Malaria is a disease that has killed more humans than any other during our history; it still infects some 500 million people each year, killing about 2 million. Although a bite from a female Anopheles mosquito is central to the transmission of malaria, it is in fact a Plasmodium pathogen harboured within the mosquito that causes the disease. There are five distinct species of Plasmodium known to cause malaria – of these P. falciparum is the most significant both in terms of quantity and severity of infection.
Having entered the body via a bite, the pathogen migrates in the bloodstream to the liver. From here it will re-enter the blood stream and colonise red blood cells. When sufficient multiplication of the pathogen has occurred, the blood cells will burst, releasing more pathogens into the blood. The bursting occurs on a 48-72 hour cycle and is accompanied by peaks in fever and other symptoms at these times.
Persistent and repeated damage to the red blood cells leads to a shortage of erthyocytes and associated symptoms of anaemia. Rarely, P. falciparum can cause blockage of blood vessels supplying the brain, resulting in insufficient supply of oxygen, leading to brain damage, seizure and/or coma (all data taken from the NHS Choices website).
New evidence undermines traditional view on the origins of P. falciparum
In a September 2010 paper in the journal Nature, Beatrice Hahn and colleagues investigated the evolutionary origin of P. falciparum (Liu et al; doi:10.1038/nature09442). Prior to publication of this research, it was believed that P. falciparum infecting humans had evolved from P. reichenowi in chimpanzees. Until recently this had also been considered as a classic case of “host-parasite co-divergence”, i.e. that when the human line had diverged from the chimpanzee line – between five and seven million years ago – they were both already infected with the same ancestral Plasmodium species which had subsequently evolved apart at the same time that the hosts had evolved. However, three studies already published in the previous 18 months had questioned this chronology (whilst still believing that chimpanzees were the reservoir from which the ancestral version of P. falciparum had passed to humans).
In the new research, Liu et al analysed about 2700 faecal samples from four apes; western and eastern gorillas, chimpanzees and bonobos. The majority of this material had been collected from 57 locations in central Africa for an earlier study on the molecular epidemiology of simian immunodeficiency virus.
The study revealed high infection rates of Plasmodium species, up to 48%, in chimpanzees and western gorillas (implying that these species are better able than humans to tolerate Plasmodium infection). No trace of Plasmodium was found in bonobos or eastern gorillas, though it remains to be seen whether this is genuinely a complete absence (which would be interesting in its own right), or reflect levels of infection below those detectable in the current research.
The major finding of the study, however, was a refutation of the a priori belief that P. reichenowi was the closest relative of P. falciparum. P. falciparum, it turns out, is most closely related to a previously undiscovered species of Plasmodium in western gorillas, not chimpanzees (nor bonobos and eastern gorillas).
What made this study different?
Why was it that previous studies had failed to see the connection to western gorillas? According to Hahn, a major explanation is the fact that other research (possibly steered inappropriately by knowledge of HIV transmission from chimpanzees) simply hadn’t looked for a connection to gorillas. Faced with the difficulty of collecting blood from wild apes, sample sizes tend to be small and/or involved captive apes. The latter point might introduce confusion into the epidemiology since close contact for a number of years might have allowed a pathogen to pass from human to ape rather than vice versa.
By collecting faecal samples, Liu et al were able to significantly enhance the number and variety of apes in their study. They also applied an important technological improvement over previous studies by diluting the samples of DNA extracted from the faeces prior to PCR amplification. In the absence of this step, DNA sequences can originate from more than one species of Plasmodium simultaneously infect the same individual ape. In consequence the sequencing data can be muddled, making it hard to determine exactly which species are present in each sample and therefore tricky to establish the order of transmission of Plasmodium between host organisms.
In order to skew the samples towards each sequencing reaction having only one genome present, the researchers chose dilution ratios such that over 70% of the tubes actually had no pathogen DNA. Although this seems wasteful, it was necessary to ensure that most of the tubes that did have Plasmodium DNA had only one set. Despite this safeguard, statistical variation also meant that some tubes still ended up containing DNA from two or more species. Where this could be confirmed, these samples were excluded from the analysis. Repeated PCR analysis of the same faecal samples revealed that many contained several different species.
The principal PCR analysis to identify species was actually carried out using the cytb gene, part of the mitochondrial DNA. Since an individual primate might be responsible for more than one faecal sample, the research team also conducted microsatellite analysis of genomic DNA to identify cases of redundant sampling and hence give a more accurate picture of the total number of organisms represented (this analysis was only conducted at a subset of the field trial sites).
Contrary to earlier beliefs, none of the cytb sequences in Plasmodium taken from chimpanzees were closely related to P. falciparum affecting humans. Additionally, there is good reason to believe that the four examples of P. falciparum identified in captive bonobos, and reported in an earlier publication (Krief et al, 2010), had in fact passed from human to ape. The main evidence for this is the identification of antimicrobial resistance mutations within the parasite genome.
The study appears to be the death-knell for a simplistic “host-parasite co-divergence” model regarding Plasmodium evolution. The current study did not isolate pathogens from any human subjects, however all P. falciparum sequences published to date form a tight grouping within the ‘G1’ gorilla clade of Plasmodium sequences and appear to represent a single lineage. The absence of a suitable ‘molecular clock’ (of the kind used to calibrate evolution of viral species) makes determination of the chronology of malaria’s development more difficult, however, this phylogenetic tree may mean that cross-species transmission to humans has occurred on only one historic occasion.
On the whole the coverage of this story in the general press was actually handled pretty well. There is no justification, however, for the Daily Mail’s claim that the new research offers any specific advance in the development of a vaccine against malaria. Indeed, Edward Holmes (in a Nature News and Views article accompanying the September 2010 research) is sceptical about any claims to medical significance associated with this kind of survey of biodiversity.
In commenting on the news coverage of this research, the NHS Choices Behind the Headlines site notes that prevention of infection remains the most significant strategy for combating malaria.
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