Thursday, February 18, 2016

Cryptosporidium parvum: Pathogenesis of Cryptosporidiosis

            Cryptosporidiosis (is also called Crypto) is a diarrheal disease mainly caused by an obligate intracellular protozoan parasite, Cryptosporidium parvum, in which the parasite cannot complete its life cycle and reproduce in the absence of a suitable host. C. parvum acquired an ability to infect the intestinal epithelial cells in gastrointestinal (GI) tract of the hosts and then undergo both asexual and sexual cycles for their replications. According to the Center for Disease Control and Prevention (CDC) report, Crypto is one of the most common waterborne diseases in the United States. Between 2001 to 2010, Crypto was the leading cause of waterborne disease outbursts, which linked to recreational water in the United States (1). Most of the Crypto can be spread by drinking recreational water contaminated with Crypto, eating raw food, exposure to stool from an infected person or animal, but it is not spread through a contact with blood. Due to C. parvum high tolerance to chlorine, they can survive in chlorinated environment for a long period of time, and this is why people swallowing recreational water (such as water in swimming pool) have higher risks to get Crypto infection.
            Cryptosporidium parvum is one of several protozoan parasite that cause Crypto in both animals and humans. It has a monoxenous life cycle that is mainly stay in the GI tract of a single host. Also, C parvum lacks of host and organ specificity, ability for autoinfection, and resistance to antimicrobial (2), which result in many aspects of the nature and pathogenic mechanisms of C. parvum remains unclear due to its characteristics.
            For the Cryptosporidium life cycle, it has both asexual and sexual cycles to allow themselves to divide and replicate in host epithelial cells. The life cycle begins with ingestion of sporulated oocysts from the host, and excystation (emerge from a cyst) occurs once the oocysts enter to the GI tract, and they will release four sporozoites which will parasitize epithelial cells in GI tract (Figure 1). Within these infected epithelial cells, C. parvum can undergo two additional asexual replication and release merozoites, which then they will undergo sexual cycle and produce microgametes (male) and macrogametes (female), which give arise to the zygote (fusion of both male and female gametes) and form oocysts (Figure 1). Then the cycle repeats from the ingestion of oocysts from the hosts.
Figure 1. Life cycle for Cryptosporidiosis caused by C. parvum.   
            The pathogenesis of Cryptosporidium in which causing diarrhea is still poorly understand. The suggested mechanism may be involving a host-parasite interaction in which the attachment of C. parvum surface protein on the host surface cell is the initial step for Crypto to occur. One of the C. parvum surface protein has been identified that plays a role in mediating attachment and invasion on intestinal epithelial cells of the hosts (3). It is believed that C. parvum surface protein acts like a ligand that binds to a receptor on the surface of the host epithelial cell and initiates attachment and invasion process to allow oocysts enter the GI tract and begins cell divisions.
            Cryptosporidial infection can be transmitted from contaminated food and water, from animal to person contact, and via person to person contact. The infection mainly infects children due to their incomplete development of immune system. The major transmission pathway for cryptosporidial infection is going through the fecal-oral route from infected hosts directly or indirectly via contaminated water or ingestion of contaminated food. Crypto is one of the frequent cause of waterborne diarrhea because small infectious dose is enough to cause infection. Also, due to their oocysts’ high resistance to disinfectants and other chemicals used in recreational and drinking water, Cryptosporidium has emerged frequently in most of the waterborne diseases in the United States. In fact, the source for Crypto mainly comes from wild animals such as bovines, dogs, or cats that ingested C. parvum oocysts in the intestines. Once the humans accidently make a contact with the stools of infected animals or humans, including swallowing unsterilized water or eating uncooked food contaminated with Crypto. In addition, individuals with immunodeficiency (failure of the immune system) also have high risks for Crypto, such as the patients with AIDS (acquired immunodeficiency syndrome) or cancer.
            The major diagnosis of Crypto is going through an examination of stool samples from the patients or animals. Because of the detection of Crypto can be challenged, several techniques have developed to identify Cryptosporidium such as acid-fast staining, is one of the reliable and traditional method to detect the presence of cryptosporidial oocysts. Also, in a view of immunology, using enzyme-linked immunosorbent assay (ELISA) and antibody immunofluorescence assay (IFA) are the two alternative methods that using antibodies to detect Cryptosporidium. Both methods are used to detect protein-protein binding interaction between C. parvum surface protein and the host cell surface protein, which allows healthcare providers to identify the infection more efficient.
            From a genetic view of C. parvum infection, a complete genome of C. parvum has been recently identified (Abrahamsen, 2004). Several novel proteins of C. parvum cell surface and secreted proteins have been identified, and it is believed these proteins have crucial roles in host interaction and pathogenesis (4). In addition, targeting Cryptosporidium metabolic pathway or enzymes may also have potentials for drug development (5). Still, due to lack of sufficient information about C. parvum pathogenesis, more genetic analysis are required to identify what genes or proteins have contributions in attachment and invasion processes, and the virulence of Crypto.
            C. parvum is one of the parasite that cause waterborne diseases in humans, and the health problem have become a major concern in the United States. Because of C. parvum oocyst’s high resistance to common disinfectants, sterilizing of recreational and drinking water still becomes a challenge work today. Accurate detection with Cryptosporidial infections is also a major challenge to healthcare providers, and more research is needed to focus on pathogenesis of Crypto and development of drugs or therapies against C. parvum infection. Knowing mechanisms and transmission of C. parvum infection would allow researchers to be able to target the interaction between C. parvum surface protein and host cell surface protein, which will prevent the first crucial step of Cryptosporidial infection, attachment and invasion process. Therefore, a combination of both genetic and pathological analyses are the essential approaches to treat Crypto in future.

