CBC Ideas - Worthy Parasites

Have you ever found a tiny parasitic pea crab living
inside your oyster? Some folks like to eat these too.
Image: NOAA

My CBC Ideas documentary, Worthy Parasites: A Villain's Silver Lining, will air for the first time on Jan 8, 2013, at 9 PM.

Regular listeners of CBCs Ideas, hosted by Paul Kennedy, know that the program can explore virtually any topic in science, history, the arts, culture, religion or anything else you can think of. Interesting, provocative, and contemporary, it's been on the air for decades and it's always worth a listen.

That's why I'm thrilled and honored to be the contributor of an episode that explores how species almost everyone loathes - parasites - are actually beneficial in many ways, even essential in our world. If you think parasites have no worth, think again, and listen to my one-hour episode in January.

CBC Ideas, Worthy Parasites: A Villain's Silver Lining is available on CBC Radio One across Canada: Tuesday, January 8 at 9 p.m. (9:30 p.m. NT) and Monday, January 28 at 2 p.m. (2:30 p.m. NT). It will also be live streamed and streamed on demand on the CBC website, and available as a podcast.

You'll be surprised by all the good things parasites do for you.

Ivermectin for Bedbugs

Would you take a drug to make yourself poisonous to mosquitoes, or black flies, or wasps? How about taking Ivermectin for bedbugs? I'm not so sure about this - the most obvious problem is that one would have to be bitten before it could work!

Ivermectin for Bedbug Infestation

 

Bedbug bites can be very uncomfortable.
Taking Ivermectin for bed bugs would
only work if every bug bit at least
once more!

A recent article on Bloomberg.com suggested that giving people oral Ivermectin for bedbugs might be an effective way of dealing with a bedbug infestation. A very small study (three people) found that most bedbugs died if they fed on someone within a day of a dose of Ivermectin, and that 54 hours after the dose, 42% of bugs died after feeding.

Treating people who aren’t sick with drugs has precedent: it’s common for people traveling in places where mosquitoes carry malaria, for example, to take an anti-malarial drug to avoid infection. But while malaria can easily kill you, bedbugs have never been shown to transmit disease to humans.

Ivermectin is an antiparasitic and obviously an insecticide. Like all drugs, it comes with a risk of side effects, some of them quite serious. Would it really make sense to expose large numbers of people – people who aren’t infected with anything - to this drug? It seems to me that treating a dwelling with insecticides is one thing – sometimes not a very good thing – but turning people into insecticide laden bug traps is another.

Bedbug Feeding Habits

Would it even work? Past research has indicated that bedbugs don’t feed every day. A 2009 study indicated that they might feed every two to three days and that they might synchronize their feeding (in other words, the bugs in a colony all tend to feed at the same time). Ivermectin is typically given as a single dose; how would we determine when it’s feeding day for the bedbugs? If the first twenty-four hours is crucial, you’d want to make sure you took the drug on the right day. And what if some survived or didn’t feed that day? And you’d need 100% participation from people staying in the dwelling. Imagine trying to do this in an apartment building. How many doses of Ivermectin would it take?

Finally, I suspect resistance would arise fairly quickly. If 42% of bugs died after feeding at the 54 hour mark, that means 58% survived – and they’d all been exposed to the drug. If their survival was due to them having more natural resistance than the other bugs, and they passed that along to subsequent generations, we’d see more and more resistance.

Before very long, the days of using Ivermectin for bedbugs would be over.

 

Sources

Gale, J. (2012) “Bed Bugs Dying After Merck Drug Suggests Possible Weapon.” Bloomberg.com

Reinhardt, K., Isaac, D. and Naylor, R. (2010), Estimating the feeding rate of the bedbug Cimex lectularius in an infested room: an inexpensive method and a case study. Medical and Veterinary Entomology, 24: 46–54. doi: 10.1111/j.1365-2915.2009.00847.x

Thoughts on Naegleria fowleri, "Brain Eating Amoeba"

Naegleria fowleri: a protist that can be a cyst, an amoeba squelching along, or a whirling swimming flagellate; an organism found all over the world that loves warm water, a free living organism that can adopt a parasitic lifestyle; an organism that will almost certainly kill you if it gets into your brain. Beautiful. Fascinating. Deadly.

Naegleria fowleri takes various forms. When it invades a human central
nervous system, it is found as an amoeba or a flagellate.
 Image CDC Image library.

