Baylisascaris procyonis Caused Meningitis.

by CL

Society today has become overly sterilized. Antibiotic hand soaps and disinfectants can be found in many American households and business and it seems that people are increasingly afraid of getting "dirty". This trend has become severe enough that in the late 1980's Dr. Strachan formulated the "Hygiene Hypothesis", which suggests that a decrease in exposure to infectious agents causes an increase in atopic diseases [1]. A good way for people, especially children, to become exposed to microorganisms is for them to eat food that has been on the ground since there can be up to 2,000 bacterial species in half a gram of Minnesotan soil [2]. That being said there are times when eating the cookie that fell on the ground is a very bad idea. One of the worst times for this would be when there is a chance that raccoons have recently defecated on that spot. The ingestion of raccoon fecal matter should be avoided because raccoons are the definitive host for Baylisascaris procyonis. More commonly known as the raccoon roundworm B. procyonis is a helminth that can be found in up to 70% of raccoons and can cause severe disease in humans [3].

Figure 1. Basic diagram of the B. procyonis

lifecycle, including other host organisms.

B. procyonis lives and mates inside the intestines of raccoons and when the animals defecate millions of unembryonated eggs are released into the environment; in two to four weeks larvae form within the eggs and they become infectious [3, 4]. B. procyonis eggs tend to be resistant to environmental degradation and given adequate moisture they can remain viable and infectious for years. This increases the chances for the organism to complete its life cycle by infecting another raccoon; however, it also increases the likely hood of a person becoming infected after accidental ingestion [3]. Once inside a human host larvae can disseminate throughout the body in as little as three days and seven percent or more of larvae can cross the blood brain barrier to infect the CNS. Once inside the CNS the larvae will molt and grow, which tends to produce irritability, fever, lethargy, and other symptoms similar to bacterial meningitis, in the infected person [3]. It is only through the discovery of high levels of eosinophils in the CNS that a positive diagnosis of eosinophilic meningitis is made. Eosinophils are white blood cells that are commonly found in parasitic infection associated with helminths and other worm infections. The use of antihelminth drugs early in the infection may help to reduce the severity of disease, but poor outcomes are still common and living larvae have been recovered from brain autopsies of treated animals and humans [3]. The severity of meningitis caused by B. procyonis infection is partly due to the fact that the larvae molt and grow as they migrate, which leads to an increased risk of permanent neurological damage in treated cases [5].  The amount of inflammation caused by the organism and the high levels of eosinophils found in the CNS also contribute to the poor prognosis of the disease [3]. Due to the fact that humans are dead end hosts for B. procyonis the organism cannot complete its lifecycle and cannot be spread between people. Also since the organism cannot reproduce inside humans the parasite cells will die and the infection will eventually clear itself, but permanent neurological damage and/or death is likely to occur before this can happen [1].

Figure 2. Adult worms next to a

dime for scale. The size of the

worms is part of the reason for

the disease severity and neurological

complications. Female worm is

 on left, male is on right.

The limited means of transmission keeps Baylisascaris procyonis infections down; however, public knowledge about this organism and its potential to cause severe disease in children will help to minimize infection rates. With an alarmingly high rate of up to 70% of raccoons carrying B. procyonis in some areas and an estimated 800,000 to 1,000,000 raccoons in the state of Minnesota alone the environment is saturated with raccoon feces and B. procyonis eggs [3, 6]. This doesn't mean that exposure to the outside should be limited or that children should not be allowed to get dirty. Instead it means that the general population should be aware of the risks and take steps to prevent the accidental ingestion of raccoon fecal matter. The prevention of infection with Baylisascaris procyonis is as simple as discouraging raccoons from spending a lot of time if residential yards; which is simply done by covering trashcans, not indirectly feeding raccoons by leaving out pet food, or directly feeding raccoons or encouraging them in any way. Those steps alone won't guarantee that an infection never happens, but they will go a long way to decrease the chances of one. That being said if the raccoon population is particularly high in a residential area it is probably a good idea to wash off any food that has been on the ground before eating it.

Citations

1. Bloomfield, S.F., Stanwell-Smith, R., Crevel, R.W.R., Pickup, J. (April 2006). Too clean, or not too clean: the Hygiene Hypothesis and home hygiene. In Clinical and Experimental Allergy. (Vol. 36). (pp. 402-425). Blackwell Publishing Ltd.

