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Pythium insidiosum:
Diagnosis and Treatment
Robert L. Glass
Pan American Veterinary Laboratories
Pythium insidiosumPythium insidiosum
Kingdom StramenipilaKingdom Stramenipila
Oomycete “aquatic fungus”Oomycete “aquatic fungus”
Hyphal elements similar to FungiHyphal elements similar to Fungi
Primarily an infectious agent of plantsPrimarily an infectious agent of plants
PythiosisPythiosis
PhycomycosisPhycomycosis
Florida Horse LeechesFlorida Horse Leeches
Swamp CancerSwamp Cancer
Species Infected bySpecies Infected by Pythium insidiosumPythium insidiosum
CanineCanine Australia, Brazil, Korea, USA and VenezuelaAustralia, Brazil, Korea, USA and Venezuela
EquineEquine Australia, Brazil, Colombia, Costa Rica, New Zealand,Australia, Brazil, Colombia, Costa Rica, New Zealand,
New Guinea, USA and VenezuelaNew Guinea, USA and Venezuela
FelineFeline USAUSA
BovineBovine Brazil, USA and VenezuelaBrazil, USA and Venezuela
HumanHuman Australia, New Zealand, Haiti, India ThailandAustralia, New Zealand, Haiti, India Thailand
and the USAand the USA
BearBear USAUSA
CamelCamel USAUSA
Incidence of Pythiosis in the USIncidence of Pythiosis in the US
1995-2003 158 cases (all species)
2004 72 cases (all species)
2005 (Jan-Aug) 102 cases (all species)
2010 752 cases (all species)
EpidemiologyEpidemiology
Oospore
Life Cycle
Skin
SkinSkin
injuryinjury
Skin
PythiosisPythiosis
ClinicalClinical Characteristics of pythiosisCharacteristics of pythiosis
- CutaneousCutaneous KeratinitisKeratinitis
NecrosisNecrosis
- SubcutaneousSubcutaneous LymphaticLymphatic
PeriorbitalPeriorbital
- ArterialArterial Large arteriesLarge arteries
AneurysmAneurysm
- IntestinalIntestinal Intestinal tractIntestinal tract
- SystemicSystemic LiverLiver
LungsLungs
FelineFeline PythiosisPythiosis
CanineCanine PythiosisPythiosis
Intestinal pythiosis in canines is often misdiagnosed as lymphoma.
Bovine PythiosisBovine Pythiosis
VenezuelaVenezuela
EquineEquine PythiosisPythiosis
““kunkers”kunkers”
Chronic PythiosisChronic Pythiosis
Osteo infectionOsteo infection
Human pythiosis in the USAHuman pythiosis in the USA
Tennessee 1998Tennessee 1998 Texas 1989Texas 1989
Human Pythiosis, ThailandHuman Pythiosis, Thailand
IgGIgG
IgMIgM
Pythiosis
Experimental
Laboratory Diagnosis
1) Very few laboratories are familiar with
P. Insidiosum
2) Difficult to culture
3) Difficult to identify in histopath studies
4) Difficult to identify by clinical
presentation
Conidiobolus coronatusConidiobolus coronatus Basidiobolus ranarumBasidiobolus ranarum
Fungal zygomycetesFungal zygomycetes
Pythium insidiosumPythium insidiosum
OomyceteOomycete
Differential
Diagnosis
Wet mountWet mount
10% KOH10% KOH Immune-fluorescenceImmune-fluorescence
CultureCulture
HistopathologyHistopathology
immunoperoxidaseimmunoperoxidase
Tissue Biopsy Studies
Serologic DiagnosisSerologic Diagnosis
Agar Gel Immunodiffusion
Western Blot
Classical treatments used
against Pythium insidiosum
Surgery ~40%
Iodines ~35-40%
Amphotericine B <30%
Itraconazole <30%
What is Pythium Immunotherapy?
Allergy desensitization via Subcutaneous
presentation of purified P. insidiosum antigens
Switching of the of the immune response
from TH2 (allergic) to TH1 mode
(protective and curative)
ImmunotherapyImmunotherapy
T helper 2 responseT helper 2 response
EosinophilsEosinophils
Mast cellsMast cells
IgEIgE
IL5IL5
IL4IL4
T helper 1 responseT helper 1 response
MacrophagesMacrophages
T lymphocytesT lymphocytes
(cytotoxic?)(cytotoxic?)