Annual Deaths Due to Naegleria fowleri

Every year during the sweltering days of summer we hear of deaths caused by the “brain eating amoeba.” This year a man died after teaching his daughter how to swim in an Indiana lake, and several children in other American states died after swimming in warm fresh water. Children have died after playing in bath water at home, and the use of neti pots to rinse the sinuses, or ritual inhalation of water into the sinuses, has resulted in deaths as well. The disease is called primary amoebic meningoencephalitis, or PAM.

How Does Naegleria flowleri Infect People?

Naegleria fowleri is just one of more than 20 Naegleria species found in the environment, but to date it is the only one found in human cases of PAM. What’s so special about N. fowleri? Perhaps it has something to do with N. fowleri being a thermophile – in other words it loves warmth. It can survive at temperatures as high as 45ºC, which would make it very comfortable at a normal human body temperature, and impervious to the highest fever. But many of the other species like high temperatures as well, so that’s not the whole answer.

Perhaps it’s important that N. fowleri adapts easily to axenic conditions – meaning that it doesn’t need a community of other organisms around to be happy; it can thrive all by itself. This does make it stand out from the other species, but living inside another organism isn’t exactly axenic, and strains of N. fowleri grown axenically in the lab lose their ability to produce disease. How this characteristic might help it invade the brain in the first place, then, and thrive there, is a tantalizing question – at least to me.

Studies have shown that N. fowleri isolated in the environment contain food vacuoles full of bacteria, whereas those isolated from cases of PAM contain vacuoles full of cell debris. So, when the organism is parasitic, it uses host cells as a food source instead of bacteria. It produces an enzyme that enables it to do this (Chang). This is clearly important, but do we know whether other Naegleria species produce a similar enzyme?

Hot Weather Means Water Sports and Naegleria fowleri

Perhaps it’s a combination of all these factors, and possibly others, that make N. fowleri uniquely equipped to be a “brain eating amoeba.” The question remains to be answered. What’s easier to understand is why it’s so rare, and yet so predictable. In order for N. Fowleri to get into a human brain, very warm water containing the organism must be inhaled into the nasal sinuses. This event is relatively uncommon, but can be expected to happen in the summer months when people – particularly young people – play in the water to cool off.

Sources

Chang SL. “Pathogenisis of Pathogenic Naegleria amoeba.” Folia Parasitol (Praha), 1979; (26)3:195-200.

De Jonckheere JF. “A Century of Reasearch on the Amoeboflagellate Genus Naegleria.” Acta Protozool, 2002; 41: 309-342.

MOLT: An Internet Game for Diagnosing Malaria

Twenty-four small images of red blood cells appear on the screen. Your job is to click on any that have a malarial parasite inside, removing the image. When you’ve removed all the infected cells, click on “Label all Negative” and another twenty-four cells appear. At the end, you’ll get a score and some information about how many correct choices you made.

You Can Help Diagnose Malaria

Blood films are used to diagnose malaria. The species of Plasmodium
present in the blood can be determined based on the appearance of the
parasites inside red blood cells.
Image: CDC - Public Health Image Library (PHIL) #5942

The game is called MOLT, and it was designed by the Ozcan Research Group at UCLA. Anyone can register and play. The idea is that anyone can be given some basic information about what malarial parasites look like in red blood cells and then be part of an accurate means of correctly diagnosing the disease without having to rely on experts in the field. This would be a huge improvement for malaria diagnosis in parts of the world where malaria kills millions each year and people skilled in diagnosis are rare.

A pilot study of the game using 20 gamers produced results that were within 1.25% of the accuracy of actual experts adept at recognizing malaria, which is pretty impressive. One can imagine an arrangement where someone puts a blood film on a microscope somewhere in Asia or Africa, the images are sent out electronically to potentially millions of gamers around the world, and the answer comes back, positive or negative, in a very short time. If the pilot is any indication, the answer would agree, most of the time, with what an expert would have said.

Crowd-sourcing Games Can Diagnose Malaria and Other Diseases

This has implications for lots of other things that are done by microscopy or other types of imagery: pap smears, fecal smears for parasites, pathology slides etc. It could be improved upon by adding automated scanning techniques and actual experts to the crowd of gamers. These things, plus a larger number of gamers would likely be even more accurate than the gamers used in the pilot. It’s exciting.

