Rocketboom: Fighting Malaria in Kenya

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BILBO and the trypanosomes

Trypanosoma brucei is a pathogen which causes epidemics of human and animal sleeping sickness in central and Southern Africa, a fatal disease if untreated. A new paper published this week in PLoS Biology investigates the formation of a trypanosome structure called the Flagellar Pocket (FP). The paper describes a newly identified protein called BILBO1 that is crucial for Flagellar Pocket formation. Experimental inhibition of the protein BILBO1 is fatal to Trypanosoma brucei. The Flagellar Pocket is a part of the single-celled pathogen that has several functions. As well as being the site where the flagellum attaches the flagellum being a structure that enables the bug to propel itself the FP is the site of endo- and exocytosis, processes that transport material in and out of the pathogen from the outside world. The FP also plays an important role in allowing the trypanosome to avoid the immune system of the host. The FP permits the trypanosomes surface proteins (the structures seen by the immune system) to be changed, so that the bug can remain camouflaged from the host’s defences.

The Flagellar Pocket is a multi-tasking organelle but we can now consider it as the parasite’s Achilles heel. How the trypanosome forms this important structure has previously been unknown. The new paper shows that BILBO1 is crucial for the formation of the FP, specifically, it is an important component of a part of the FP called the Flagellar Pocket Collar. BILBO1 is a cytoskeletal protein (the cytoskeleton being a network of proteins that provide internal structure for a cell), thus BILBO1 is involved in creating the cytoskeletal framework that supports the FP. This paper describes the effect of preventing BILBO1 expression in the trypanosome. Without BILBO1, the trypanosome is unable to create a new FP; with the FP disrupted, it is unable to regulate endo- and exocytosis, therefore preventing it from taking up nutrients. These factors prove fatal to the trypanosome. Therefore, this work provides a target for anti-sleeping sickness drugs: knock out BILBO1 function and you will kill the trypanosome.

Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated. 2008 PLoS Biol 6(5): e105

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Guinea Worm Disease

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Humans are hosts to nearly 300 species of parasitic worms and over 70 species of protozoa, some derived from our primate ancestors and some acquired from the animals we have domesticated or come into contact with during our history (History of human parasitology. Clin Microbiol Rev 2002 15: 595-612). The best-documented parasitic disease known from ancient times is caused by the nematode worm Dracunculus medinensis. The earliest description is from an Egyptian papyrus from 1500 BC that refers to both the nature of the infection and to techniques for removing the worm. Confirmation of the presence of this worm in ancient Egypt comes from the finding of a well-preserved worms in Egyptian mummies. Dracunculiasis, or Guinea worm disease, is one of the few diseases unambiguously described in the Bible, and most parasitologists accept that the “fiery serpents” that struck down the Israelites in the region of the Red Sea after the Exodus from Egypt somewhere between 1250 to 1200 BC were actually Guinea worms.

The adult worms live in the subcutaneous connective tissues of their victims, from which the females emerge to release thousands of larvae into water, where they are taken up by intermediate hosts, tiny aquatic crustaceans called Cyclops. In these hosts they mature into infectious larvae that infect humans when the crustaceans are accidentally swallowed in contaminated drinking water. On maturity, the large female worm, up to nearly a metre in length, protrudes from the skin, usually of the leg, and causes intense inflammation and irritation. The effects of the disease are crippling. Its victims develop large ulcers, usually in the lower leg. The ulcers swell, sometimes to the size of a tennis ball, and burst, releasing the spaghetti-like parasitic worm. Victims experience a pain so excruciating that they say it feels as if their leg is on fire. The searing pain compels people to jump into water, often the community’s only source of drinking water, to relieve the pain. When the infected person immerses his or her leg in the water, the worm in the leg releases thousands of larvae. The larvae are then ingested by Cyclops that live in the water. Thus the cycle begins again - when people drink the water, they are in effect drinking in the disease.