IFNIFN
IL2IL2
ImmunotherapyImmunotherapy
Pythium
Infection
Infection
Cleared
Before ImmunotherapyBefore Immunotherapy Day 4Day 4 Day 8Day 8 Day 15Day 15
Pythium antibody in Canines participating in FieldPythium antibody in Canines participating in Field
Trial Training and CompetitionsTrial Training and Competitions
Result #Dogs % of TotalResult #Dogs % of Total
Negative 105 61.4Negative 105 61.4
Borderline 43 25.1Borderline 43 25.1
PositivePositive 23 13.523 13.5
Total 171 100Total 171 100
* Normal Dogs <1 % Positive ,<5% Borderline, >95% Negative* Normal Dogs <1 % Positive ,<5% Borderline, >95% Negative
Pythium Associated Symptoms in Field Trial/HuntPythium Associated Symptoms in Field Trial/Hunt
CaninesCanines
VOMITING/DIARRHEAVOMITING/DIARRHEA
IN LAST 12 MONTHSIN LAST 12 MONTHS
Blood testBlood test
Negative 7 (6.7%)Negative 7 (6.7%)
Borderline 3 (7.0%)Borderline 3 (7.0%)
Positive 13 (26.1%)Positive 13 (26.1%)
Efficacy of Pythium Immunotherapy
Studies Conducted 2004-2005Studies Conducted 2004-2005
Species # Treated # Successes # Failures %Success
Equine 58 52 6 90%
Canine 28 15 13 53%
Bovines 65 65 0 100%
Efficacy of Pythium V2 Immunotherapy
In Canines
Studies Conducted 2007-2008Studies Conducted 2007-2008
# Treated # Successes # Failures %Success
14 10 4 71%
ImmunotherapyImmunotherapy
BeforeBefore
AfterAfter
BeforeBefore
AfterAfter
Treatment
Regimen
1cc Sub Q
Day 1
Day 7
Day 21
8/22/03 9/27/03
11/25/0
3
3/17/04
Immunotherapy
Treatment
Progression
Surgery + ImmunotherapySurgery + Immunotherapy
Immunotherapy
♦ Purified protein extract of P. insidiosum
♦ Early treatment increases success rate
♦ Use as first option treatment
♦ >85% effective in equines, 50% effective in
canines
♦ Combine with surgical de bulking in chronic
cases
• Pythium insidiosum is endemic in
tropical and subtropical regions
• Differential diagnosis includes
subcutaneous mycosis, lymphoma,
parasitic and bacterial infections
• Serologic diagnosis is fast and accurate
• Itraconazole, Amphotericin B, Iodine and
surgery have shown limited success
• Immunotherapy alone or in conjunction with
other therapies demonstrates highest success
rate
Lagenidium gigantumLagenidium gigantum
Similar to Pythium in classSimilar to Pythium in class
Similar to Pythium in diseaseSimilar to Pythium in disease
characteristicscharacteristics
Essentially identical to Pythium inEssentially identical to Pythium in
tissue sectionstissue sections
Does not respond to PythiumDoes not respond to Pythium
ImmunotherapyImmunotherapy
Accurate Diagnosis is EssentialAccurate Diagnosis is Essential
Clinical presentation is not goodClinical presentation is not good
enoughenough
Histopath examination is not goodHistopath examination is not good
enoughenough
Pythium test alone is not good enoughPythium test alone is not good enough
Accurate diagnosis requires blood testAccurate diagnosis requires blood test
for both Pythium and Lagenidiumfor both Pythium and Lagenidium

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Pythiosis General

  • 1. Pythium insidiosum: Diagnosis and Treatment Robert L. Glass Pan American Veterinary Laboratories
  • 2. Pythium insidiosumPythium insidiosum Kingdom StramenipilaKingdom Stramenipila Oomycete “aquatic fungus”Oomycete “aquatic fungus” Hyphal elements similar to FungiHyphal elements similar to Fungi Primarily an infectious agent of plantsPrimarily an infectious agent of plants PythiosisPythiosis PhycomycosisPhycomycosis Florida Horse LeechesFlorida Horse Leeches Swamp CancerSwamp Cancer