I’ve played the game – a number of times. I have lots of experience with reading blood films for malaria, and my biggest issue with the game is that the resolution – the sharpness – of the images is often not good enough

for me to feel completely comfortable with my choices. Platelets sitting on top of red blood cells can look like a parasite. So can debris on the slide. A red cell that’s damaged, or crunched up against another cell, or too darkly stained, or abnormal in some way, etc. etc., doesn’t look like it should to begin with.

I always want to look around a bit, see what the rest of the slide looks like, look for those particular features of a malarial parasite that leave no doubt. In other words, I have a very difficult time deciding whether something is positive or negative on the basis of only one cell (unless the resolution is very good).

My other complaint is with the scoring. I find it ambiguous. When they say “Correct Positive Diagnosis 91%” does that mean 91% of the cells marked as positive were actually positive (false positives), or 91% of positive cases were identified (false negatives). For anyone trying to improve at the game, clarification on this is important.

Play MOLT on BiioGames

Of course I understand that the point is that people who are not experts, and not demanding in terms of excellent microscope optics and parasite features, can still get the right answer if there are enough people providing input. From that perspective, I think the game is brilliant, and I hope it changes the world.

Play the game on Biogames

Read the paper:

Mavandadi S, Dimitrov S, Feng S, Yu F, Sikora U, et al. (2012) Distributed Medical Image Analysis and Diagnosis through Crowd-Sourced Games: A Malaria Case Study. PLoS ONE 7(5): e37245. doi:10.1371/journal.pone.0037245

Do Mosquitoes Transmit Lyme Disease?

While it takes a blood meal, a mosquitoe might
transmit a disease-causing organism. Thankfully,
mosquitoes are not known to transmit Lyme disease.
 Image: US Department of Agriculture

We’ve known for years that Lyme disease is transmitted to humans by ticks. In Europe, it’s usually I. ricinus, the sheep tick, and any of a group of closely related organisms: Borrelia burgdorferi, B. garinii, or B. afzelii; while in my area it’s the deer tick, Ixodes scapularis, and B. burgdorferi. This is enough to worry about as the woods are full of deer and the deer are full of ticks. In some areas more than 30% of deer ticks carry Borrelia, and the ticks are not fussy: they’ll jump onto deer, dogs, cats, and people without hesitation. One hates to think that other biting arthropods could also be transmitting Lyme.

But studies going back as far as the 1980s, and perhaps even farther have found Borrelia in the guts of mosquitoes. Websites devoted to Lyme disease state that mosquitoes are transmitting the disease to humans. Why, then, does the CDC website say “There is no credible evidence that Lyme disease can be transmitted… from the bites of mosquitoes, flies, fleas, or lice” (Lyme Disease Transmission)?

Lyme Disease Organisms (Borrelia) in Ticks

It’s not a matter of a mosquito or tick sucking Borrelia out of one host and then simply injecting it into another like a flying (or crawling) syringe, not like pouring liquid from one glass to another with no change in the contents. Here we are dealing with interactions between living things. Research has shown that things happen in the tick, things that are important in transmission.

In the tick’s gut, Borrelia produces a protein that enables it to persist there for long periods of time, likely aiding survival until the tick feeds again. When the tick is feeding, the spirochete cuts back on this protein and produces a different one instead, one that enables it to invade the tick’s salivary gland and then be transmitted to the new host in the tick’s saliva.

Similarly, Borrelia is able to enhance a tick protein that protects both tick and spirochete from attack by the host immune system: “Borrelia burgdorferi, the Lyme disease agent, is critically dependent on the presence of the tick protein Salp15 when infecting the host” (Schwalie and Schultz). The extended time that a tick spends feeding (days) provides plenty of time for this interaction to take place.

Lyme Disease Organisms (Borrelia) in Mosquitoes

In contrast, while Borrelia has been detected in mosquito guts and saliva, it doesn’t appear to survive there very long, probably because the proteins that support it in ticks don’t work in mosquitoes. Salp15, too, is a tick protein that won’t be available to help out in a mosquito, and mosquitoes take only minutes to obtain a blood meal, compared to days for a tick. Put simply, mosquitoes are not competent vectors of B. burgdorferi; they just don’t have the right stuff. While it’s not impossible that a mosquito bite could contain the spirochetes, it’s unlikely, and it’s even more unlikely Borrelia would succeed in setting up an infection. Mosquitoes are not significant vectors of Lyme disease.