The most common way to treat Guinea worm disease involves wrapping the worm around a stick. This treatment has been employed for millennia and may have inspired the Rod of Asclepius which historically has symbolized the medical profession. As the adult worm begins to emerge from the patient’s skin, it is wound around a stick, then further extracted by a few centimeters per day. This slow process can take days or even weeks, but it is required to avoid breakage and leaving behind a portion of the worm. Leaving a portion of the dead worm remain within the host’s body increases the risk of infection, and can trigger immune responses resulting in pain and swelling. In many countries, a broken worm is immediately removed surgically, or the worm can be excised surgically from the very beginning if health care facilities are available. Antihelminthic drugs such as metronidazole or thiabendazole are sometimes used in conjunction with physical extraction. However, one study found that antihelminthic therapy was associated with aberrant migration of worms, resulting in infection in areas other than the lower extremity.

Dracunculiasis is a classic example of a neglected tropical disease, a symptom of poverty and disadvantage. Those most affected are the poorest populations often living in remote, rural areas, urban slums or in conflict zones. With little political voice, neglected tropical diseases have a low profile and status in public health priorities. In 1997 the World Health Assembly pledged to completely eradicate Guinea worm disease. This is no small task, but there are several factors which make eradication a possibility. Dracunculiasis is the first parasitic disease targeted for eradication because:

• Diagnosis is easy and unambiguous (presence of an emerging adult worm).
• The transmission agent, Cyclops, is not a mobile vector as is a mosquito.
• The incubation period in both Cyclops and humans is of limited duration.
• Interventions are effective, low cost, and relatively simple to implement.
• The disease has a limited geographic distribution and is seasonal in nature.
• Success in eliminating the disease has been demonstrated in several countries in Asia and the Middle East.
• There is no known animal reservoir.

Is Dracunculiasis eradication close? In 2007 the WHO announced that Guinea worm disease now affects around 25,000 people in nine countries, compared with an estimated 3 million people were infected in over 20 countries in the early 1980s. Twelve countries were declared Guinea worm-free in early March. If progress continues at this rate, the disease could be eradicated in less than two years. It is probable that complete eradication will take quite a few years yet, although it should be possible to eliminate the disease from seven countries in a couple of years, leaving only two endemic countries, Sudan and Ghana (Dracunculiasis eradication by 2009: will endemic countries meet the target? Tropical Medicine & International Health 2007 12: 1403-1408). One lesson to be drawn from the problems of local ownership and the experience of cash rewards is that there are dangers in throwing money at the problem. While the eradication initiative badly needs additional resources, it needs them at such a level and managed in such a way that they do not distort the priorities of the health care system, or exceed the capacity of local staff to manage them. The amounts needed are not large, but their continuity and flexibility is important. Given the highly seasonal transmission of dracunculiasis, the resources must be available at very specific times of the year, which is not always achieved. In spite of the difficulties, complete worldwide eradication of this ancient disease is drawing nearer.

New Drugs for Nasty Nematodes

The nematodes, or roundworms, comprise a large number of pathogens of man and domestic animals. Gastrointestinal nematodes, such as the blood-sucking Haemonchus contortus, are major parasites of ruminants that cause substantial economic losses to livestock production worldwide. In the absence of vaccines for gastrointestinal nematodes, control of infections relies mainly on chemotherapy. Drug resistance in human and animal pathogenic helminths has been spreading in prevalence and severity to a point where multidrug resistance against the three major classes of anthelmintics - the benzimidazoles, imidazothiazoles and macrocyclic lactones - has become a global phenomenon in gastrointestinal nematodes of farm animals. Hence, there is an urgent need for an anthelmintic with a new mode of action. This paper report the discovery of the amino-acetonitrile derivatives (AADs) as a new chemical class of synthetic anthelmintics and describes the development of drug candidates that are effective against various species of livestock-pathogenic nematodes. These drug candidates seem to have a novel mode of action involving a unique, nematode-specific clade of acetylcholine receptor subunits. The AADs are well tolerated and of low toxicity to mammals, and overcome existing resistances to the currently available anthelmintics.
These optimized AAD compounds meet the following requirements for an urgently needed new anthelmintic for livestock: low toxicity, favourable pharmacokinetic properties and broad-spectrum efficacy against sheep and cattle nematodes. Moreover, this efficacy includes multidrug-resistant parasites owing to a presumed activation of signalling. However, nematodes will ultimately develop resistance to any new drug, including the AADs. To secure the maximum lifespan of the AADs as well as the current anthelmintic drugs, monitoring of drug resistance and rational exploration of combinations with current or future drugs will be necessary. If the excellent tolerability of the AADs in ruminants can be proven for humans, the class may offer an alternative anthelmintic for human medical practice.

