Challenges in the diagnosis of Invasive Mould Diseases What are we looking at? Challenges in the diagnosis of Invasive Mould Diseases Alessandro C. Pasqualotto pasqualotto@santacasa.tche.br Porto Alegre, Brazil

Potential conflicts of interest Research Grants Myconostica, Pfizer, Merck, Sigma-Tau, CAPES, CNPq, Fungal Research Trust Travel Grants Pfizer, United Medical, Schering (now Merck), Bagó, Merck Speaker honoraria Pfizer, United Medical, Merck, Schering (now Merck), Biometrix Este slide e’ rapido

IFD are highly lethal diseases First assumption: IFD are highly lethal diseases Este slide fala por si, em especial se considerarmos infeccoes por fungos filamentosos

Incidence 13.3% in lung transplant recipients Apresento entao a sobrevida entre tx de pulmao que tiveram ou nao aspergilose. Incidence 13.3% in lung transplant recipients Xavier MO, Pasqualotto AC, et al. ECCMID 2009

Rapidly evolving diseases Dizer entao que a evolucao e’ muito rapida, em paciente imunocomprimido, particularmente neutropenicos. Usar o termo agressivo ja desde o inicio, para acostumar a plateia com o que vira a seguir (a importancia de sermos agressivos). 4 days later www.aspergillus.org.uk

Disseminated infection Sem tratamento e com persistencia de neutropenia, as infeccoes podem disseminar para o SNC, momento em que a mortalidade e’ entre 90-100%. www.aspergillus.org.uk

We need to intervene asap Second assumption: We need to intervene asap Este slide fala por si

Early versus late intervention Mortality rate (%) Entao precisamos de um dx precoce, o que pode mudar o desfecho. No entanto, e’ preciso que saibamos o que estamos tratando Von Eiff, et al. Respiration 1995; 62: 241-7

Early versus late intervention Mortality rate (%) Von Eiff, et al. Respiration 1995; 62: 241-7

But how can we achieve such an early diagnosis? O que queremos e’ a verdade! Dizer com certeza que o paciente tem IFD. Mas isto requer biopsia e cultura, o que nao e’ alcancavel em geral na pratica clinica

A small black scar + serum GM Patient died 2 days earlier 1 day after this picture was taken Vejam por ex este caso, de um paciente com leucemia aguda. Doenca com rapida evolucao para obito. GM positiva, mas que adianta, uma vez que o paciente ja esta morto neste estagio?

Zygo + A. flavus A small black scar + serum GM Patient died 2 days earlier + serum GM Patient died 1 day after this picture was taken Zygo + A. flavus

MD Anderson Cancer Centre Necropsy study over a 15-years period IFD detected in 31% over 1,017 necropsies Antemortem diagnosis in only 25% Em hospital de referencia, e’ incrivel que a maioria dos dx ocorram pos-mortem apenas Chamilos G, et al. Haematologica 2006; 91: 986-9

MD Anderson Cancer Centre Necropsy rate has reduced over time % Mas infelizmente estamos fazendo menos e menos necropsia Chamilos G, et al. Haematologica 2006; 91: 986-9

By the way, what is the necropsy rate in your institution? >40% 10-39% 1-10% <1% Are you kidding? Comentar aqui que histopatologia e’ a unica maneira de comprovar a presenca da infeccao

MD Anderson Cancer Centre Trends in the prevalence of IFD % Chamilos G, et al. Haematologica 2006; 91: 986-9

CT scan and galactomannan No need to worry! CT scan and galactomannan are there to help us out!

