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Pythium insidiosum

History of the Infections Caused by Pythium insidiosum

Equine Pythium insidiosum (Pre-treated)

    The first reports of infections caused by P. insidiosum were published last century in equines with cutaneous granulomas in Australia, Florida (USA), India and Indonesia. Its true etiology, however, was not established. The first well documented cases in which a "fungal-like" etiology was suggested were published by Smith, Fish, and Drouin (J. Mycol Med. 6:151; 1996). Perhaps the important event in the history of pythiosis occurred when two Dutch investigators, (de Haan and Hoogkamer) isolated for the first time (1901) the organism from several cases of equine pythiosis. Their isolated could not be identified as it did not sporulate (a requirement for its identification). These investigators did not suggest a name for the organism, but they named the disease hyphomycosis destruens (mycotic disease of destructive character). Another Dutch investigator, J. Witkamp (1924) in Indonesia, isolated the organism for the second time, developed several serological tests for it diagnosis, and published the most comprehensive paper on this disease at that time (Ned. Ind. Bland. Diergenaeskd Dierenteelt 36:229;1924).

     Sporadic reports of the disease in equines were made during the beginning of the 20th century. This in part was due to the finding that cutaneous granulomas in equines may also be cause by nematodes species of the genus Habronema. This is why the term used to describe pythiosis were also used for equine cutaneous habronemiasis. The early finding on the "fungal nature" of cutaneous granulomas in equines, therefore, was obscured by the new hypothesis of equine habronemiasis, and the theory fell into oblivion. Thirty seven years after J. Witkamp's studies, Bridges and Emmons report the disease in several horses in Texas. They isolated, for the third time in the history of this disease, a sterile filamentous organism. They believe it was close to the fungus Mortierella. However, the name Hyphomyces destruens (coined from the name previously proposed by de Haan and Hoogkamer) was proposed to describe this sterile filamentous isolate (JAVMA 38:579; 1961).

    In 1974, Austwick and Copland reported that an organism isolated from equine cutaneous granulomas in New Guinae produced biflagellated zoospores in water and identified it as Pythium spp. A similar finding was reported later by Ichitani and Amemiya in Japan from cases of granular dermatitis in equines. They identified the isolate as Pythium gracile. In 1987, the binomial Pythium insidiosum was proposed by de Cock et al., (J. Clin. Microbiol. 25:344;1987). These investigators found that all P. insidiosum isolated from humans and animals, including the Japanese isolate, belonged to the single species P. insidiosum. A strain isolated from an Astralian horse with the disease was later named P. destruens by Shipton  (J. Med. Vet. Mycol.25:137;1987). However, this strain proved to be a synonym of P. insidiosum.

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Biology of Pythium insidiosum

Figures 1 and 2  show Pythium insidiosum hyphae drawn by Jan Witkamp, a Dutch investigator in 1924.  He was the second researcher to isolate P. insidiosum in pure cultures.

    The genus Pythium comprises about 200 species. Pythium species are common pathogens causing disease in plants and fishes. The species of this genus are among the most destructive plant pathogens, inflicting serious economic losses of crops by destroying seed, storage organs, roots, and other plant tissues. So far, Pythium insidiosum is the only species reported to cause infections in mammals. The disease caused by this unique microorganism has been termed pythiosis (insidiosi) and can cause life threatening infections in mammals and birds.

    Members of the genus Pythium have been described as "aquatic fungi". However, they are not true fungi (Kingdom Fungi), they belong to the Kingdom Stramenopila, Phylum Oomycota, Class Oomycetes (Peronosporomycetes), Family Pythiaceae. In culture, P. insidiosum develops sparsely septate fungal-like hyphae similar to those produced by the Zygomycetes (true fungi). Like other Oomycetes, P. insidiosum produces motile zoospores (asexual stage) when exposed to damp conditions. The zoospores are single cells with two lateral flagella that swim to find a new plant host where it completed its life cycle. Once in contact with the host the zoospores lose their flagella and encyst. It is believed that zoospores act as infecting units once in contact with a mammalian host (J. Mycol. Med. 6:151; 1996). Under conditions, still under investigation, P. insidiosum develops globose oogonia (sexual stage) typical of this species, but they very difficult to obtain  in most isolates.