  • 3. Species Infected bySpecies Infected by Pythium insidiosumPythium insidiosum CanineCanine Australia, Brazil, Korea, USA and VenezuelaAustralia, Brazil, Korea, USA and Venezuela EquineEquine Australia, Brazil, Colombia, Costa Rica, New Zealand,Australia, Brazil, Colombia, Costa Rica, New Zealand, New Guinea, USA and VenezuelaNew Guinea, USA and Venezuela FelineFeline USAUSA BovineBovine Brazil, USA and VenezuelaBrazil, USA and Venezuela HumanHuman Australia, New Zealand, Haiti, India ThailandAustralia, New Zealand, Haiti, India Thailand and the USAand the USA BearBear USAUSA CamelCamel USAUSA
  • 4. Incidence of Pythiosis in the USIncidence of Pythiosis in the US 1995-2003 158 cases (all species) 2004 72 cases (all species) 2005 (Jan-Aug) 102 cases (all species) 2010 752 cases (all species)
  • 8. ClinicalClinical Characteristics of pythiosisCharacteristics of pythiosis - CutaneousCutaneous KeratinitisKeratinitis NecrosisNecrosis - SubcutaneousSubcutaneous LymphaticLymphatic PeriorbitalPeriorbital - ArterialArterial Large arteriesLarge arteries AneurysmAneurysm - IntestinalIntestinal Intestinal tractIntestinal tract - SystemicSystemic LiverLiver LungsLungs
  • 10. CanineCanine PythiosisPythiosis Intestinal pythiosis in canines is often misdiagnosed as lymphoma.
  • 13. Chronic PythiosisChronic Pythiosis Osteo infectionOsteo infection
  • 14. Human pythiosis in the USAHuman pythiosis in the USA Tennessee 1998Tennessee 1998 Texas 1989Texas 1989
  • 15. Human Pythiosis, ThailandHuman Pythiosis, Thailand
  • 17. Laboratory Diagnosis 1) Very few laboratories are familiar with P. Insidiosum 2) Difficult to culture 3) Difficult to identify in histopath studies 4) Difficult to identify by clinical presentation
  • 18. Conidiobolus coronatusConidiobolus coronatus Basidiobolus ranarumBasidiobolus ranarum Fungal zygomycetesFungal zygomycetes Pythium insidiosumPythium insidiosum OomyceteOomycete Differential Diagnosis
  • 19. Wet mountWet mount 10% KOH10% KOH Immune-fluorescenceImmune-fluorescence CultureCulture HistopathologyHistopathology immunoperoxidaseimmunoperoxidase Tissue Biopsy Studies
  • 20. Serologic DiagnosisSerologic Diagnosis Agar Gel Immunodiffusion Western Blot
  • 21. Classical treatments used against Pythium insidiosum Surgery ~40% Iodines ~35-40% Amphotericine B <30% Itraconazole <30%
  • 22. What is Pythium Immunotherapy? Allergy desensitization via Subcutaneous presentation of purified P. insidiosum antigens Switching of the of the immune response from TH2 (allergic) to TH1 mode (protective and curative)
  • 23. ImmunotherapyImmunotherapy T helper 2 responseT helper 2 response EosinophilsEosinophils Mast cellsMast cells IgEIgE IL5IL5 IL4IL4 T helper 1 responseT helper 1 response MacrophagesMacrophages T lymphocytesT lymphocytes (cytotoxic?)(cytotoxic?) IFNIFN IL2IL2 ImmunotherapyImmunotherapy Pythium Infection Infection Cleared
  • 24. Before ImmunotherapyBefore Immunotherapy Day 4Day 4 Day 8Day 8 Day 15Day 15
  • 25. Pythium antibody in Canines participating in FieldPythium antibody in Canines participating in Field Trial Training and CompetitionsTrial Training and Competitions Result #Dogs % of TotalResult #Dogs % of Total Negative 105 61.4Negative 105 61.4 Borderline 43 25.1Borderline 43 25.1 PositivePositive 23 13.523 13.5 Total 171 100Total 171 100 * Normal Dogs <1 % Positive ,<5% Borderline, >95% Negative* Normal Dogs <1 % Positive ,<5% Borderline, >95% Negative