A new class of anthelmintics effective against drug-resistant nematodes
Nature 2008 452: 176-180

Malaria, mosquitoes and the legacy of Ronald Ross

Malaria is a vector-borne infectious disease caused by protozoan parasites of the genus Plasmodium. It is widespread in tropical and subtropical regions, including parts of the Americas, Asia, and Africa. Each year, it infects approximately 515 million people and kills between one and three million people, the majority young children in Sub-Saharan Africa.

Malaria is thought to have infected humans for over 50,000 years, and may have been a human pathogen for the entire history of our species. Close relatives of the human malaria parasite are common in chimpanzees. References to the unique recurring fever of malaria are found throughout recorded history, the earliest from China in 2700 BC. The term malaria originates from the Medieval Italian: mala aria meaning “bad air”, and the disease was also formerly called ague or marsh fever due to its association with swamps, the home of the mosquitos which transmit the parasite.

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In 1880, a French doctor Charles Laveran was the first to observe parasites inside the red blood cells of people suffering from malaria. This was the the first time that protozoan was identified as causing a disease. In 1908, Carlos Finlay, a Cuban doctor treating patients with yellow fever in Cuba, first suggested that mosquitoes were transmitting the disease to and from humans. However, Sir Ronald Ross, working in India, finally proved that malaria is transmitted by mosquitoes in 1898. He did this by showing that certain mosquito species transmit malaria to birds and by isolating malaria parasites from the salivary glands of mosquitoes that had fed on infected birds:

For the last two years I have been endeavouring to cultivate the parasite of malaria in the mosquito. The method adopted has been to feed mosquitos, bred in bottles from the larva, on patients having crescents in the blood, and then to examine their tissues for parasites similar to the haemamoeba in man. The study is a difficult one, as there is no a priori indication of what the derived parasite will be like precisely, nor in what particular species of insect the experiment will be successful, while the investigation requires a thorough knowledge of the minute anatomy of the mosquito. Hitherto the species employed have been mostly brindled and grey varieties of the insect; but though I have been able to find no fewer than six new parasite of the mosquito, namely a nematode, a fungus, a gregarine, a sarcosporidium, a coccidium, and certain swarm spores in the stomach, besides one or two doubtfully parasitic forms, I have not yet succeeded in tracing any parasite to the ingestion of malarial blood, nor in observing special protozoa in the evacuations due to such digestion.

Apart from combating malaria, what else do Ross’s experiments teach us?

How about the value of persistence? Ross records that before the reported successful experiment, work in the preceding two years involving the examination of approximately a thousand mosquitoes had failed to reveal any parasites. It also shows the benefit of sharing data before publication so as to put forward possibly conflicting interpretations of the results. Today, many a journal editor may have rejected such a speculative, uncontrolled and unreplicated study as Ross’s original paper. And if they had, we might still be waiting to discover the infectious agent responsible for malaria.

Malaria, mosquitoes and the legacy of Ronald Ross.
Bull World Health Organ. 2007 85: 894-896

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New target for anti-malaria drugs

Malaria parasites must invade the erythrocytes of the host to be able to grow and multiply. Having depleted the host cell of its nutrients, the parasites break out to invade new erythrocytes. Researchers have discovered that a new organelle, the exoneme, which contains a protease SUB1, helps the parasite to escape from old erythrocytes and invade new ones. By scanning thousands of compounds, the researchers also found a plant-derived molecule that was able to block the SUB1 enzyme preventing the merozoites from escaping.

Subcellular Discharge of a Serine Protease Mediates Release of Invasive Malaria Parasites from Host Erythrocytes
Cell 2007 131: 1072-1083
The most virulent form of malaria is caused by waves of replication of blood stages of the protozoan pathogen Plasmodium falciparum. The parasite divides within an intraerythrocytic parasitophorous vacuole until rupture of the vacuole and host-cell membranes releases merozoites that invade fresh erythrocytes to repeat the cycle. Despite the importance of merozoite egress for disease progression, none of the molecular factors involved are known. Just prior to egress, an essential serine protease called PfSUB1 is discharged from previously unrecognized parasite organelles (termed exonemes) into the parasitophorous vacuole space. There, PfSUB1 mediates the proteolytic maturation of at least two essential members of another enzyme family called SERA. Pharmacological blockade of PfSUB1 inhibits egress and ablates the invasive capacity of released merozoites. Our findings reveal the presence in the malarial parasitophorous vacuole of a regulated, PfSUB1-mediated proteolytic processing event required for release of viable parasites from the host erythrocyte.