    Like other oomycetes, P. insidiosum grows relatively well on a variety of media. On corn meal agar and Sabouraud dextrose, colonies are colorless to white, submerged with short aerial mycelium and a finely radiate pattern. The coenocytic hyphae range between 4 and 12 mm in diameter with 90° perpendicular lateral branches. Septation is only occasionally observed in early hyphae, but they become abundant in old viable hyphae. Hyphal swellings, that mimic sporangia measuring 12 to 28 in diameter, are common in laboratory cultures.

    Production of zoospores can be induced in water cultures containing minimal quantities of different ions and grassleaves at 37°C. Zoospores induction in water without ions is rare. Early sporangia cannot be differentiated from normal hyphae. This early sporangia, at maturity, flows their cytoplasm into a discharge tube and form a globose sporangium. The cytoplasmic content of the sporangium goes through progressive cleavage and biflagellated zoospores are formed inside a vesicle. The zoospores mechanically break the sporangium vesicle wall and swim approximately 20-30 minutes and then encyst. It is believe that P. insidiosum use a plant to complete its life cycle in nature (lily or grass), but this is yet to be confirmed.

    LIFE CYCLE. Pythium insidiosum, like other Pythium spp, need wet environments to carried out their life cycle in nature. It also can grow in wet soil with grass because its ability to produce resistant spores. Several investigators have shown that this pathogen requires some plants to complete its life cycle in nature (lily and gramineae (grass). It is believed that the zoospores are the infecting units. After exposure to water containing zoospores they will be attracted by small wounds in the host's skin, intestinal tract and other sites.  The zoospores will encyst and then produced hyphae that will, mechanically, penetrate the tissue and cause the disease. The majority of cases in equines occur through open wounds on the skin of the extremities and areas in contact with water or grass. In Dogs, the disease is observed in the skin and the intestinal tract. This is due to the fact that dogs may drink water and/or eat grass contaminated with P. insidiosum.

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Epidemiology of Pythium insidiosum

    In areas where the disease is commonly observed, pythiosis occurs during the summer months especially after periods of high pluvial precipitation. It was noted that horses that had grazed for a period of time in stagnant water frequently developed pythiosis. However, cases of pythiosis in equines, dogs, and humans never exposed to stagnant water, indicated that the infection can also be acquired after contact with soil and grass containing resting structures of P. insidiosum (perhaps oogonia).

    Due to its occurrence in wet environments and summer months the disease has been termed: swamp cancer, Florida horse leeches, summer sores, burusattee (means rain in India), espundia, granular dermatitis and others.

    Pythium insidiosum has been reported more frequently in tropical and subtropical regions of the world. However, cases in temperate areas of Japan and USA indicate that this organism can be found in cooler environments as well. Well documented cases of the disease have been reported in Australia, the Pacific islands, Asia, and the Americas. It would seem that the tropical climate of much of Africa would make it suitable for P. insidiosum. But so far only one case of dog pythiosis in Bamako, Mali has been recorded (Emerg. Infect. Dis. 11:479;2005).

    In the Americas, the disease is known to occur in North, Central and South America as well as the Caribbean islands. In the United States, the disease is more commonly reported in the states along the Gulf of Mexico: Alabama, Florida, Louisiana, Mississippi, and Texas. However, several cases in dogs equines, and humans have occurred in states such as Georgia, Illinois, Missouri, North, Oklahoma, and South Carolina, Tennessee (human), New Jersey, New York, and as far north as Wisconsin.

    Cases in Central America have also been reported in Guatemala, Nicaragua, Costa Rica, and Panama. In South America, Argentina, Colombia, Venezuela, and Brazil had also reported the disease.

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Diagnosis of Pythiosis

    The early diagnosis of pythiosis is of importance for its successful treatment. Common mistakes in diagnosis of P. insidiosum occurs frequently because it mimics other infections, and most healthcare professionals will treat with antibiotics, which does not cure this disease.  Several methodologies have been used for its diagnosis: wet mount preparations, culture, histopathology, and serology.

    Wet Mounts. The collected tissue from the infected areas is sent to the laboratory in sterile distilled water at room temperature. Pieces of the tissue is placed with 10% KOH. The finding of sparsely septate hyphae may be indicative of P. insidiosum, or other fungal pathogen (zygomycetes).