‘Halo sign’ surrounding a nodule Day 0: halo Day 4: size, halo Day 7: air crescent Tanto o halo como o sinal do ar crescente sao manifest altammente sugestivas. No entanto, o halo e’ fugaz e precoce. Se nao tivermos politica agressiva de TC, nao faremos o dx Caillot, et al. J Clin Oncol 1997; 15: 139-47

The sign is not specific for IA Vasculitis Metastasis Pseudomonas infections Zygomycosis and other angio-invasive infections Esta tambem presente em muitas lesoes vasculares, incluindo zigomicose! Greene RE, et al. Clin Infect Dis 2007; 44: 373-9

Absence of typical findings at chest CT scan COPD Steroids Other non-neutropenic patients / ICU Lung transplant recipients ? Monoclonal antibodies

Organising cryptogenic pneumonia ‘Reversed halo sign’ Organising cryptogenic pneumonia Importante lembrar tambem do sinal do halo reverso, que pode sugerir zigo Wahba H, et al. Clin Infect Dis 2008; 46: 1733-7

‘Reversed halo sign’ Review of 189 cases of invasive mould disease Overall frequency 4% Wahba H, et al. Clin Infect Dis 2008; 46: 1733-7

‘Reversed halo sign’ Review of 189 cases of invasive mould disease Overall frequency 4% Zygomycosis 19% Aspergillosis <1% Fusariosis 0% (p<0.01) Wahba H, et al. Clin Infect Dis 2008; 46: 1733-7

Other predictors of zygomycosis >10 nodules Pleural effusion Concomitant sinusitis Treatment with voriconazole Lembrar do paciente que tivemos – bem como da nossa estatistica do HCPA Chamilos G, et al. Clin Infect Dis 2005; 41: 60-6

Meta-analysis of GM testing Low PPV High NPV Pfeiffer CD, et al. Clin Infect Dis 2006; 42: 1417-27

Caution with low +ve indexes! Reproducibility Caution with low +ve indexes! Ja acumulamos alguma experiencia com o teste e observamos variacao importante … nao se pode usar na presenca de ictericia … Upton A, et al. J Clin Microbiol 2005; 43: 4796-800

GM release by non-Aspergillus fungi Penicillium marneffei Geotricum capitatum Acremonium species Alternaria alternata Rhodotorula rubra Trichophyton species Paecilomyces variotii Botrytis tulipae Cladosporium species Exophiala dermatitidis Agora discutir GM Aquino VR, Goldani LZ, Pasqualotto AC. Mycopathologia 2007; 163: 191-202

Cross-reaction with GM testing % Xavier MO, Pasqualotto AC, Severo LC. Clin Vaccin Immunol 2009; 16: 132-3

Clinical case 19 year-old man, refractory leukaemia Febrile neutropenia Amox-clav for E. Coli bacteremia Maertens J, et al. Clin Infect Dis 2004; 39: 289-90

Clinical case 19 year-old man, refractory leukaemia Febrile neutropenia Amox-clav for E. Coli bacteremia Daily GM determination D1 after antibiotic: GM index of >1.5 5 +ve tests afterwards Maertens J, et al. Clin Infect Dis 2004; 39: 289-90

Clinical case 19 year-old man, refractory leukaemia Febrile neutropenia Amox-clav for E. Coli bacteremia Daily GM determination D1 after antibiotic: GM index of >1.5 5 +ve tests afterwards Fluoroquinolone: gradual reduction in GM index Maertens J, et al. Clin Infect Dis 2004; 39: 289-90

Clinical case 1 wk later Pipe-tazo for appendicitis GM >2.5; bilateral nodular infiltrate Maertens J, et al. Clin Infect Dis 2004; 39: 289-90

Clinical case 1 wk later Probable IA (EORTC / MSG) Pipe-tazo for appendicitis GM >2.5; bilateral nodular infiltrate Probable IA (EORTC / MSG) Antifungal therapy + meropenem Gradual ↓ in GM index Maertens J, et al. Clin Infect Dis 2004; 39: 289-90

Clinical case 1 wk later Probable IA (EORTC / MSG) Pipe-tazo for appendicitis GM >2.5; bilateral nodular infiltrate Probable IA (EORTC / MSG) Antifungal therapy + meropenem Gradual ↓ in GM index Necropsy: leukaemia infiltrate Absence of IA Maertens J, et al. Clin Infect Dis 2004; 39: 289-90

Meta-analysis of GM testing Proven or probable IA Sensitivity Specificity Haematological malignancies 0.58 (52-64) 0.95 (94-96) Solid organ transplantation 0.41 (21-64) 0.85 (80-89) Para estes casos e’ preciso testar LBA; e ai nao temos ponto de corte Pfeiffer CD, et al. Clin Infect Dis 2006; 42: 1417-27

