1. Fascinoma Rounds Penicillium marneffei October 26th, 2005 Sharmistha Mishra, Vanessa Allen, And with great thanks to Subash Mohan

2. Case #1: Penicillium marneffei What are the clinical risk factors for acquiring disseminated penicillium marneffei? What are the laboratory features of this organism (courtesy of Subash Mohan)? What precautions should be taken in the laboratory environment?

3. Clinical Case 32 M originally from Vietnam, no known PMH Moved to Canada 16 years ago Travel to Vietnam every year, –last trip in January/ February 2005 Illness since March/April 2005 –Cough, SOB and 18 lb weight loss –went to family MD and to a local hospital treated for CAP with azithromycin –V/Q scan negative –CT scan consistent with CAP Also had two month history of non-pruritic papular rash on face which he attributed to lobster allergy

4. Case continued Sept 22nd –Presented with SOB, fever, and bilateral chest infiltrates –Started on Ceftraixone and azithromycin for CAP Laboratory values –Hgb 95 (MCV 75), Leuk 4.4 (0.3 lymphs), platelets 233 –Electrolyes and creatinine normal –AST 113, ALT 109, ALP 100, bili 5 –LDH 514

7. Case #3 Bronchoscopy Sept 23rd –Positive for H. influenza –PCP –And ….Penicillium marneffei –Blood cultures from Sept 22nd became positive for Penicillium marneffei –Subsequent HIV+, CD4 32

8. Discovery 1956  bamboo rats in Vietnam First human case = accidental innoculation in the lab (1959) from a needle Then 1973  in pt with Hodgkin’s disease 1985 – pt with HIV in tropical medicine course  suspicion of inhalation of spores! 1988 onwards  rising # of cases in HIV+ pts from endemic areas

9. Epidemiology Bamboo rat & association unclear Soil exposure felt to be a risk factor Inhalation, ingestion, skin puncture - postulated Geographical distribution –Thailand, Myanmar (Burma), Vietnam, Cambodia, Malaysia, northeastern India, Hong Kong, Taiwan, and southern China –Imported disease from patients from these endemic regions

11. Clinical Risk Factors More commonly seen in immunocompromised (also occurs in immunocompetent individuals) –29 cases diagnosed 1959-1988 prior to HIV –Now  80% have HIV CD4 < 70 cells/mm 3 –Among HIV + pts in N. Thailand 1. TB 2. Cryptococcal meningitis 3. Penicillium marneffei –Other risk factors are lymphoproliferative disorders, bronchiectasis and tuberculosis, autoimmune diseases and corticosteroid therapy Supparatpinyo K, et al.. Lancet. 1994;344:110-113.

12. Clinical Features Localized disease Disseminated disease –Fever (99%) –Anemia (78%), –Pronounced weight loss (76%) –Generalized lymphadenopathy (58%) –Hepatomegaly (51%). –Skin lesions, most commonly papules with central necrotic umbilication (71%) –Pneumonia (CXR abnormalities)

15. Penicillium marneffei Penicillium marneffei is a dimorphic fungus of RES Grows as mycelium at RT and yeast at 37 0 C Usually attacks immunocompromised hosts P. marneffei can also attack immunocompetent hosts Reservoir – bamboo rat in south east Asia Laboratory safety precautions similar to Coccidioides Direct microscopy frequent confusion with Histoplasma

16. Penicillium marneffei Direct Microscopy Specimen: BAL (E1232193) Stain: Fungi-Fluor x400 Structure: small, round, oval cells non-budding cells Compare with yeast. Rule out histoplasma. Differentiation difficult.

17. Penicillium marneffei Direct Microscopy Specimen: BAL (E1232193) Stain: Gram stain x1000 Structure: few oval cells non-budding mimic yeast Observe septum

18. Penicillium marneffei Direct Microscopy Specimen: BAL (E1232193) Stain: KOH x400 Structure: cluster of cells poorly differentiated appear non-budding Rule out yeast Interpretation difficult

19. Penicillium marneffei Direct Microscopy Specimen: Blood Stain: Gram stain x1000 (E1221874) Structure: septate hyphae right angle branching fragmenting Arthroconidia Appear converted Observe branched pattern Not dichotomous

20. Penicillium marneffei Direct Microscopy Specimen: Sputum Stain: GMS x1000 (E1222049) Structure: cluster of small, round, oval cells non-budding bisected cells Septum rules out yeast Suspect P. marneffei A second type yeast not P. marneffei

21. Penicillium marneffei Direct Microscopy Specimen: Sputum Stain: GMS x1000 (E1222049) Structure: bisected cells no budding seen no septate hyphae no pseudohyphae Go ahead and call it P. marneffei non-budding bisected cells

22. Penicillium marneffei Macroscopic Morphology Medium: IMA Morphology: rapid growth mycelial phase red pigment suspect P. marneffei Not all red pigment producing Penicillium species are Penicillium marneffei

23. Penicillium marneffei Microscopic morphology Stain: Lactophenol x400 Structures: phialides metulae bi- or univerticiallate brush type ID: Penicillium sp. Compare with Paecilomyces

24. Penicillium marneffei Conversion to Yeast Phase Medium: Blood agar Incubation: 37 0 C glabrous & matted no mycelium yeasty consistency pigmentation lost Conversion phase at higher temperature is essential for confirmation DNA probe not available

25. Penicillium marneffei Confirming conversion Wet Preparation: BA 37 0 C Magnification: x1000 Structures: arthroconidia multiplies by fission bisected cells Compare with yeast and pseudohyphae

26. Treatment Sensitive to Itraconazole, Ketoconazole and Ampho B Failure rates in a study of 86 HIV-infected patients were as follows: amphotericin B, 8 of 35 patients (22.8%); itraconazole, 3 of 12 (25%); and fluconazole, 7 of 11 (63.6%) Current recommendation –Amphotericin B, 0.6 mg/kg/day for 2 weeks, followed by itraconazole, 400 mg/day orally in two divided doses for the next 10 weeks –97.3% effective in 74 HIV + individuals Sirisanthana T, Clin Infect Dis. 1998;26:1107-1110.

27. Lab Safety and Penicillium marneffei No formal guidelines Inhalation and direct inoculation are possible mechanisms of transmission in lab.