OPPORTUNISTIC FUNGAL INFECTIONS Smilja Kalenic, MD, PhD Clinical Hospital Centre Zagreb, Croatia My everyday job is clinical microbiology and hospital infections in a 1670 bed University hospital. We have many compromised patients suffering from opportunistic fungal infections, and every year we can see more and more such patients and more fungal species involved. So, these infections are for me a great challenge in diagnosis and in prevention.

LEARNING AND PERFORMANCE OBJECTIVES to learn about the most frequent opportunistic fungi and to understand main risk factors for developing infection to be able to predict the most probable agent of invasive fungal infection in a particular compromised patient state and to be able to act preventively Additional Internet readings: http://www.cdc.gov/ncidod/dbmd/mdb http://www.nfid.org/publications/clinical updates/fungal http://www.doctorfungus.org/mycoses/human http://www.med.sc.edu:85/book/mycol-sta

FUNGI EUCARIOTIC ORGANISMS TWO BASIC FORMS: - YEASTS - MOLDS Yeasts are unicellular organisms, reproducing by budding and division; molds are multicellular organisms growing in filaments called hyphae, forming mycelium and reproducing by conidia and spores. Both yeasts and molds have sexual and asexual reproduction. Rare fungi have other forms (cysts, spherulas). Fungi have a rigid cell wall with mannans, glucans and chitin in it, and with ergosterol in the cell membrane.

MYCOSES 1. SUPERFICIAL 2. CUTANEOUS 3. SUBCUTANEOUS 1. Affects stratum corneum only; Malassezia furfur rarely causes opportunistic fungemia 2. Affects superficial keratinized tissue only (about 40 related fungi) 3. Fungi from soil or vegetation, reach subcutaneous tissue by traumatic inoculation; rarely cause systemic disease.

4. ENDEMIC (PRIMARY, SYSTEMIC): MYCOSES 4. ENDEMIC (PRIMARY, SYSTEMIC): Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis 4. Fungi geographically restricted to specific endemic areas. They are primary pathogens (true pathogenic fungi), living in soil mixed with guano (H. capsulatum, C. immitis), or not clearly defined soil (B. dermatitidis, P. brasiliensis). They cause pneumonia and systemic disease in a previously healthy persons.

- Candida (different species) - Pneumocystis carinii (?) MYCOSES 5. OPPORTUNISTIC endogenous - Candida (different species) - Pneumocystis carinii (?) “Endogenous” means that the fungus is a part of a normal human flora. “Exogenous”(see next slide) means that the fungus does not normaly live in/on human body, although it can transiently contaminate human body surfaces (especially respiratory tract). P.carinii is most probably a part of normal flora of lungs of many mammals, including humans.

- Cryptococcus neoformans MYCOSES 5. OPPORTUNISTIC exogenous - Cryptococcus neoformans - Aspergillus (different species) - Zygomycetes - MANY OTHER FUNGI Some of many other opportunistic fungi are: Penicillium marneffei, Fusarium, Bipolaris, Exophiala, Scedosporium, Sporothrix, Pseudallescheria. True pathogenic (endemic) fungi cause more severe infections in compromised host than in otherwise healthy people. If a patient, undergoing therapy which will compromise him/her, was exposed, he/she has to take fluconazole for the reactivation prevention.

Candida albicans and other Candida species Harmless inhabitants of the skin and mucous membranes of all humans Normal immune system keeps candida on body surfaces As Candida is present in practically all humans, it has many opportunities to cause endogenous infections in compromised host - so, Candida infections are the most frequent opportunistic fungal infections. Other Candida species are: C.tropicalis, C.krusei, C.parapsilosis, C.glabrata, C.gullermondii, C.lusitaniae, C.kefyr and many more.

MAIN DEFENSE MECHANISMS AGAINST CANDIDA I. skin and mucous membranes integrity presence of normal bacterial flora If normal bacterial flora is disturbed by antimicrobial therapy, then Candida overgrowths on mucosal surfaces, and with its pseudohyphae internalizes (translocates) itself to deeper layers and causes mucosal infections. The same happens if skin and mucous membranes integrity is broken.

