The importance of epidemiology in the diagnosis of invasive fungal infections J Peter Donnelly BSc PhD Department of Haematology University Medical Centre St Radboud Nijmegen, The Netherlands

Some issues Because Hence Microscopy and culture are essentially unavailable to microbiologists with respect to invasive fungal infections (IFI) Because IFI commonly affects the lungs initially but cases can easily go unnoticed Even when recognized early, suitable specimens can be difficult to obtain Tests for detecting fungal pathogens in clinical material (particularly blood) are available, but there is no consensus about their clinical utility Hence many expect nothing from diagnosis and do not even attempt to make one. The resulting lack of adequate diagnoses makes estimating the prevalence and incidence of IFI unreliable.

Epidemiology This dismal state of affairs serves only to emphasize the importance of epidemiology since, in order to determine the value of any diagnostic test or battery of tests, one has to know the underlying prevalence of the disease, particularly when this is low. The first questions are:- Who gets IFI? What do they get and how? When do they get?

Who gets IFI?

Candidaemia in French hospitals Incidence/ 1000 admissions Total 0.29 General hospital 0.17 Teaching hospital 0.38 Cancer center 0.71 tropicalis glabrata krusei albicans parapsilosis origin central line 26% digestive tract 11% unknown 43% Andremont et al, ICAAC 1998, San Diego

Risk factors for invasive candidosis Risk factor Cancer ICU Neutropenia, HSCT, chemotherapy, GvHD, mucosal barrier injury Candida colonisation Broad-spectrum antibiotics Haemodialysis, azotemia Central venous catheter Severity of illness Hyperalimentation Recurrent/persistent GI tract perforation Prior surgery Neonatal ICU (age, low APGAR, LOS, shock, H2 blockers, intubation) Rex & Sobel Clin Infect Dis 2001 32;1191

Incidence of invasive fungal infections among solid organ transplant recipients Transplant IFI Renal 1.4 - 14 Heart 5 - 21 Liver 7 - 42 Lung & heart/lung 15 - 35 Small bowel 40 - 59 Pancreas 18 - 38 Aspergillus Candida Singh Clin Infect Dis. 2001 31: 545

Timing of fungal infections after solid organ transplant CMV Candida Aspergillus Cryptococcus Endemic fungi Pneumocystis 1 2 3 4 5 6 7 8 Snydman Clin Infect Dis. 2001 33: S5

Mc Neil et al 2001 Clin Infect Dis 33;641 Incidence of fatal fungal infections amongst patients other than those with HIV in the USA Candidiasis Aspergillosis Mc Neil et al 2001 Clin Infect Dis 33;641

Invasive candidiasis, colonisation and bacteraemia 81 patients NO YES 46 35 Bacteraemia 0% 53% Colonisation - + ++ - + ++ 14 24 8 7 13 15 Colonization is a very prominent factor particularly when it follows treatment of a bacteremia (with antibiotics) Acute Invasive Candidiasis 1 1 8 Guiot et al, Clin Infect Dis 1994; 18:525-32 641

MBI and invasive Candida infections Risk group Colonisation Mucosal barrier injury Treatment Low no no no Intermediate no yes no yes no yes or no* High risk yes yes yes * depending on other predisposing factors

Invasive aspergillosis and underlying disease Condition range (%) Chronic granulomatous disease 25-40 Lung ± heart transplant 19-26 Liver transplant 1.5-10 Heart & renal transplant 0.5-10 AIDS 0-12 SCID 3.5 Burns 1-7 SLE 1 Acute leukaemia 5-24 Allogeneic HSCT 4-9 Autologous HSCT (no growth factors) 0.5-6 Autologous HSCT (with growth factors) <1 Denning Clin Infect Dis 2001 26 pp781-805

Incidence of invasive aspergillosis under various conditions heart/heart-lung transplant chronic granulomatous disease acute leukemia allogeneic haematopoietic stem cell transplant autologous + growth factor solid organ transplant AIDS 0 10 20 30 40% Denning. Clin Inf Dis 1998

Incidence of invasive aspergillosis in transplant recipients Transplant type incidence (%) Lung 8.4 Haematopoietic stem cell 6.4 Autologous 2.6 Allogeneic Related donor 6.7 Unrelated donor 10.3 Heart 6.2 Liver 1.7 Pancreas 1.3 Kidney 0.7 Paterson et al. Medicine 1999;78:123-38.