References

Fontaine et al: Implication of haematophagous arthropod salivary proteins in host-vector interactions. Parasites & Vectors 2011 4:187 doi:10.1186/1756-3305-4-187

Hovius, JWR. Tick-host-pathogen interactions in Lyme borreliosis. Dissertation, Academic Medical Center, University of Amsterdam 2009

Kosik-Bogacka D, Bukowska K, Ku?na-Grygiel W. Detection of Borrelia burgdorferi sensu lato in mosquitoes (Culicidae) in recreational areas of the city of Szczecin. Annals of Agricultural and Environmental Medicine 2002, 9, 55–57

Schwalie PC, Schultz J.  Positive Selection in Tick Saliva Proteins of the Salp15 Family. Journal of Molecular Evolution Volume 68, Number 2 (2009), 186-191, DOI: 10.1007/s00239-008-9194-1

Magnarelli LA, Anderson JF. Ticks and Biting Insects Infected with the Etiologic Agent of Lyme Disease, Borrelia burgdorferi. Journal of Clinical Microbiology Aug. 1988, p. 1482-1486

Toola, Sea Otters, and Toxoplasma gondii

Reports in March 2012 of the death of Toola, a Toxoplasma gondii-infected sea otter who lived out her days at the Monterey Bay Aquarium, reminded me of the threat that T. gondii poses to marine mammals. Toola suffered from neurological damage thought to have been caused by the parasite and required daily anti-seizure medication. Among other things, she was the poster otter for legislation and other efforts to protect marine mammals from various health risks. And she was cute too.

Sea otters frequent the California coast, where they may
become infected with T. gondii. The consequences can be deadly.
Image by Mike Baird, Morro Bay, USA. CC BY 2.0

How do Sea Otters Get Toxoplasma gondii?

My impression has been that the risk of acquiring T. gondii has been rising in marine mammals, and that this is likely to be the result of runoff – oocysts being washed off the land into coastal waters. This made sense to me when considering the number of feral and roaming domestic cats, and the quantity of cat feces that must be carried into coastal waters by runoff (this has actually been studied: “domestic feline faecal deposition in communities adjacent to Estero Bay was conservatively estimated at 107 metric tonnes/year, or 26 kg/ha:” Miller et al.) I was surprised; therefore, to read that the majority of California sea otters tested in the 2008 study reported by Miller et al had a unique strain (dubbed Type X) that is not typically found in domestic cats.

Rather, the paper by Miller et al. reports that Type X T. gondii was found in wild felids (mountain lion, bobcat) and foxes. While foxes might be doing relatively well in urban areas, the number of wild felids is down from what it must have been before humans covered the west coast of North America with concrete and asphalt. So if domestic cats aren’t to blame, why are there more infected marine mammals now than before?

Humans and Mollusks Spread Toxoplasma to Marine Mammals

One answer apparently lies in all that concrete and asphalt. Hardscaping of the coast reduces the amount of runoff that’s absorbed into the ground before it spills into the sea. In addition:

• Human development has reduced wetlands, which provide natural filtration for runoff.


• Bivalves such as mussels flourish near storm sewers and have been shown to filter organisms, including T. gondii oocycts out of the water and concentrate them in tissue.


• Sea otters feed on mussels and other bivalves, consuming at least 76 mussels each day.

Studies done on land mammals have shown that a single oocyst can potentially be the source of chronic toxoplasmosis. Given those odds, its not surprising that Toola, and lots of other California sea otters (and other marine mammals) are infected with T. gondii.

Read the paper:

Miller, M.A., W. A. Miller, P. A. Conrad et al. "Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: New linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters." International Journal for Parasitology: 38(11), 2008

Sanitation Prevents Intestinal Worm Infections

A pit latrine in Haiti. Even a simple design
such as this will do much to reduce
contamination of the environment, and result
in fewer intestinal parasites.
Image by Rémi Kaupp. CC BY-SA 3.0

A paper in PLOS Medicine (January 24, 2012) reports that “sanitation is associated with a reduced risk of transmission of helminthiases to humans.” The authors looked at 36 previously published studies that measured prevalence of intestinal helminths (A. lumbricoides, large intestinal roundworm; T. trichiura, whipworm; and hookworm) compared to availability and use of sanitary facilities. They found that “people who either had or used a latrine were half as likely to be infected with a soil-transmitted helminth as people who neither had nor used a latrine.”