1.         General Information for the Public | Cryptosporidium | Parasites | CDC. (n.d.).      Retrieved November 23, 2015, from           info.html
2.         S. Tzipori, Cryptosporidiosis in animals and humans, Microbiol. Rev. 47 (1983), 84–96.
3.         M.W. Riggs, Immunology: host response and development of passive        immunotherapy and vaccines, in: R. Fayer (Ed.), Cryptosporidium and             Cryptosporidiosis, CRC Press Inc., New York, 1997, pp. 129–162.
4.         Abrahamsen, M. S., Templeton, T. J., Enomoto, S., Abrahante, J. E., Zhu, G., Lancto, C. a, … Kapur, V. (2004). Complete genome sequence of the       apicomplexan, Cryptosporidium parvum. Science (New York, N.Y.), 304(5669),     441–445.
5.         G. H. Coombs, Parasitol. Today 15, 333 (1999).     

Don’t go in the water.

by AM
Brain eating amoeba claim another victim
The most terrifying creature lurking below the water’s surface does not have fins or razor sharp teeth.  In fact, you can’t see it at all. The microscopic amoeba, Naegleria fowleri, is best known for its grotesque ability to digest and consume its victim’s brain.  This ‘brain-eating’ amoeba may sound like Eli Roth’s latest stomach-turning horror flick, but it’s no work of fiction and it’s in the water.  Each year, this amoeba kills more people than sharks do in the United States1.  The single-celled organism can cause the deadly brain infection called primary amebic meningoencephalitis (PAM), a disease that is nearly always fatal2.  Here is what you should know about the deadly infection and the current research desperately seeking to find a treatment.
Infection occurs when a rush of contaminated water enters the nose and the amoeba moves up the nasal cavity and into the brain3.  These tiny organisms swim by twitching fingerlike extensions called, pseudopods2.  Once inside the brain, the parasite begins releasing proteins that cause nerve and red blood cells to lyse, or break open, spilling the cell’s innards for the organism to devour2.  The parasite can also use ‘sucking structures,’ known as ‘food-cups,’ located on the outside of the organism to rip open cells and feed on the brain’s grey-matter4.   This process is shown in the figure below which was captured using a scanning electron microscope capable of viewing these microscopic organisms.  The amoeba can be seen tearing apart and devouring a cell5.  Destruction of these cells leads to severe swelling and necrosis, or tissue death, resulting in devastating damage to the brain and often brain hemorrhaging6.  Symptoms of the PAM progress quickly, often involving fever, nausea, seizures, severe frontal headaches, and vomiting6.  Death generally occurs within 7-10 days of infection6.
N. fowleri attacking and devouring cell observed using SEM. (Bar is equal to 10 µm)5    
While uncommon, the disease is virtually always fatal6.   Between 0 and 8 infections are reported every year in the United States7.  Only three people have ever survived6.  This is in part due to the difficulty detecting and treating the infection6.  Early symptoms often resemble the flu or other viral illnesses resulting in misdiagnosis for 74% of initial healthcare visits during the beginning stages of the disease6.  As the disease progresses, incorrect diagnosis of bacterial meningitis results because the symptoms are nearly identical consequently leading to 94% of patients of patients receiving inappropriate treatment prior to diagnosis with PAM6.  Diagnosing PAM requires visually examining cerebrospinal fluid for motile amoebas6.  For timely identification, it is important for doctors to ask about possible risk of exposure, such as recent freshwater swimming, in order to begin treatment as early as possible6.
Exposure to the amoeba typically occurs while swimming, diving, or during other water-related activities8.  The amoeba is present in freshwater sources including lakes, rivers, and untreated swimming pools8. Public health authorities are encouraged to regularly monitor recreational waters at risk of contamination and post appropriate warnings where high amounts of the amoeba have been identified9.  Proper chlorination of public pools can eliminate risk of infection9. Despite these attempts, infections do still occur and the only known way to prevent infection is by avoiding nasal contact with untreated fresh water7.