Aspergillosis at autopsy - sites of infection 1187 autopsies 1993 - 1996 48 (4%) aspergillosis aspergillosis Lungs only Disseminated CNS only Disseminated (not lungs) Vogeser et al Eur J Clin Microbiol Infect Dis 1999;18; 42-45

Explaining the current trends in opportunistic fungal infections Increase in number of susceptible hosts New medical methods HSCT - CD 34+ selection Advances in surgical techniques for solid transplant immunesuppressive regimens for solid transplant More conservative approach Less use of corticosteroids Use of novel agents Antimicrobial prophylaxis Fluoroquinolones for Gram negative bacilli Fluconazole for Candida Ganciclovir for CMV Improved laboratory expertise detection identification Singh Clin Infect Dis 2001 33;1692

Haematological malignancy Who gets IFI? ICU Haematological malignancy Allogeneic HSCT Liver Lung HIV Invasive fungal infections CGD Heart Transplant Burns Renal

What do they get and how?

Huh. You guys get all the attention The main players Opportunity knocks! Huh. You guys get all the attention Fusarium Aspergillus Candida Hi Bud! Hi pal!

How do they get it?

Candida - colonisation Candida albicans Candida tropicalis Candida parapsilosis Candida albicans Candida albicans Candida glabrata Candida krusei

Model for invasive candidiasis GI tract insult antibiotics Normal commensal flora injury selection infection Candida species Disease translocation

Aspergillosis

Aspergillus from the breeze or the bucket Graybill Clin Infect Dis 2001 26 pp781-805

What do they get and how ? Mainly Candida albicans or Aspergillus fumigatus prior colonisation with Candida species is a prerequisite for infection Spores of Aspergillus and other moulds are inhaled directly through the air or indirectly from aerosols of contaminated water

When do they get it?

Time to diagnosis of aspergillosis after BMT 20 18 16 14 12 10 8 6 4 2 Cases 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 >180 days after transplant Wald et al J Infect Dis 1997:175;1459

Aspergillosis following HSC transplant PRE-TRANSPLANT ENGRAFTMENT neutropenia corticosteroids EARLY POST-ENGRAFTMENT LATE POST-ENGRAFTMENT 41 40 39 Temperature °C 38 37 36 10 Stem cells acute GvHD chronic GvHD low IgG Granulocytes (log10 1x 106/L) 1 0.1 -7 7 14 21 12 6 9 -14 28 8 10 Transplant Days Weeks Months

Source of stem cells and GVHD Cutler et al 2002 J Clin Oncol 19:3685-3691

Source of stem cells and GVHD Cutler et al 2002 J Clin Oncol 19:3685-3691

HEPATOSPLENIC CANDIDIASIS FEVER ALKALINE PHOSPHATASE NEUTROPHILS DISSEMINATION MICROCOLONIES "BULLS EYE"

when do they get? Both candidiasis and aspergillosis occur during neutropenia but also manifest themselves later after bone marrow recovery. Patients are at risk of aspergillosis for as long as they have active GvHD or are receiving high dose corticosteroids

Diagnosis

Sites of infection Yeast Moulds Aspergillus fumigatus Mucor Candida parapsilosis Candida albicans Fusarium species Candida albicans Candida tropicalis Candida albicans Candida glabrata Candida krusei

Defining invasive fungal infection Host factor Clinical feature Mycology Ascioglu et al 2002 Clin Infect Dis 34:7-14 Invasive Fungal Infections Cooperative Group Mycoses Study Group

Defining infection - Host factors neutropenia > 4 days unexplained fever despite broad spectrum antibiotics Graft versus Host Disease > 3 weeks corticosteroids <36°C or > 38°C and prior mycosis AIDS Immunosuppressives > 10 days neutropenia Host factor Ascioglu et al 2002 Clin Infect Dis 34:7-14 Invasive Fungal Infections Cooperative Group Mycoses Study Group

Defining infection - Clinical features Halo sign Air-crescent sign cavity Lower respiratory tract infection Sinonasal infection CNS infection Disseminated fungal infection Chronic disseminated candidiasis Radiological evidence Unexplained papular or nodular skin lesions Chorioretinitis endophthalmitis Bull’s eye lesions in liver or spleen MAJOR Clinical feature Ascioglu et al 2002 Clin Infect Dis 34:7-14 Invasive Fungal Infections Cooperative Group Mycoses Study Group