  • 26. Pythium Associated Symptoms in Field Trial/HuntPythium Associated Symptoms in Field Trial/Hunt CaninesCanines VOMITING/DIARRHEAVOMITING/DIARRHEA IN LAST 12 MONTHSIN LAST 12 MONTHS Blood testBlood test Negative 7 (6.7%)Negative 7 (6.7%) Borderline 3 (7.0%)Borderline 3 (7.0%) Positive 13 (26.1%)Positive 13 (26.1%)
  • 27. Efficacy of Pythium Immunotherapy Studies Conducted 2004-2005Studies Conducted 2004-2005 Species # Treated # Successes # Failures %Success Equine 58 52 6 90% Canine 28 15 13 53% Bovines 65 65 0 100%
  • 28. Efficacy of Pythium V2 Immunotherapy In Canines Studies Conducted 2007-2008Studies Conducted 2007-2008 # Treated # Successes # Failures %Success 14 10 4 71%
  • 32. Immunotherapy ♦ Purified protein extract of P. insidiosum ♦ Early treatment increases success rate ♦ Use as first option treatment ♦ >85% effective in equines, 50% effective in canines ♦ Combine with surgical de bulking in chronic cases
  • 33. • Pythium insidiosum is endemic in tropical and subtropical regions • Differential diagnosis includes subcutaneous mycosis, lymphoma, parasitic and bacterial infections • Serologic diagnosis is fast and accurate • Itraconazole, Amphotericin B, Iodine and surgery have shown limited success • Immunotherapy alone or in conjunction with other therapies demonstrates highest success rate
  • 34. Lagenidium gigantumLagenidium gigantum Similar to Pythium in classSimilar to Pythium in class Similar to Pythium in diseaseSimilar to Pythium in disease characteristicscharacteristics Essentially identical to Pythium inEssentially identical to Pythium in tissue sectionstissue sections Does not respond to PythiumDoes not respond to Pythium ImmunotherapyImmunotherapy
  • 35. Accurate Diagnosis is EssentialAccurate Diagnosis is Essential Clinical presentation is not goodClinical presentation is not good enoughenough Histopath examination is not goodHistopath examination is not good enoughenough Pythium test alone is not good enoughPythium test alone is not good enough Accurate diagnosis requires blood testAccurate diagnosis requires blood test for both Pythium and Lagenidiumfor both Pythium and Lagenidium

Editor's Notes

  1. Subcutaneous No draining tracts
  2. Cutaneous Intestinal infection occurs in +/- 50% of infected dogs Peri-anal cutaneous lesions develop secondary to intestinal lesions
  3. Usually cutaneous Up to 5% annual herd loss in herds studied in Venezuela
  4. Results in 1-2 days vs 2-3 weeks for histopath Sensitivity &gt;95% Specificity &gt; 99% 1 cc serum required Cost $40 per sample
  5. Surgery must be done in first 30 days of infection with complete removal of Pythium “ Wide and Deep” Equine Immunotherapy &gt;90% at 30 days &gt;75% at 60 days &gt;60% at 90 days Canines probably similar, but most intestinal cases present at &gt;90 days
  6. Intense allergic/inflammatory response leading to tissue death and necrosis
  7. Top Eosinophil/mast cell changing to Macropahge/lymphocyte Bottom Hyphae death and removal
  8. Historical Equines overall 65-75% (90% in 30 day dropping to 50% in 90 day) (in this study 3 of 6 failures were &gt;90 day infections) Canines overall 35% Lower due to advanced Intestinal infections
  9. 8/22/03 initial trmt 9/27/03 90 days 11/25/03 120 days 3/17/04 240 days
  10. In advanced cases de-bulking has proven helpful. Removes a large part of the Pythium allowing immuno destruction of remaining inection.