2. Schloss, P.D., Handelsman, J. (2006). Toward a census of bacteria in soil. In PLoS Computational Biology. (Vol. 2). (Issue 7). (pp. 786-793). Retrieved online from http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.00 20092

3. Park, S.Y., Glaser, C., Murray, W.J., Kazacos, K.R., Rowley, H.A., Fredrick, D.R., Bass, N. (2000). Raccoon Roundworm (Baylisascaris procyonis) Encephalitis: Case Report and Field Investigation. In Pediatrics: The Official Journal of The American Academy of Pediatrics. (Vol. 106). (Number 4.) Elk Grove Village, Illionis: American Academy of Pediatrics.

4. CDC. (Oct. 11, 2012). Parasites - Baylisascaris infection. In Centers for Disease Control and Prevention. Retrieved Nov. 16, 2012, from http://www.cdc.gov/parasites/baylisascaris/biology.html.

5. Dangoudoubiyam, S., Kazacos, K.R. (Nov. 2009). Differentiation of Larva Migrans Caused by Baylisascaris procyonis and Toxocara Species by Western Blotting. In American Society for Microbiology: Clinical and Vaccine Immunology. (Vol. 16). (Number 11.) (pp. 1563-1568). Washington DC: American Society for Microbiology.

6. Minnesota DNR. (2012). Raccoon. In Minnesota Department of Natural Resources. Retrieved Nov. 16, 2012, from http://www.dnr.state.mn.us/mammals/raccoon.html.

Naegleria fowleri “The Brain-Eating Amoeba”

by TC

Naegleria fowleri also known as the “brain-eating amoeba” is the only known human pathogenic Naegleria species. Nonetheless, this free-living, microscopic amoeba is the cause of primary amoebic meningoencephalitis (PAM), a fatal disease of the central nervous system (CNS). The typical environmental niche consists of warm fresh-water such as ponds, lakes, rivers, or hot springs with a bacterial food source [8]. However, N. fowleri has also been isolated from under chlorinated swimming pools, heated tap water, and soil deposits [3,4,8].

Figure 1: N. fowleri life cycle. [8]

There are three stages in the N. fowleri lifecycle: cyst, flagellate, and ameboid trophozoite (Fig. 1). Of all three stages only trophozoites are capable of causing infection. Within natural warm-water environments trophozoites replicate via promitosis, a special form of binary division keeping the nuclear membrane intact at all times. In times of environmental stress due to nutrient limitation or cold temperatures trophozoites may regress back to non-feeding flagellates or cysts in extreme cases. Cysts are spherical approximately 7-15μm in diameter, significantly more resilient than the other forms and may remain dormant for prolonged periods of time [8]. Trophozoites, however, are granular single-nuclei cells approximately 10-35μm long, and may be inhaled during water activities such as swimming. It is worthy to note that an infection will not ensue if the contaminated water is ingested [8].

Trophozoites once in the nasal cavity trigger the host innate immune system by inducing heavy mucosal secretions and leukocyte recruitment via a reactive oxygen species (ROS) signaling pathway [5]. ROS activates epidermal growth factor receptors (EGFR) eliciting the recruitment of interluken IL-8 and MU5AC, the most abundant mucin (inhibit N. fowleri trophozoites) in human airway epithelium [4,5]. IL-1β is also recruited to the zone of infection but is not regulated by EGFR [5]. Chemokines IL-8, MU5AC, and IL-1β signal neutrophils to the site of infection inducing mild inflammation of the nasal cavity [5]. Some trophozoites evade or escape mucosal engulfment. While the complete mechanism is unknown research has shown mucinolytic activity of N. fowleri by cysteine proteases that degrade the surrounding mucus [4]. Free of engulfment trophozoites penetrate the olfactory neuroepithelium followed by invasion of the olfactory bulb (Fig. 7) [3,4,5,8,9]. Once trophozoites gain access to the central nervous system (CNS) they migrate to the brain causing a massive inflammation response in addition to lysis of inflammatory cells and leukocytes [3,4,5,8]. Lytic-necrotic tissue areas and hemorrhaging develop, all of which likely contribute to the significant tissue degradation seen primarily in the frontal brain [5,7,8,10].