Defining infection - Clinical features Cough, chest pain, haemoptysis, dyspnoea Physical finding of pleural rub Any new infiltrate not fulfilling major criterion Lower respiratory tract infection Sinonasal infection CNS infection Nasal discharge, stuffiness Nose ulceration, eschar or epistaxis Periorbital swelling Maxillary tenderness Black necrotic lesions or perforation of the hard-palate CSF No pathogens no malignant cells abnormal biochemistry abnormal cell count Focal neurological seizures hemiparesis cranial nerve palsy Mental changes Meningeal irritation MINOR Clinical feature Ascioglu et al 2002 Clin Infect Dis 34:7-14 Invasive Fungal Infections Cooperative Group Mycoses Study Group

Defining infection - Mycology Culture of mould from tissue, aspirate BAL or sputum mould seen in sinus aspirate Fungi seen in tissue or sterile body fluids Mycology antigen in BAL, CSF or blood Ascioglu et al 2002 Clin Infect Dis 34:7-14 Invasive Fungal Infections Cooperative Group Mycoses Study Group

Proven invasive fungal infective disease Host factor Clinical features + Tissue + Mycology + Ascioglu et al 2002 Clin Infect Dis 34:7-14 Invasive Fungal Infections Cooperative Group Mycoses Study Group

Probable invasive fungal infective disease Host factor Clinical features + Mycology + Ascioglu et al 2002 Clin Infect Dis 34:7-14 Invasive Fungal Infections Cooperative Group Mycoses Study Group

Possible invasive fungal infective disease Clinical features + Host factor Mycology OR Ascioglu et al 2002 Clin Infect Dis 34:7-14 Invasive Fungal Infections Cooperative Group Mycoses Study Group

EORTC/MSG definitions of fungal infections -aspergillosis Host factor Clinical features Mycology + + GVHD antigenaemia Probable disease Halo sign on chest CT scan OR cough + pleural rub

EORTC/MSG definitions of fungal infections -candidosis Host factor Clinical features Mycology Neutropenia + Small, peripheral, target-like abscesses (Bull’s eye) in liver and/or spleen demonstrated by CT, MRI or ultra sonogram. Probable disease +/- elevated alkaline phosphatase

Strategy

Aim of the strategy include all patients likely to have aspergillosis and treat them pre-emptively with the safest and most effective drug AND exclude all patients unlikely to have the disease and adopt a WAIT-and-SEE policy By using techniques that offer a high negative predictive value i.e. a low false-negative rate

Risk factor selection + Risk factors diagnosis AML HSC transplant febrile Radiology Mycology febrile Pulmonary Infiltrate Antigen +

Screening test for a potentially fatal disease with a low prevalence ruled out withhold treatment Controls Tests + - - - ± + not ruled out start treatment

A strategy for managing pulmonary aspergillosis At risk 3 x weekly GM-ELISA no ELISA GM positive OR > 5 d unexplained fever abnormal chest X-ray yes CT scan EORTC/MSG criteria

Microbiological evidence A strategy At risk Clinical features yes no Microbiological evidence yes probable no possible yes no unlikely eg. CT scan halo-sign or air-crescent sign Prevalence ≥ 10% yes no Pre-emptive therapy therapy wait-and-see

Obtaining a specimen - tools of the trade Bronchoscopy Sputum Bronchoalveolar lavage Fine needle aspirate Biopsy Brush Lung biopsy

Bronchoscopy specimen - processing BAL 10-20 mL Culture Aerobic bacteria S. pneumoniae, Ps aeruginosa Enterobacteria Klesiella spp Legionella spp Mycobacteria M. tuberculosis Nocardia spp Mycoplasma spp Yeasts Candida spp Moulds A. fumigatus Virus CMV, HSV, RSV 10-20 mL cytology Cytospin Gram Giemsa Silver Acid-fast stain IFA -Legionella Centrifuge 500 g 5 min Shell vial culture IFA -CMV -HSV - RSV influenza A & B Martin et al 1987 Mayo Clin Proc 62:549-557

Invasive Fungal Infection Mycology Clinical features Host factors + tissue = Proven Mycology Clinical features Host factors + Probable = Clinical features + Host factors Negative or Not done = Possible Mycology + Host factors Negative or Not done

Conclusions Patients at risk are better known The timing of the risk is better understood Diagnosis is improving Criteria now exist for defining invasive mycosis