    Culture. To isolate this organism is important to remember that P. insidiosum is highly inhibited by low temperatures. Those, transportation of the biopsied tissue in ice will decrease the chance to isolate this pathogen in culture. Samples, therefore, should be send to the laboratory in water and at room temperature. Small pieces of the biopsied tissue should be placed onto Sabouraud dextrose agar plates and incubated at 37°C. This temperature is ideal for P. insidiosum primary isolation. Incubations at room temperature delay its growth rate. After 24 to 48 hours small colonies should observed around the tissue. Pythium insidiosum growth rapidly at 37°C, thus in 5 days the plate would be cover by a submerged filamentous fungi-like colony.

    Microscopically, hyphae without sporulation is observed. To identify P. insidiosum the formation of  zoospores has to be induced in water cultures with, grass leaves, and some ions. Sporangia containing zoospores (asexual stage) will be observed at edges of the grass. The production of oogonium (sexual stage) is difficult and rarely found in the media commonly used in clinical labs.

    Histopathology. Tissue sections of the biopsied samples in H&E shows the typical eosinophilic inflammatory reaction discussed in animal and human disease. The hyphae of P. insidiosum, however, is difficult to observed with this stain. Silver stain and Periodic Acid-Schiff (PAS) are suggested for the proper identification of the hyphal elements of P. insidiosum in tissue. A differential identification of P. insidiosum and fungi developing filamentous structures, especially the zygomycetes, has to be performed.

     Serology.  Several serologic tests have been developed to diagnose pythiosis in humans and animals. They are: Complement fixation, immunodiffusion, Enzyme-Linked Immunosorbent  Assay, immunoperoxidase assay, fluorescent antibodies, and western blot.

     Complement fixation. Was developed in Australia for the diagnosis of equine pythiosis. It is a sensitive test but it is not specific. This test is no longer in use in laboratories dealing with P. insidiosum.

    Immunodiffusion ID. This is the most popular serological test to diagnose pythiosis in humans and animals. The test is entirely specific, but it is highly sensitivity. The finding that the sera from some humans and dogs with proven pythiosis gave negative results in this test indicated that the diagnosis using ID has to be confirmed with a more sensitive test. Still, the ID test is recommended as an screen test for the putative diagnose of pythiosis in humans and animals.

    Enzyme-Linked Immunosorbent Assay ELISA. This assay was introduced to be overcome the drawbacks of the ID test. The ELISA for P. insidiosum is specific and sensitive. This assay detected all proven cases of pythiosis in humans and dogs negative by the ID. It also showed to be helpful in detecting cases of cat, cattle, and equines with the disease. Our laboratory offers this ELISA test service to the national and international community. In addition, Pan American Veterinary Labs (www.pavlab.com, Tel: 800-856-9655) a Texan company offers ELISA, culture, fluorescent antibodies in fixed tissues, and molecular tools, such as PCR, to diagnose pythiosis.

    Immunoperoxidasel assay. This assay was developed to detect specifically the hyphae of P. insidiosum in the biopsied tissue from humans and animals. The technique use polyclonal antibodies against P. insidiosum and the Ag-Ab reaction is detected with a  peroxidase assay. The technique is as specific as the culture.

    Fluorescent antibodies. This technique was developed to diagnosis pythiosis from fixed tissue samples and to identify P. insidiosum from culture. The technique specifically detected P. insidiosum hyphae and gave negative results when tested against Entomophthorales and Mucorales zygomycetes. Our laboratory offers this service to the national and international community. For details go to www.pavlab.com, Tel: 800-856-9655.

    Western blot. This test have been mainly used for research purposes. It detects different antigenic proteins of P. insidiosum with sera from patients with Pythiosis. It is sensitive and specific.

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Cat Pythiosis

Dog Pythiois

    Two main features of dog pythiosis are the cutaneous and subcutaneous lesions and the gastrointestinal involvement. Dogs acquire the infection through open wounds on the skin and mucous membranes. The fact that dogs frequently drink stagnant water and eat grass that may contain elements of P. insidiosum explains the high number of intestinal pythiosis cases in canines.