MAIN DEFENSE MECHANISMS AGAINST CANDIDA II. phagocytosis killing, mostly in polymorphonuclear cells, less in macrophages T-cells (CD4) Neutrophil leukocytes are probably responsible for resistance to invasive candidiasis, and CD4 T-cells are responsible for resistance to mucocutaneous candidiasis. If neutrophil number or function is disturbed, after translocation, Candida goes to lymphatic and blood, spreading throughout the body and causing infection in virtually all organs.

THE MOST IMPORTANT RISK FACTORS 1. Neutropenia 2. Diabetes mellitus 3. AIDS 4. SCID 5. Myeloperoxidase defects 6. Broad-spectrum antibiotics 1. The most profound neutropenia (less than 100 neutrophils/L) is seen in bone marrow and hematopoietic stem cells transplant patients, but occurs also in patients with malignancies treated with intensive chemotherapy. 2. In diabetic patients, fusion of lysosome in phagocytes is greatly impaired. 3.,4. CD4 T-cells defects are important risk factors.

THE MOST IMPORTANT RISK FACTORS 7. Indwelling catethers 8. Major surgery 9. Organ transplantation 10. Neonates 11. Severity of any illness 12. Intravenous drug addicts

CLINICAL FORMS OF CANDIDIASIS 1. Cutaneous and mucosal candidiasis Oral trush, oezophagitis (AIDS patients, diabetic patients, patients on corticosteroid therapy, T-cell deficiency); vulvovaginal infection (diabetes, pregnancy, antibiotic therapy); cutaneous candidiasis (skin trauma, burns, maceration); onychomycosis; mucocutaneous candidiasis in SCID patients

CLINICAL FORMS OF CANDIDIASIS 2. Invasive (systemic, disseminated, hematogenous) candidiasis Treatment: amphotericin B (neutropenic patients), fluconazole (nonneutropenic patients), caspofungin (a new drug - inhibitor of glucan synthesis - very promising because of low toxicity and good spectrum). The bigest threat to the therapy is a development of strains resistant to fluconazole.

If phagocytic system is normal, invasive infection stops here INVASIVE CANDIDIASIS Usually begins with candidemia (but in only about 50% of cases candidemia can be proven) If phagocytic system is normal, invasive infection stops here Most cases of candidemia in surgical patients end this way. C.albicans is found in over 75% in blood culture of non-neutropenic and non-cancer patients, while other species are found in more than 50% of neutropenic and cancer patients.

mortality of candidemia is 30-40% INVASIVE CANDIDIASIS If phagocytic system is compromised, infection spreads to many organs and causes focal infection in these organs mortality of candidemia is 30-40% The most frequent organs involved are kidney, skin (maculonodular lesions), eye, heart, liver, meninges. Prevention of candida infections in severely immunocompromised patient can be done by use of peroral fluconazole (azole antifungal drug) during deepest immunosupression

DIAGNOSIS OF INVASIVE CANDIDIASIS Gram stain and isolation from blood, CSF or peritoneal fluid isolation and/or pathology positive of organ involved other tests are of lower significance for the diagnosis CSF - cerebrospinal fluid Other tests: isolation from urine, respiratory secretions, wound secretion; isolation from organs without pathology slide positive; serology; antigens and metabolites detection PCR - promising, not yet standardized

EPIDEMIOLOGY Although candidiasis is endogenous in most cases, cross infections are described, especially in intensive care unit patients. Handwashing is the most important activity to prevent spread of many hospital pathogens, and of Candida too.

Pneumocystis carinii Present in lungs of many mammals, including humans, in persistent but harmless infection P.carinii is present worldwide and seroepidemiological studies show that most humans are infected in early childhood. Natural reservoir, the source and mode of transmission are not known, possibly P.carinii spreads by aerosols.