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Toxoplasma gondii and heart transplant recipients

A quarter of the world population is infected with Toxoplasma gondii, a long-lived and common brain parasite. The normal host for this organism is the domestic cat, so maybe its not so surprising that human infections are also common. New research points to the potential impact of this normally slient pathogen:

Abstract: Chronic Toxoplasma gondii infection is known to trigger potentially adverse immunoregulatory changes, but the long-term implication for heart transplant recipients has not been assessed previously. We evaluated the risk of mortality, development of cardiac allograft vasculopathy (CAV), and acute cellular rejection among T. gondii-seropositive heart transplant (HTx) recipients and the 4 donor/recipient seropairing groups. Frozen pre-HTx serum samples of 288 recipients and 246 donors were evaluated for T. gondii serostatus. Patients had undergone prospective serotesting using alternative assays, and results determined by the 2 methods were compared. Overall, 211 recipients (73%) were seronegative and 77 (27%) were seropositive. In total, 82 recipients died, 76 developed CAV, and 82 had 1 or more episode of treated cellular rejection. Recipient seropositivity was associated with a significantly higher risk of mortality and CAV. Seropositivity did not influence the number of rejection episodes, and donor/recipient seropairing was not a risk factor for any end point.

Pre-Transplant Toxoplasma gondii Seropositivity Among Heart Transplant Recipients Is Associated With an Increased Risk of All-Cause and Cardiac Mortality. J Am Coll Cardiol. 2007 50: 1967-1972

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Blood group O protects against severe malaria

Malaria has been a major selective force on the human population, and several erythrocyte polymorphisms have evolved that confer resistance to severe malaria. Plasmodium falciparum rosetting, a parasite virulence phenotype associated with severe malaria, is reduced in blood group O erythrocytes compared with groups A, B, and AB, but the contribution of the ABO blood group system to protection against severe malaria has received little attention. We hypothesized that blood group O may confer resistance to severe falciparum malaria through the mechanism of reduced rosetting. In a matched case-control study of 567 Malian children, we found that group O was present in only 21% of severe malaria cases compared with 44-45% of uncomplicated malaria controls and healthy controls. Group O was associated with a 66% reduction in the odds of developing severe malaria compared with the non-O blood groups. In the same sample set, P. falciparum rosetting was reduced in parasite isolates from group O children compared with isolates from the non-O blood groups. Statistical analysis indicated a significant interaction between host ABO blood group and parasite rosette frequency that supports the hypothesis that the protective effect of group O operates through the mechanism of reduced P. falciparum rosetting. This work provides insights into malaria pathogenesis and suggests that the selective pressure imposed by malaria may contribute to the variable global distribution of ABO blood groups in the human population.

Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting. PNAS 2007 104: 17471-17476

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Trypanosomes and immune evasion

African trypanosomes cause sleeping sickness, a fatal disease of humans and livestock in Africa. During their life cycle, these protozoan parasites cycle between the bloodstream of mammals and tsetse flies. Their two main developmental stages are the bloodstream form and the procyclic form in the tsetse fly. Bloodstream trypanosomes thwart their host’s immune response by periodically switching their major surface protein, the variant surface glycoprotein (VSG). When bloodstream trypanosomes are ingested by a tsetse fly, they must quickly shed the VSG and replace it with an unrelated invariant protein more suited to their survival as procyclic organisms in the fly midgut. This paper examines the mechanisms used by trypanosomes to remove the VSG during their differentiation from bloodstream to procyclic form in culture. Scientists deleted the genes for one of the trypanosome’s protease enzymes from the trypanosome genome and found that bloodstream trypanosomes could still differentiate to the procyclic form, but VSG removal was diminished. Deleting the genes for a phospholipase enzyme had a similar effect - they could still differentiate but VSG removal was impaired. When the genes for both the protease and the phospholipase were deleted, bloodstream trypanosomes could not differentiate to the procyclic form, they retained about 60% of the VSG on their surface, and they died in the differentiation medium. These results highlight the synergistic roles of these two enzymes in the differentiation process.