Contaminated Soil and Intestinal Worms

I submit that there are no surprises here. One acquires hookworm by coming in contact with hookworm larvae from feces contaminating the soil. They penetrate skin. Trichuris trichiura and A. lumbricoides eggs, infective a week or so after being deposited in warm moist soil in feces, must be swallowed. Obviously if feces were deposited in a pit latrine, septic system or other sanitary arrangement, instead of on the ground, those eggs and larvae would not be available to infect new hosts.

The fact that intestinal helminthes are much less common, even rare, in developed countries is no mere accident of climate, especially for the tough A. lumbricoides. It is because the majority of people in developed countries don’t defecate outside on the ground.

Parasite Prevention: Sanitation Works

The best point in this paper, though understated, is that periodically treating people for intestinal worms is perhaps not the best long term approach to getting rid of these parasites. Without good sanitation, people will quickly be reinfected due to contamination of their environment. Lets build toilets.

Read the paper:

Ziegelbauer K, Speich B, Mäusezahl D, Bos R, Keiser J, et al. (2012) "Effect of Sanitation on Soil-Transmitted Helminth Infection: Systematic Review and Meta-Analysis." PLoS Med 9(1): e1001162. doi:10.1371/journal.pmed.1001162

Are We Beating Malaria?

No human parasitic disease has ever been eradicated (although we may be close to eradicating Guinea Worm), but if ever there was a target, it would be malaria. Keeping an eye on the news and medical journals convinces me that there has never been more activity in scientific research aimed at understanding the parasites that cause malaria and finding ways to thwart them. Researchers have tried to develop malaria resistant mosquitoes. They’ve uncovered how the parasites invade red blood cells. They’ve investigated enzymes and proteins essential to the parasite’s survival. They’ve developed novel drugs. Hardly a day goes by when there is not something new. One might think we’re on the cusp of success.

Progress in Eradicating Malaria

But the reality is still grim. If you look at the statistics, you see that things are not really changing, at least not yet. Maps released by the Malaria Atlas Project allow us to roughly compare 2007 with 2010, and though there are clearly some changes (WHO statistics do indicate that the total number of malaria cases has dropped over the last decade), in the big picture they are minor changes that could easily reverse themselves.

This is discouraging. I wonder how long it will take for all this new research to provide us with a successful (and likely multi-pronged) approach to loosening the grip of this terrible disease.

Clinical burden of Plasmodium falciparum map in 2007 globally. CC BY 3.0

The spatial limits of Plasmodium falciparum malaria transmission
map in 2010 globally. CC BY 3.0

There are many maps on the Malaria Atlas Project site. Anyone interested in this should have a look.

Parasitic Worms and Wound Healing

Parasites can do you good. I didn’t discuss it at length in Parasites: Tales of Humanity’s Most Unwelcome Guests (I touched on it in chapter six), but I’m fascinated by the growing evidence that at least some of our parasites have good things to contribute to our health. The latest piece of that puzzle is research showing that hookworms can initiate an immune response in the host that actually speeds healing of tissue damage.

The business end of a hookworm.
You may not like the idea of hosting a few
hookworms, but they might return the favor
with better health. CDC; CC BY-SA 3.0

In their paper “An essential role for T_H2-type responses in limiting acute tissue damage during experimental helminth infection,” Fei Chen et al report that in mice “IL-17 initially contributed to inflammation and lung damage, whereas subsequent IL-4 receptor (IL-4R) signaling reduced elevations in IL-17 mRNA levels, enhanced the expression of insulin-like growth factor 1 (IGF-1) and IL-10 and stimulated the development of M2 macrophages, all of which contributed” to healing (Nature Medicine, published online 15 January 2012). In other words, there was an inflammatory response to the worms at first, then inflammation was suppressed while healing was enhanced.

It’s reasonable that a healing response may have evolved to help the host’s body deal with damage done by the worms themselves, but of course the potential exists for us to use that response to help heal tissue damage from other causes. This new report also ties in with previous work that suggests the ability of some of our parasites to suppress inflammation may protect us from autoimmune diseases.