Despite the low risk of an infection with the amoeba, water monitoring programs have shown that the organism is wide-spread.  A study examining water sources in Arizona found the amoeba present in 17 out of the 19 samples collected10.  The question many experts have asked, is why then, do infections seem to be so rare? “Almost every single person has antibodies in their blood which indicate they were exposed at some point in time to the amoeba, but they didn’t die from it,” said expert, Dr. Francine Marciano-Cabral, a professor of microbiology and immunology at Virginia Commonwealth University who has studied N. fowleri for more than 30 years11.  She explains it could be that infection requires an encounter with a large number of amoebas all at one time or, there may be a particular subspecies which causes infection in humans while other subspecies are nonthreatening11.  Nearly all cases in the United States had occurred in southern states (see map below), but recent expansion has brought the disease northward12.
Reported cases of PAM
In August 2010, the first confirmed case of PAM in a northern state occurred in Minnesota and it was associated with local freshwater exposure6,12.  Since then, three additional cases have been reported in the Midwest necessitating better awareness among providers across the US6,12.  The amoeba is thermophilic meaning it thrives and rapidly multiplies in warm temperatures9.  The projected increase in temperatures due to global temperature change could result in the infection being introduced to more areas that were previously unaffected9.  Recently, the number of infections reported annually appear to be rising which could be a cause for concern6,13.  While this may be due to improvements in surveillance, it is believed that cases are still largely underreported6. Whether the increasing temperature will lead to an increase in infections remains to be seen.  Overall, the increase of infections, expansion of the amoeba’s geographical range, and high fatality associated with the disease makes finding an effective treatment for the infection more pressing. 
One area of current research, aimed at finding a treatment for the disease, involves understanding the amoeba’s ‘brain-eating’ abilities.  How the amoeba destroys brain tissue is not fully known.  Studies, focused on the potential causes for these deadly effects, suggest proteins released by the amoeba may be involved.  Dr. Kenneth Aldape believes cysteine proteases may be contributing to the tissue destruction caused by the amoeba14.  Cysteine proteases are enzymes that work by cutting up proteins into pieces9.  This damage can eventually result in the death of the cell14.  These conclusions were based on experiments where an irreversible cysteine protease inhibitor was applied to the amoeba14.  The inhibitor inactivates the amoeba’s enzyme preventing it working properly.   When the inhibitor was added to the amoeba? They were no longer able to kill cells14.  These results suggest that these specific enzymes are required in order for the amoeba to kill cells during infection14.  While non-disease causing amoeba produce similar proteases, the ability to function at human body temperature is unique to this particular species15. 
          One specific set of these cysteine proteases investigators are focusing on is, naegleriapores, which poke holes into the cell-membrane exposing the cell’s interior16.  Two of these proteins, named naegleriapore A and B, were discovered by Dr. Rosa Herbst at the Bernhard Nocht Institute for Tropical Medicine in Germany16.  The studies by Dr. Herbst identified these proteins by measuring their ability to pierce the cell-membrane16.  Examining the structure of these proteins unveiled that they were actually fragments of the same, larger protein16,17.  A process leading to a large arsenal of protein forms capable of destroying cells16,17.
            The identification of these proteins is an important step towards understanding this deadly disease.  Scientists are hopeful that one of these proteins may lead to a treatment.  Until then, education for rapid diagnosis and treatment are essential. 