PAM while rare is extremely fatal. Within the U.S. there is only one documented survivor of 123 reported cases dating 1962-2011. This sole survivor contributes to the 2 total survivors recorded in North America [8]. The symptoms, which set in approximately 1-7 days after exposure, are very similar to bacterial meningitis rendering proper diagnosis difficult. There are two distinct stages of symptomatic infection. The initial stage is characterized by fever, nausea, vomiting, and severe frontal lobe headaches. Progression leads to stiff necks, altered mental status causing hallucinations, seizures, and a coma. Death generally ensues 1-12 days post-symptom development giving victims roughly 2-19 days to live post-exposure [8]. As N. fowleri is a worldwide microbe, isolated from more than a dozen different countries spanning five continents, PAM too contracted worldwide [6].

There are no current rapid detection methods known for determining contamination concentrations of water sources or individual infection [5,8]. However, extensive diagnostic methods such as staining, immunohistochemistry (IHC), polymerase chain reaction (PCR), and isolated amoeba cultures may be in conjunction to detect N. fowleri organisms, nucleic acids, or antigens in cerebrospinal fluid (CSF) or tissue samples [8]. Such methods are only available at select U.S. laboratories due to the rarity of PAM, and preformed post-mortem approximately 75% of the time due to rapid fatality rates [8].

Additionally, no successful treatments are currently known, although, the two survivors in North America both received Amphotericin B, a polyene antifungal drug [8]. Amphotericin B binds to ergosterol, an essential sterol in fungal membranes, forming transmembrane channels allowing monovalent ion (Na⁺, K⁺, H⁺, and Cl^-) leakage resulting in cell death [1]. Additional antifungal drugs typically given congruently include azoles such as miconazole, fluconazole, and ketoconazole [8]. Azoles inhibit the lanosterol to ergosterol converting enzyme, lanosterol 14 α-demethylase, thus decreasing ergosterol concentrations leading to cell death [2].

Work Cited:

1. "Amphotericin B." Wikipedia: The Free Encyclopedia. Wikimedia Foundation, Inc, 11 2012. Web. 14 Nov 2012. http://en.wikipedia.org/wiki/Amphotericin_B
2. "Azole antifungals." Drugs.com. Drugs.com, n.d. Web. 14 Nov 2012. http://www.drugs.com/drug-class/azole-antifungals.html
3. Cervantes-Sandoval, Isaac, Jose de Jesus Serrano-Luna, Ethel Garcia-Lattore, Victor Tsutsumi, and Mineko Shibayama. (2008). “Characterization of brain inflammation during primary amoebic meningoencephalitis.” Parasitology International. 57:307 – 313. Web 14 Nov. 2012. www.sciencedirect.com
4. Cervantes-Sandoval, Isaac, Jose de Jesus Serrano-Luna, Ethel Garcia-Lattore, Victor Tsutsumi, and Mineko Shibayama. 2008. "Mucins in the host defence against Naegleria fowleri and mucinolytic activity as a possible means of evasion." Microbiology. 154: 3895-3904. Print.
5. Cervantes-Sandoval, Isaac, Jose de Jesus Serrano-Luna, Patricia Meza-Cervantez, Rossana Arroyo, Victor Tsutsumi, and Mineko Shibayama. 2009. "Naegleria fowleri induces MUC5AC and pro-inflammatory cytokines in human epithelial cells via ROS production and EGFR activation." Microbiology. 155(11):3739-3747. Web. 14 Nov. 2012. http://mic.sgmjournals.org/content/155/11/3739.short
6. De Jonckheere, Johan F. “Origin and evolution of the worldwide distributed pathogenic amoeboflagellate Naegleria fowleri.” 2011. Infection, Genetics and Evolution. 11(7):1520–1528. Print.
7. Marciano-Cabral, Francine, Guy A. Cabral. “The immune response to Naegleriafowleri amebae and pathogenesis of infection.” 2007. FEMS Immunol Med Microbiol. 51:243–259. Print.
8. "Naegleria fowleri - Primary Amebic Meningoencephalitis (PAM)." CDC: Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, n.d. Web. 14 Nov 2012. http://www.cdc.gov/parasites/naegleria/index.html
9. Olfactory System. 2012. Neuro News & Cosmo CluesWeb. 14 Nov 2012. http://protoplasmix.wordpress.com/2012/03/
10. Serrano-Luna, Jose de Jesus, Isaac Cervantes-Sandoval , Victor Tsutsumi, and Mineko Shibayama. (2007). “A Biochemical Comparison of Proteases from Pathogenic Naegleria fowleri and Non-Pathogenic Naegleria gruberi.” J. Eukaryot. Microbiol., 54(5): 411–417. Print.