A Function for a Specific Zinc Metalloprotease of African Trypanosomes
PLoS Pathogens 2007 3 (10) e150

Tough Choices - DDT or Malaria?

The organochlorine compound DDT (Dichloro-Diphenyl-Trichloroethane) was first synthesized in 1874, but its insecticidal properties were not discovered until 1939 by the Swiss scientist Paul Muller, who was awarded the 1948 Nobel Prize in Physiology and Medicine for his efforts. DDT kills by opening sodium ion channels in insect neurons, causing the neuron to fire spontaneously. This leads to spasms and eventual death. Insects with mutations in their sodium channel gene or with up-regulation of genes expressing cytochrome P450 may become resistant to DDT and similar insecticides.

In the early years of World War II DDT was used with great effect to combat mosquitoes spreading malaria, typhus, and other insect-borne human diseases among both military and civilian populations. After the war, DDT was made available as an agricultural insecticide, and its production and use skyrocketed.

In 1955 the World Health Organization began a program to eradicate malaria worldwide, relying largely on DDT. Though this effort was initially highly successful (reducing mortality rates from 192 per 100,000 to a low of 7 per 100,000), resistance soon emerged in many insect populations as a consequence of the widespread agricultural use of DDT. In the 1960s, the environmental impacts of indiscriminate spraying of DDT became known. As a persistent organic pollutant, DDT accumulated in the food chain and had severe effects on fish, amphibians, birds, and rather less well known impacts on mammals, including humans. DDT can still be found in the fat reserves of polar bears, penguins, and possibly you, thousands of miles away from where it was ever sprayed. In 1987 the US EPA classified DDT as a probable human carcinogen. DDT is also known to be an endocrine disruptor and to cause developmental problems in infants.

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In the 1970s and 1980s, agricultural use of DDT was banned in most developed countries, in 1970 in Scandinavia, 1972 in the USA, but not until 1984 in the UK. The Stockholm Convention which came into effect in 2004 outlawed several persistent organic pollutants, and restricted the use of DDT to the control of insect vectors of human diseases. After these bans, the populations of many severely threatened species, such as the American bald eagle, rebounded.

In September 2006, the World Health Organization announced that DDT will be used as one of the three main tools against malaria, and recommended indoor spraying in epidemic areas and places with high malaria transmission. USAID now funds the use of DDT overseas. DDT sprayed inside a home provides protection from mosquitoes for up to six months. New studies show that despite mosquito resistance to DDT, it also acts as a powerful insect repellent.

Malaria afflicts between 300 million and 500 million people each year. The World Health Organization estimates that around 1 million people die of malaria and malaria-related illness every year, with 90% of these deaths in Africa, mostly in children under the age of five. To put that in perspective, that is equivalent to the death toll of around ten of the nuclear bombs dropped on Hiroshima during World War II. Malaria also weakens the economies of poor countries. People who become infected cannot work or die. Infected children can suffer brain damage. The World Bank estimates that malaria costs Africa more than US$100 billion annually and this cost is growing by 1.3 per cent each year. In 2004, when Uganda publicly contemplated reintroducing DDT to fight malaria, the European Union made threats that the country’s US$32 billion agriculture exports could be at risk if tough new measures were not taken to ensure DDT residues did not find their way into food crops.

As a result of the WHO program, the number of African countries spraying DDT inside houses has exploded. Eritrea, Madagascar, Ethiopia, Swaziland, Senegal, Ghana, Angola, South Africa, Mauritius, Mozambique, Zimbabwe, Namibia, Zambia and Burkina Faso are all using the chemical. Uganda, where more than 100,000 people died from malaria in 2006, began spraying it this year in a pilot project, and Tanzania and Malawi may follow. But Rwanda, Burundi and Kenya (a major producer of pyrethrum, the main alternative to DDT) are so far refusing to adopt the use of the chemical. In 1995, South Africa stopped spraying DDT to control malaria, citing international pressures, but as soon as the ban started, the incidence of malaria rose.

DDT is cheap. Safer pyrethrum-based insecticides are 20 times more costly, often too expensive for developing countries. The price of controlling malaria in Africa has been estimated at US$1 billion per year, but foreign aid targeting the disease has never topped US$200 million.

So my question to you is this: imagine you are the president of the world, but with a limited budget. What would you do?

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