Works Cited

1.         Human Shark Bait. National Geographic Channel - Videos, TV Shows & Photos - Canada at <>
2.         John, D. T. Primary Amebic Meningoencephalitis and the Biology of Naegleria Fowleri. Annu. Rev. Microbiol. 36, 101–123 (1982).
3.         Jarolim, K. L., McCosh, J. K., Howard, M. J. & John, D. T. A light microscopy study of the migration of naegleria fowleri from the nasal submucosa to the central nervous system during the early stage of primary amebic meningoencephalitis in mice. J. Parasitol. 86, 50–55 (2000).
5.         John, D. T., Cole, T. B. & Marciano-Cabral, F. M. Sucker-like structures on the pathogenic amoeba Naegleria fowleri. Appl. Environ. Microbiol. 47, 12–14 (1984).
6.         Capewell, L. G. et al. Diagnosis, Clinical Course, and Treatment of Primary Amoebic Meningoencephalitis in the United States, 1937-2013. J. Pediatr. Infect. Dis. Soc. (2014). doi:10.1093/jpids/piu103
7.         Primary Amebic Meningoencephalitis (PAM) | Naegleria fowleri | CDC. at <>
8.         Heggie, T. W. Swimming with death: Naegleria fowleri infections in recreational waters. Travel Med. Infect. Dis. 8, 201–206 (2010).
9.         Visvesvara, G. S., Moura, H. & Schuster, F. L. Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol. Med. Microbiol. 50, 1–26 (2007).
10.      Marciano-Cabral, F., MacLean, R., Mensah, A. & LaPat-Polasko, L. Identification of Naegleria fowleri in Domestic Water Sources by Nested PCR. Appl. Environ. Microbiol. 69, 5864–5869 (2003).
11.      21, P. S. | 10:15 a m A., Updated, 2015 |, 24, 5:21 p m | Aug & 2015. Rare brain-wasting amoeba suspected in San Diego death. The San Diego Union-Tribune at <>
12.      Kemble, S. K. et al. Fatal Naegleria fowleri infection acquired in Minnesota: possible expanded range of a deadly thermophilic organism. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 54, 805–809 (2012).
13.      Krainik, F., Merle, G. & Bertin, M. [Will Naegleria fowleri become a public health problem?]. Sem. Hôp. Organe Fondé Par Assoc. Enseign. Méd. Hôp. Paris 59, 775–782 (1983).
14.      Aldape, K., Huizinga, H., Bouvier, J. & Mckerrow, J. Naegleria fowleri: Characterization of a Secreted Histolytic Cysteine Protease. Exp. Parasitol. 78, 230–241 (1994).
15.      Jamerson, M., da Rocha-Azevedo, B., Cabral, G. A. & Marciano-Cabral, F. Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins. Microbiology 158, 791–803 (2012).
16.      Herbst, R., Marciano-Cabral, F. & Leippe, M. Antimicrobial and pore-forming peptides of free-living and potentially highly pathogenic Naegleria fowleri are released from the same precursor molecule. J. Biol. Chem. 279, 25955–25958 (2004).
17.      Herbst, R. et al. Pore-forming polypeptides of the pathogenic protozoon Naegleria fowleri. J. Biol. Chem. 277, 22353–22360 (2002).