A Crappy Parasite

by Anuj Saluja

I love Jurassic Park. If you haven’t seen it^[1] or need a refresher, here is the synopsis: a rich old man makes a park with living, breathing dinosaurs. Several experts and the old man’s grandkids come for a test visit. Naturally, [crap] hits the fan and the dinosaurs start wreaking havoc. When I was a kid, it was terrifying because of the velociraptors and T-Rex chasing the hapless humans. It still scares me as an adult but for a whole different reason. I was watching it recently and one scene had me screaming at my tv. No, not the kitchen raptor scene^[2] but rather a much more subtle scene: Dr. Grant, the protagonist of the film, washing and drinking from a fresh water stream. As a microbiology major, this made me cringe – hasn’t Dr. Grant heard of Giardia lamblia?! Running from velociraptors is pretty bad, but running after being infected with G. lamblia cysts? Well, consider yourself literally [crap] out of luck.

Figure 1 – A Giemsa stain of G. lamblia.

Giemsa helps visualize chromosomes.

Here you can see the two nuclei of

G lamblia as well as another organelle

across the middle. 

Most campers are familiar with G. lamblia; the parasitic protozoon is the most common cause of epidemic waterborne diarrheal disease (Giardiasis). This usually happens because of fecal contamination of water. The parasite was first discovered by an ambitious Antony van Leeuwenhoek when he decided to check out his diarrheic feces. From afar, the protozoon looks similar to other human parasites, which often leads to misdiagnosis (2). However, upon closer scrutiny, it has several characteristic features that can distinguish it from other members of the same family, Hexamitdae. They are flagellated (meaning they are mobile organisms) and have a unique structure: they have paired organelles, including two active nuclei, as well as an attachment organelle known as the ventral disc. (2). Another distinguishing feature of G. lamblia is its characteristic “smiley face”^[3] when stained (see figure 1). Potential hosts for the parasite include a variety of animals aside from humans. This includes cats and dogs as well as beavers (hence Giardiasis known colloquially as “beaver fever”) (1)

G. lamblia begins as a cyst (a dormant or non-active cell) that has been excreted with the feces of an infected individual. When the cyst is ingested by a host, a trophozoite (a young, active cell essentially) emerges. It is motile and absorbs nutrients from the host’s small intestine, causing dehydration and diarrhea. During this stage, the parasite replicates through binary fission, meaning new daughter cells are genetically identical to mother cells. Eventually, they too become cysts and are excreted out of the system (some trophozoites are excreted out too but do not survive – only cysts can survive outside the host) (2).  The cysts are tough buggers – they can survive pretty extreme environmental pressures like fluctuating climate and most predators - they’ll just infect them and multiply.

G. lamblia cysts have also been found in pools (3). In 1988, several swimmers contracted Giardiasis after swimming in a public pool. Cysts are normally quite chlorine resistant. According to a formula provided by the paper, a pool must have .3 mg of chlorine for 50 minutes if it is at 25 degrees Celsius in order to kill any cysts (3). On the day of occurrence, a handicapped child who happened to be infected with the parasite had, let us say, “an accident” in the pool. By an inconvenient coincidence, the pool’s chlorine tank ran out, which led to a small outbreak of Giardiasis. Ultimately, this may have been due to bad luck. However, a slightly more alarming publication was released by the CDC in 2008 (4). In it, they describe several instances where cysts of the similar species, Giardia intestinalis, were found in several chlorinated pools in Atlanta. The authors note that even though the cysts were taken from a small sample size, the findings were statistically significant. Given the similarity between the two species, it would not be too surprising to see cases of Giardiasis from G. lamblia popping up from folks at swimming pools. If you own one, it’s probably a good idea to make sure no vermin have “an accident” and to make sure your chlorine levels are up to par.