    The cutaneous and subcutaneous lesions are denuded of hair and perforated by fistulous sinus tract, that discharge a serosanguineous exudate. They are usually located on the dog’s tail, legs, thorax, and abdomen. The hard stony masses (kunkers) observed in equine pythiosis are not present in dogs with the disease. Microscopically, multifocal areas of necrosis with eosinophils and a moderate number of neutrophils and macrophages is the main feature of the infection. The hyphae of P. insidiosum are found in the center of eosinophilic microabcesses.

    Canine gastrointestinal pythiosis is characterized by vomiting, weight loss, and sporadic diarrhea. Formation of hard gastrointestinal tumor-like masses, areas of mural thickness, and mucosal ulceration are common. The organism can spread to adjacent tissue such as pancreas, uterus, and mesenteric lymph nodes. Histopathologically, the mucosa shows ulceration, atrophy, and hyperplasia. Eosinophils, plasma cells, macrohages, epithelioid cells and giant cells are detected in those tissues. The hyphae of P. insidiosum, however, is difficult to detect. Special stain, such as silver stain are required to visualized the hyphae of this pathogen in the infected tissues.

    TREATMENT. Because dog pythiosis is relatively a new disease in this specie, most small animal veterinary practitioners are not familiar with its clinical features. Most cases of cutaneous pythiosis are initially diagnosed and treated as bacterial or parasitic infections, whereas gastrointestinal pythiosis is often missdiagnosed as intestinal tumoral-like disease (neoplastic) and treated by surgery (removal of the tumoral masses).

     -SURGERY. Because pythiosis lesions progress rapidly a quick diagnosis is essential for animal survival. Once the diagnosis of pythiosis have been established, wide surgical removal of the infected tissues is the only effective treatment for cutaneous, subcutaneous, and intestinal dog pythiosis.

     -DRUGS. Antifungal therapy using amphotericin B, itraconazole, ketoconazole, or trebinafine has been unsuccessful in most cases.

     -IMMUNOTHERAPY. Treatment with the therapeutic vaccine used to cure cases of equine pythiosis has been only 55% effective in dogs with pythiosis. This may be due in part that all vaccinated cases were dogs with chronic pythiosis more than two months old. The vaccine has yet to be evaluated in more cases of dog pythiosis to determine its true cure rate in this species. Immmunotherapy has also been used in humans with the disease. About 55% of the treated human patients with pythiosis in Thailand, some of them with arterial involvement, did respond to this approach. This procedure was used once in a child with orbital pythiosis in the USA without success (unpublished data).

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Equine Pythiosis

    Equine pythiosis is characterized by the development of cutaneous, subcutaneous, lymphatic vessels, intestinal, granulomatous lesions and less frequently by the involvement of bones and lungs (chronic pythiosis). Lesions caused by P. insidiosum can occur on any anatomical area, but it is more common on the lower limbs because they first come in contact with the organism in infested environments (stagnant water, grasses). The lesions often occur as a single lesion, but cases with multiple granulomas have been encountered. There are no reports of animal to animal, or animal to human transmission of this pathogen. If the disease is not treated in the early stages (usually surgical removal of the granulomas) it becomes life threatening. In most cases chemotherapy with antifungal drugs is not helpful.

    Lesions on the limbs are characterized by the formation of tumoral-like masses with fistules and a serosanguineous discharge. Lesions on the thorax, abdomen, and shoulders, tend to be circular, 5 to 500 mm in diameter. Ulceration and pruritus (itch) is commonly associated with large lesions on these sites. The formation of small hard coral-like masses termed "kunkers" is an interesting characteristic of the disease in equines. These stony masses contained the viable hyphae of P. insidiosum surrounded by cell detritus from degranulated eosinophils. Metastasis (spread) from distant lesions, through lymphatic vessels to regional lymph nodes, lungs, or bones have been reported.  As in dog pythiosis, intestinal equine pythiosis is more likely to be acquired from direct inoculation of the organism through ingestion, than spread from distant lesions.

    Histopathologically, in early equine pythiosis, abundant microabcesses with eosinophils a few neutrophils, lymphocytes, and macrophages are present. In chronic cases, an eosinophilic granuloma with giant cells is observed. In the center of the microabcesses, stony masses (kunkers) are often present. With PAS and Silver stains P. insidiosum appears as sparsely septate hyphae 6 to 10 mm in diameter.

