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  1. Fungal Case Studies:New Lessons Karin L. McGowan, Ph.D., F(AAM), M.S. SM(NRCM) Professor Emeritus, Departments of Pathology & Laboratory Medicine and Pediatrics Perelman School of Medicine University of Pennsylvania

  2. Disclosure • I have no affiliations with commercial organizations or their products, research, or services related to this presentation. • At this time, I am not receiving commercial support of any kind including stock options, nor research support.

  3. Case #1 • The patient is a 22 year old white male with CGD who we have followed since he was 11. • He presented to the Immunology clinic with a mass pressing on his mediastinum. • Since early January, 1 month prior, he had been suffering from chest pain which was relieved with motrin….because of the constant pain he was brought in 3 months early for his yearly chest MRI & CT.

  4. Case #1 • His chest CT revealed new nodules in his right & left upper lobes plus a right middle lobe infiltrate. • We have known for the past 2 years that he has had a mediastinal mass but on his current CT & MRI the mass has not changed. • He has no fever or rhinorrhea but does have a mild cough.

  5. CGD: Chronic Granulomatous Disease • Chronic granulomatous disease (CGD) is a rare genetic disorder, it occurs in 1 in every 200,000 people (Amish). CGD results from an inability of phagocytes to produce bactericidal superoxide anions (O2). • The bacteria get phagocytized but not killed. This leads to recurrent life-threatening bacterial and fungal infections. The rest of their immune system functions normally.

  6. Chronic Granulomatous Disease • CGD was first described in the 1950s as a syndrome of recurrent infections in males who all died in the first decade of life. 4 out of 5 children with CGD are boys. • CGD gets its name from the characteristic granulomas that develop in response to these patients having chronic infections and inflammation.

  7. Chronic Granulomatous Disease • Bacteria and fungi that cause most infections in CGD are catalase-positive organisms. S. aureus and B. cepacia are the most common bacteria seen early in the life of CGD patients. • Fungal infections occur in as many as 20% of patients with CGD. The most common pathogens are Aspergillusfumigatus,Candida glabrata, and Candida albicans.

  8. Chronic Granulomatous Disease • A. nidulanshas emerged as a problem pathogen in CGD. It causes disseminated disease that is more lethal than that caused by A. fumigatus. Pneumonia is the most common presentation. • In a recent review of young adults with CGD, Aspergillus was the leading cause of death from molds. As a result, most patients are kept on treatment doses of itraconazole after their first serious fungal infection.

  9. Case #1 • Past History: • Gets IVIG every 3 weeks from his local MD (to prevent infections; he’s allergic to Bactrim) • He has low T cell numbers • Since 2002 has been on ciprofloxacin for recurrent sinusitis • Has been on itraconazole for the past 9 years following Aspergilluspneumonia • Takes Zantac daily for GERDS (gastro-esophageal reflux disease)

  10. Case #1 • Physical Exam: afebrile, systolic murmur, decreased breath sounds, no acute distress. • Labs: fairly normal except for an increased WBC count (15.1/mm3, 90% polys). • Because of his history a lung biopsy was performed: • Gram Stain: Moderate WBCs, No Organisms • Pathology/Histology: All Negative, including GMS • Fungal Culture: mold growing at 48 hours

  11. Paecilomyces: Microscopic • Paecilomycesspp. show chains of single celled conidia produced in succession from a phialide.  • Conidia are formed in chains with the youngest at the base. • Phialides are elongated and taper into a long, slender tube. Phialides are more tapered than Penicilliumand are swollen at their bases, they may form a brush-like penicillus.

  12. Paecilomycesvariotti

  13. Paecilomyceslilacinus

  14. Case #1 • Because this patient has been on itraconazole for the past 9 years, the lab was asked to obtain antifungal MICs on this isolate. • Distinguishing between species is clinically important because P. variottiand P. lilacinushave marked differences in their in vitro susceptibility to antifungals. P. variottimay be resistant to voriconzole, P. lilacinusis usually resistant to itraconazole. Testing of individual strains is highly recommended.

  15. PaecilomycesvariottiMIC Results

  16. Case #1 • Despite the terrific antifungal susceptibility results, because this patient is known for being highly compliant, the team following this patient was now ‘confused’. • They asked the microbiology lab to repeat the antifungal susceptibility testing… another lab. • We said No. We suggested they consult the clinical pharmacist.

  17. Woops, Pharmacology • When the hospital clinical pharmacist was asked to consult on the case, everyone was embarrassed to be informed that GERD drugs such as Zantac decrease intraconazole levels by as much as 60%! • Whenever he was an inpatient, our pharmacy had also participated in this error and had given him both drugs.

  18. Treatment • Our patient received a month of liposomal amphotericin B and was then discharged onposaconazolewhich he will take for a full year. • Voriconazole and itraconazole resistant strains of Paecilomyces spp. have been reported…so distinguishing between the two species is clinically very important -- your lab should be able to do this!

  19. Treatment • Our patient was then scheduled for a bone marrow transplant which is expected in a year when he is proven to be culture negative. • The cure for CGD is now bone marrow transplant. Many of our patients have undergone the procedure and are expected to live long lives……yea

  20. Case 2 • BT is a 10 year old boy diagnosed in the winter of 2009 with malignant fibrous histiocytoma of bone who completed chemotherapy and radiation therapy 1 year later. •  He remained symptom-free for months and during that time he traveled with his parents to western NY and southern CA for brief vacations to visit family.  