In general if you live in a modern country, unless you are out in the middle of nowhere camping, you are fine – your home tap water doesn’t have any G. lamblia. Yes those two reports from the pools are from first world countries but they are rare instances (the papers even note that the sample size is small). However, for poor third world countries, G. lamblia is a real threat. Most don’t have proper water filtering systems, meaning most of their water is highly susceptible to contamination.

If I may get serious for a moment, Giardiasis can do some serious damage for poorer populations; severe diarrhea can lead to dehydration which is life threatening in a poorer country without available healthcare (5). That said though, treatment for Giardiasis is simple. Metronidazole, an antiprotozoal is used to combat Giardiasis and clears it up quickly. More powerful antiprotozoals can also be used if metronidazole fails (2).

Now back to Dr. Grant from Jurassic Park and his callous^[1] use of the stream water. Instead of directly applying the water, he could have boiled it. However, it’s the 21^st century and the movie is 20 years old. We are in the smartphone era now^[2]! Surely there must be some high tech method of cleaning up G. lamblia cysts from water.

Indeed it does – it’s this brilliant device called the sun. Solar Disinfection or SODIS is used to kill pathogenic microbes in drinking water. The best part is that it is simple and cheap – just use a transparent container like a plastic bag, water bottle or glass bottle and place it in direct sunlight for about 8 hours. The UV light of the sunlight will damage the cysts generally.  The sunlight also stimulates the microbes that perform aerobic (oxygen required) photosynthesis and release O₂ gas into the water.  The gas is highly reactive and can disinfect any remaining microbes (6). This has resulted in a reduction, especially with children, of diarrhea from drinking water.

Using Giardia muris instead of G. lamblia, a research group successfully showed that SODIS can work for killing cysts in water. They did this by setting up suspensions of G. muris cysts and exposing them to various conditions of light. They then exposed mice to the cysts and found that cysts that were subjected to full SODIS conditions (40 degrees Celsius and 870 W/m² light, 4 hours) rendered completely un-infectious. Granted, this is tough to achieve on a smaller scale – like say a camping trip unless it’s sunny and hot, the authors suggest that in emergency scenarios – like say a bad hurricane, SODIS can be used to clean up water from G. lamblia cysts^[3].

Even though it’s treatable, Giardia lamblia is not a microbe to be trifled with. Giardiasis is not pleasant and can cause some serious dehydration. If you camp regularly, be sure to take the proper precautions to prevent infection. This includes using clean water to clean any dishes you have. Plastic water bottles with filtering devices have recently become commercially available. Take full advantage of them – they usually say how powerful they are on the label and Giardia species are often listed amongst the list of screened microbes.

References:

1. Willey, Sherwood, Woolverton. Prescott’s Microbiology: 8^th Edition. McGraw Hill, 2011. New York, NY. Pg 584 – 586, 730
2. Ortega-Pierres et al. Giardia: A Model Organism online textbook. 1st Edition, 2011 SpringerWien New York.  Springer.com
3. Porter, et al. Giardia Transmission in a Swimming Pool. American Journal of Public Health. June 1988. Vol 78 no 6 pg 659 – 662.
4. Shields, et al. Prevalence of Cryptosporidium spp. and Giardia intestinalis in Swimming Pools, Atlanta, Georgia. Emerg Infect Dis. 2008 June; 14(6): 948–950.
5. Dewey, Daniel MD. World Altering Medicine: Breath of Life. Lecture. University of Minnesota. December 2010.
6. McGuigan et al. Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water. Journal of Applied Microbiology ISSN 1364-5072. J Appl Microbiol. 2006 Aug;101(2):453-63.

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[1] I of course realize that it’s Hollywood and worse sins have been committed in films. Also, stopping to sterilize water is probably the last thing on your mind when you are being chased by ravenous dinosaurs

[2] Smokey the Bear and PETA may have heart attack because of him starting a fire on an  animal reserve too

[3] While they didn’t use G. lamblia, G. muris acted as a model and the group notes that this can be applied to G. lamblia