Main defense mechanism is T-cell mediated Pneumocystis carinii Main defense mechanism is T-cell mediated causes interstitial pneumonitis in compromised patients treatment and prevention: cotrimoxasole or pentamidine Main risk factors are AIDS, transplantation, corticosteroid and antineoplastic therapy. About 20% of AIDS patients will develop P.carinii pneumonia despite prophylaxis, if CD4 count is >100 mm3. Diagnosis is made from bronchoalveolar fluid, induced sputum or lung biopsy - smears are stained with special stains, and the presence of cysts and trophozoits is diagnostic. P.carinii has not yet been isolated.

Cryptococcus neoformans Occurs worldwide in soil and in bird droppings Prominent feature: thick polysaccharide capsule, which causes evasion from phagocytosis Cryptococci are found in large numbers in dry pigeon feces. In macrophages Cryptococci can survive and grow easily.

MAIN DEFENSE MECHANISMS AND PATHOGENESIS T-cells responsible for defense Cryptococcus reaches humans by inhalation of aerosolized yeast cells Main risk factors are T-cell deficiency (AIDS patients), corticosteroid therapy, organ transplantation, hematological malignancy. About 10% of AIDS patients develop cryptococcal infection. Yeast cells are inhaled, and in otherwise healthy humans can cause asymptomatic or mild pneumonia; if risk factors are present, cryptococci spread through blood and have special predilection for meninges, but can disseminate in different organs too.

CHRONIC MENINGITIS IN AIDS-PATIENTS The most important clinical syndrome treatment: amphotericin B+/-flucytosine recurrence prevention: fluconazole Diagnosis is made from CSF - India ink smear (capsule!), isolation or capsular polysaccharide antigen test

EPIDEMIOLOGY OF CRYPTOCOCCOSIS Infection is always exogenous, is not transmitted from human to human

Aspergillus species Aspergilli are worldwide occurring saprophytes, living in soil and on plants; they have small conidia that form aerosols Most frequent species causing infections in compromised patients are: A.fumigatus, A.flavus, A.niger, A.terreus, A.nidulans and many other species. Especially abundant are aspergilli conidia when buildings are done and various dusts are spread around.

Main defense mechanism is phagocytosis Main risk factors are hematological malignancy, bone marrow transplantation and corticosteroid therapy In patients with heart, liver and bone marrow transplantation, invasive aspergillosis can be found in as much as 25-40% of cases (post mortem mycology). Although aspergilli can disseminate to many organs, invasive pulmonary aspergillosis (IPA) is found in 70% of cases.

The most frequent syndromes are: - aspergilloma - invasive aspergillosis (high mortality rate) Treatment: amphotericin B, itraconazole, flucytosine and surgery Prevention: avoid exposure to conidia (new buildings in the hospital!) Aspergilloma is formed when conidia are inhaled in a preexisting lung cavity (tuberculosis, emphysema); if there is not a cavity, aspergilli develop in lung tissue causing invasive infection (spreading through the tissue and involving blood vessels); then spread can occur to other organs. Diagnosis of aspergilloma is radiological (CT scan); invasive aspergillosis can be diagnosed from respiratory secretions or lung biopsy; test for circulating galactomannan is also diagnostic

Zygomycetes are ubiquitous saprophytes main host defense is phagocytosis main risk factors are diabetes, hematological malignancies, corticosteroid therapy Most frequent genera in the class Zygomycetes, causing disease in compromised host, are: rhisopus, rhizomucor, absidia, mucor, cunningamella.

Major clinical syndrome is: Rhinocerebral mucormycosis (infection of nasal passages, sinuses, eyes, cranial bones and brain) Treatment: surgery and amphotericin B Prognosis: very poor Pulmonary infection can also occur, with very high mortality rate. Diagnosis is made by direct smear and by isolation of molds from respiratory secretions or biopsy specimens.

OPPORTUNISTIC FUNGAL INFECTIONS ARE: difficult to diagnose difficult to treat difficult to prevent more and more frequent a great challenge for a future work in all fields