It does make sense to me that organisms that have been with us for millions of years would have a relationship of both give and take with the host. While it’s true that parasitic diseases are some of the worst we face, and hookworm is a nasty parasite, I think we need to set aside the idea that anything parasitic is utterly bad. Let’s get to know them properly before we send them to extinction. (Not that hookworm is in danger of going extinct any time soon.)

I think we still have a lot to learn about our relationship with our parasites, especially our "old friends."

Helping Mosquitoes Fight Off Malaria

Anybody who knows anything about malaria knows that one catches it from a mosquito bite. Mosquitoes don’t just physically carry the parasite from person to person: they are a required host for Plasmodium spp., the agents of malaria. In the mosquito, the parasites multiply sexually, producing tiny forms called sporozoites which are injected into the next person the mosquito bites.

[caption id="attachment_382" align="alignleft" width="300" caption="Anopheles stephensi feeding; CDC, public domain image"][/caption]

Clearly, the mosquitoes are infected just as people are, but we seldom feel sorry for the poor mosquitoes because, well, we hate them for all sorts of reasons. The mosquito, however, does have an immune system which tries to fight off invading Plasmodium sp. parasites; Mosquitoes don’t mean to transmit these dangerous parasites.

Humans have had a long and costly battle with malaria which, so far, we have not won. Though not self-evident perhaps, it makes sense that we might be able to enlist the help of the lowly mosquito to our mutual benefit, and that’s what some researchers at Johns Hopkins University have done. Yuemei Dong et al. have genetically modified the immune system of a mosquito species, Anopheles stephensi, giving it an enhanced ability to fight off invading Plasmodium falciparum, the worst of the malaria parasites in humans.

In order for this research to prove useful in the real world, the modified mosquitoes would have to be released into the wild and allowed to breed with wild populations (and hopefully do better than the wild type). Aside from the obvious need for caution when releasing a genetically modified organism into the wild, at this point we still don’t know whether:

• the resistant mosquitoes will do as well in the wild, faced with different A. falciparum strains


• other Anopheles spp., also malaria vectors, can be similarly modified (there are about 40)


• Plasmodium falciparum will develop resistance to the mosquito resistance


• all other species of Plasmodium infecting humans can be targeted this way

This breakthrough is not the answer to the battle against malaria yet, but it may be part of the answer.

Read the paper:

Dong Y , Das S , Cirimotich C , Souza-Neto JA , McLean KJ , et al. 2011 “Engineered Anopheles Immunity to Plasmodium Infection” PLoS Pathog 7(12): e1002458. doi:10.1371/journal.ppat.1002458

Leishmania donovani Resistance to Drugs and Host Defenses

Leishmania donovani, agent of visceral leishmaniasis, or kala-azar, has always known how to evade the human immune system. This parasite literally uses the cells of the immune system to survive and multiply: when it enters the body via a sand fly bite it is engulfed by a macrophage – part of the immune response – and then it multiplies until the cell is destroyed.

[caption id="attachment_376" align="alignleft" width="300" caption="Leishmania parasites inside macrophages"][/caption]

Nonetheless, the immune system does have some control over the infection and some infections are without symptoms of disease. An interesting paper published in 2011 (Vanaerschot et al.) reports on research that suggests that strains of L. donovani that are resistant to the commonly used antimonial drugs are also able to multiply to greater numbers in the host. So these strains not only fail to respond to antimonial drug treatment, but also cause worse disease than other strains. That is unfortunate.

It seems that antimonials work by enhancing the ability of macrophages to kill the parasites they ingest. Antimonial resistant strains appear to have evolved a way to swing things the other way somehow – they don’t do just as well as the sensitive strains; they actually do better. The authors write “all [sensitive] strains caused a similar parasite burden both in the liver and the spleen… [Resistant] strains displayed… an average 8-fold higher parasite burden in the liver and 3-fold higher parasite burden in the spleen compared to [sensitive] strains” (p. 3). That is an impressive increase, and not good news for the patient.

The authors also looked at whether these strains are also more resistant to the new drug of choice, miltefosine. They didn’t find evidence of this, but note that more study and surveillance are needed to be sure it’s not the case.

Vanaerschot M, De Doncker S, Rijal S, Maes L, Dujardin J-C, et al. (2011) "Antimonial Resistance in Leishmania donovani Is Associated with Increased InVivo Parasite Burden." PLoS ONE 6(8): e23120. doi:10.1371/journal.pone.0023120