  21. Case 2 • In August 2010 a routine chest CT scan (performed to monitor for potential metastases) revealed right upper and lower lung nodules. • In retrospect, his parents agreed that he’d had a persistent nonproductive cough for the previous 3-4 weeks. He and they denied any other signs and symptoms including fever.

  22. Because physicians were concerned that he had a metastatic tumor, lung biopsies were performed immediately. Pathology reported that no tumor cells were seen but that “yeast-like organisms” were seen in both nodules. • The cells were thinned-walled, oval and elliptical, and varied in size from 8 to 15 microns in diameter.

  23. Lung Biopsy • Because the morphology on Gomorimethamine silver stain was consistent with Cryptococcus neoformans, a mucicarmine stain to visualized the capsule was performed.

  24. When the mucicarmine stain was negative, and a serum Cryptococcal antigen test was also negative, pathology stayed with the diagnosis of “yeast-like organisms”. • Fortunately, by this time cultures in mycology were positive for a yeast….which was capsule negative but rapid urease positive.

  25. The isolate biochemically identified as Cryptococcus neoformans(Vitek 2)….. thus we had a capsule-deficient isolate. • Pulmonary infection by capsule-deficient Cryptococcus neoformans (CDCN) is a very rare form of pneumonia. • CDCN were originally described in the 1980’s in patients with AIDS and we learned then that pulmonary cryptococcosis due to CDCN does not differ clinically from pulmonary cryptococcosis due to encapsulated C. neoformans.

  26. C. neoformansGenome Project • Many genes that effect virulence have been identified and they include: making a polysaccharide capsule, melanin, urease, and phospholipase. • With the exception of capsule formation, all of the virulence factors are single-gene traits. • We now know at least 8 separate genes effect capsule formation. GPA1 = major gene encoding for a protein that effects capsule formation.

  27. Histopathologic Identification of CDCN • Diagnosis of CDCN via tissue section analysis alone is difficult because many other nonencapsulated fungi, such as C. immitis, B. dermatitidis, and H. capsulatumcan mimic CDCN morphologically. • These organisms all stain negative with mucicarmine the same as CDCN.

  28. Histopathology • The Fontana-Masson stain, originally developed in 1912 and later modified by Masson in 1928, detects melanin. • C. neoformanspossesses the enzyme phenol oxidase that converts phenolic compounds to melanin. Melanin accumulates in the organism cell wall and can be detected with this stain. • Among pathogenic fungi, only S. schenckiiand dematiacious fungi stain positive with FMS stain like Cryptococcus because of the melanin in their cell walls.

  29. Fontana-Masson Stain-Cryptococcus

  30. Treatment • Pulmonary cryptococcosis due to CDCN does not differ clinically from pulmonary cryptococcosis due to encapsulated C. neoformans…..the capsule is just one virulence factor. • Our patient completed a 2-week course of amphotericin B followed prolonged (~18M) fluconazole therapy. His cough resolved in 1M and follow-up chest CT scans reveal no signs of recurrent disease.

  31. Case 3 • The patient is a 16 year old girl admitted in September with fever (102.2oF), sweating, chills, myalgias, and a nonproductive cough. • 6 months earlier she had been diagnosed with acute lymphoblastic leukemia and is in the final phase of her chemotherapy. • She was receiving trimethoprim-sulfamethoxazole 3 x/week for prophylaxis of Pneumocystisjiroveciinfection and had received her chemotherapy 2 weeks prior.

  32. Case 3 • On admission her absolute neutrophil count was zero. Her pulse was 118, respiratory rate 26, her blood pressure was 115/65 mm Hg, and her PE was normal. IV ticarcillin-clavulanate and gentamicin was initiated. • Her chest X-ray revealed a ‘subtle opacity’ at the edge of the lower lobe of her left lung; antibiotics were immediately changed to vancomycin & cefotaxime.

  33. Case 3 • 5 days after admission, the patient’s cough produced blood-tinged sputum and a repeat chest X-ray showed that the subtle opacity had enlarged to a large nodular mass. • Amphotericin B was initiated and a BAL done the same day was unrevealing and both culture and pathology reports were negative. • She was then taken to surgery which disclosed a firm brown mass of 3 x 1.5 cm.

  34. Case: Histology • Histologically the mass showed central necrotic lung parenchyma, with large arteries containing masses of hyphae. • The hyphae were broad, “mostly” non-septate, and wavy and showed predominantly right-angled branching. • Multinucleated giant cells were also seen at the edge of the necrotic area.

  35. Case: Microbiology • In less than 24 hours, a mold was growing on chocolate agar incubated at 350C, and on inhibitory mold agar (IMA) and brain-heart infusion agar with blood plus gentamicin and chloramphenicol incubated at 250C. Agar with cyclohexamide was not showing growth. • Growth was rapid with a white/light grey surface and pushed up against the lid of the petri dish (which was sealed).

  36. Mold: Microscopic • Broad hyphae, rare septation, with sporangiophores that are long, branched, and end in swollen vesicles. • The vesicles heads are covered with oval spores (sporangiola) supported by short needle-like stalks. The spores are often covered with crystals. • No rhizoids are present with this organism.

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