasite products will be important for our understanding of pathogenesis and protection in malaria.
Speculations on the origins of Plasmodium vivax malaria.
It is likely that Plasmodium vivax diverged approximately 2 million years ago from a group of malaria parasites which are now endemic in monkeys and apes in southern Asia. In those times, primates were spread throughout most of Eurasia and Africa, indicating an Old World location, but nothing more precise, for the place of divergence of P. vivax. From approximately 1 million years ago, the Ice Ages would have isolated human malaria, including P. vivax, into humid temperate or warm climate refuges around the Mediterranean, in sub-Saharan Africa and in south and east Asia. As there appears to be no record of humans in south and east Asia from 100,000 to 60,000 years ago, they might not have passed on their parasites, including P. vivax, to modern humans entering the region after this time. Today, all P. vivax might be descended from parasites which infected human populations in the Mediterranean region and in sub-Saharan Africa during the last Ice Age, between 100,000 and 20,000 years ago. Evidence for the latter is provided by the presence of very high frequency RBC Duffy negativity in sub-Saharan Africa.
Spatial simulation of malaria transmission and its control by malaria transmission blocking vaccination.
A simple, visual representation of spatial aspects of malaria transmission in successive snap-shots in time, is presented. The spatial components of the simulation involve (i) the identification of mosquito vector breeding sites of defined shape and area, (ii) the identification of a zone of malaria transmission determined by the shapes and areas of the vector breeding sites and the distance from these sites that the mosquitoes disperse, (iii) a human population dispersed in relation to the malaria transmission zone, (iv) perimeters around each individual human within which his or her infection can be transmitted by the local vector mosquitoes. The intensity of transmission within a malaria transmission zone is given by a number which is the number of new cases of malaria that each existing case will distribute through the human population within the duration of an infection. The simulation has been used here to examine the effects of vaccination against malaria transmission. Different levels of vaccine coverage are represented under endemic and epidemic malaria. The consequences of full or partial coverage of a zone of malaria transmission are also examined. The results are numerically compatible with the predictions of previous simple mathematical simulations of malaria transmission and interventions. The present simulation allows the nature of malaria transmission and the effects of interventions to be communicated easily and directly to an audience. It could have practical value in discussions of malaria control strategies with health planners.
Numerous, robust genetic markers for Plasmodium chabaudi by the method of amplified fragment length polymorphism.
We have used the method of amplified fragment length polymorphism (AFLP) to identify genetic polymorphisms between two cloned isolates of the rodent malaria parasite Plasmodium chabaudi chabaudi. The method employs polymerase chain reaction (PCR)-amplification of genomic DNA fragments cut with specific combinations of restriction endonucleases; we used EcoRI and Tru1I (isoschizomer of MseI). We have identified 819 parasite clone-specific AFLPs between P. c. chabaudi clones AS and AJ. Of these, 403 fragments were specific to AS and 416 to AJ. In preparing blood stage parasites for DNA, nucleated host cells were removed by successive filtration of infected blood through powdered cellulose and Plasmodipur filters. This reduced nucleated host cell contamination to around 1-10 per million parasite nuclei and reduced host DNA to below the limit of detection by the AFLP method. Analysis of our results showed that the total number of PCR-amplified fragments of parasite DNA was consistent with the predicted number of EcoRI sites in the parasite genome. 19.4% of all amplified fragments were P. c. chabaudi clone-specific. From this figure we estimated that the diversity between clones AS and AJ, measured as the probability of a sequence difference, was between about 8 x 10(-3) and 4.6 x 10(-4) per base pair. This is consistent with the sequence diversity found between alleles of candidate drug resistance genes from P. c. chabaudi clones AS and AJ identified and sequenced in this laboratory.
Evolutionary and historical aspects of the burden of malaria.
Malaria is among the oldest of diseases. In one form or another, it has infected and affected our ancestors since long before the origin of the human line. During our recent evolution, its influence has probably been greater than that of any other infectious agent. Here we attempt to trace the forms and impacts of malaria from a distant past through historical times to the present. In the last sections, we review the current burdens of malaria across the world and discuss present-day approaches to its management. Only by following, or attempting to follow, malaria throughout its evolution and history can we understand its character and so be better prepared for our future management of this ancient ill.
The effectiveness of magnet therapy for treatment of wrist pain attributed to carpal tunnel syndrome.
We conducted a double-blind placebo-controlled randomized clinical trial in which 30 patients with pain attributed to carpal tunnel syndrome had either a 1000 gauss magnet or a placebo metal disk applied to the carpal tunnel area using a Velcro wrap for a period of 45 minutes. Pain was measured on a visual analogue scale using 0 and 10 as anchors. Presenting symptoms including numbness, tingling, burning, and pain did not differ significantly between the 2 groups. There was significant pain reduction across the 45-minute period for both groups. However, t test comparisons found no significant differences between the groups for beginning pain, pain at 15 minutes, pain at 30 minutes, or pain at 45 minutes. The use of a magnet for reducing pain attributed to carpal tunnel syndrome was no more effective than use of the placebo device.
Legal considerations of compulsory sickle cell screening.
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Ombudsman's eighth report.
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