    TREATMENT. -SURGERY. The most common treatment of equine pythiosis has been the surgical removal of the lesions. This method is very popular and frequently used by veterinary practitioners. In general the response to surgery is limited (40%). A common drawback of surgical treatment is its high rate of recurrence. This is mostly due to the incomplete removal of the P. insidiosum hyphae from the affected tissues. More importantly, lesions of the limbs are very difficult to treat by surgery due to the presence of key anatomical structures in these areas.

     -CHEMOTHERAPY. Two main groups of antifungal drugs have been used to treat pythiosis: Iodine and amphotericin B. Both drugs, however, have given contradictory results. For instance, some practitioners reported that iodine had cured the disease after intravenous injections, whereas others reported failures with the same procedures.  In theory, owing the fact that this pathogen does not have ergosterol in its cytoplasmic membrane (target of the drug) amphotericin B should not work on P. insidiosum. Nevertheless, the drug has been used with some success in equine pythiosis. The use of drugs in pythiosis has been limited because of cost, poor success rate, and high toxicity.

     -IMMUNOTHERAPY. In the early 80's a therapeutic (curative) vaccine against P. insidiosum infections in equines was developed in Australia and Costa Rica. This early immunotherapeutic vaccine cures 100% of the acute cases (less than 15 days after infection) but it fails to cure chronic equine pythiosis (two month and more after infection). It was found that chronic cases often become anergic due to the lost of large quantities of proteins, electrolytes and water through the open wounds. Thus, the immunotherapeutic vaccine works better in equine with intact immune system (early pythiosis).

Human Pythiosis

     Human pythiosis is characterized by the formation of subcutaneous lesions and the invasion of the main arteries. If not treated the infection is fatal. The majority of the cases have been diagnosed in Thailand. The disease has been also reported in Australia, Haiti, India, and the USA. The organism is acquired through traumatic implantation and remains localized or spreads to infect other tissues, especially arteries. The diagnosis of the disease in humans is based in culture, serology, and histopathology. In all cases hyphae of this oomycete are present in the infected tissues. Serological test such as ID and ELISA have proved to be of value for its early diagnosis.
    
     The main clinical forms of the disease includes: keratitis, orbital disease (in children), arterial pythiosis, and a subcutaneous form on the limbs and other anatomical areas. Keratitis is difficult to differentiate from cases of keratitis caused by the hyaline filamentous fungi. The clinical manifestations and the microscopic characteristic of the clinical samples sent to the laboratory are identical. Culture is the only tool to make the appropriate diagnosis. The problem is that only few laboratories are familiar with this organism. So, mast cases are misdiagnosed. Something similar had happened with orbital pythiosis in USA children. Since zygomycetous fungi can also cause orbital disease, and their filamentous hyphae are very similar to that developed by P. insidiosum in the infected tissues, some cases has been in the past misdiagnosed as cases of zygomycosis. Arterial pythiosis is more frequently diagnosed in Thailand.    

    -Treatment.   The treatment of human subcutaneous pythiosis on limbs, in which the arteries have been involved, consists of the amputation in the affected extremity. Iodides and other drugs have been used with questionable results. More recently the vaccine used to treat equine pythiosis, was successfully used in cases of human pythiosis with 55% cure rate. In addition, a combo of itraconazole and terbinafine cure a Tennessee boy with orbital disease. However, this combination had failed in other similar cases of pythiosis in humans and animals.

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Serological and Molecular Tests

800-856-9655

    The Pan American Veterinary Laboratories www.pavlab.com, Tel: 800-856-9655 offers three serological tests to diagnose the infections caused by Pythium insidiosum in cats, dogs, horses, and humans. The following serological test can be requested:  

     Immunodifussion test (ID). This is a very specific but insensitive (false negatives) test for pythiosis. Dogs and humans are usually negative in this test. It is recommended in cases of horses with suspected pythiosis. About 40% of dogs and human with active pythiosis reacted negative in ID.

    ELISA. This is sensitive and specific test for pythiosis in all species.

    Fluorescent Antibodies for tissue samples. This is a very specific and sensitive test. It detects the hyphae of P. insidiosum in the infected tissues.

    PCR for tissue samples. This is a very sensitive test recommended when culture is not possible.

    Culture, and identification of P. insidiosum from clinical samples is also available.

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