(particularly for sensitivity) Specificity 94% Sensitivity 79% Marked heterogeneity (particularly for sensitivity) BAL PCR testing Tuon FF. Rev Iberoam Micol 2007; 24: 89-94

PCR Critical points Variable sensitivity / specificity Lack of standardised targets / reagents Extraction method Platform (conventional PCR vs Real time) Poor understanding of DNA kinetics Not yet part of the EORTC/MSG criteria De Pauw B, et al. Clin Infect Dis 2008; 46: 1813-21

Beta-Glucan Detected in IFDs caused by Candida and Aspergillus Fungal cell wall Phospholipid bilayer of the fungal cell membrane -(1,3)-glucan -(1,3)-glucan synthase Ergosterol Yoshida M, et al. J Med Veter Mycol 1997; 35: 371-4

Beta-Glucan Detected in IFDs caused by Candida and Aspergillus Trichosporon Fusarium Acremonium Saccharomyces Pneumocystis Yoshida M, et al. J Med Veter Mycol 1997; 35: 371-4

speaking about a test that nobody uses in Brazil … There he is again, speaking about a test that nobody uses in Brazil …

The reality in Brazil A survey performed in collaboration with ANVISA 140 hospitals >42,000 beds 65% teaching hospitals 90% belonging to the Sentinel Network

Complexity in hospital care %

Is IFD a problem in your centre? 38.7% n=140

Do you know your local epidemiology? 40.1% n=140

Specialised media for fungi 19.7% n=140

Aspergillus identification at the species level 58.5% n=140

Fungal staining - biopsies 51.1% n=140

Biopsy specimens are sent in formalin only? 26.0% n=140

Access to high resolution CT 34.3% n=140

Galactomannan 83.6% n=140

The appropriateness or inappropriateness of feelings is relative to the ground and to the circumstances of those feelings Aristotle The Doctrine of the Mean, 384-322 BC Aristoteles ja dizia que nao ha sentimento que nao possa ser expresso … Por favor, nao se sintam culpados por usar de agressividade …

Fight fire with fire Metallica Ride the Lightning, 1984 AD Mais recentemente …

Invasive diagnostic interventions CT-guided percutaneous lung biopsy: yield of 70-100% Dizer que a Cornelia tem feito um otimo trabalho neste campo, tentando ao maximo fazer o dx ainda em vida para pacientes com infeccoes invasivas por fungos filamentosos Nosari A, et al. Haematologica 2003; 88: 1405-9 Crawford SW, et al. Transplantation 1989; 48: 266-71 Hoffer FA, et al. Pediatr Radiol 2001; 31: 144-52 Lass-Florl C, et al. Clin Infect Dis 2007; 45: e1001-4

Invasive diagnostic interventions CT-guided percutaneous lung biopsy: yield of 70-100% Platelets >60,000/ml are required Pneumothorax 18% Haemoptysis 3% Nosari A, et al. Haematologica 2003; 88: 1405-9 Crawford SW, et al. Transplantation 1989; 48: 266-71 Hoffer FA, et al. Pediatr Radiol 2001; 31: 144-52 Lass-Florl C, et al. Clin Infect Dis 2007; 45: e1001-4

Invasive diagnostic interventions Transbronchial biopsies: False-negative results are frequently seen Lass-Florl C, Freund MC. In: Aspergillosis: from diagnosis to prevention. Pasqulaotto AC, ed. Springer, 2009

Invasive diagnostic interventions Open lung biopsies: Provide larger samples of tissue with improved accuracy and specificity Lass-Florl C, Freund MC. In: Aspergillosis: from diagnosis to prevention. Pasqulaotto AC, ed. Springer, 2009

Invasive diagnostic interventions Open lung biopsies: Provide larger samples of tissue with improved accuracy and specificity Contradictory results Complication rate of 10-15% Severe thrombocytopenic patients who urgently require histological identification should undergo open lung biopsy Lass-Florl C, Freund MC. In: Aspergillosis: from diagnosis to prevention. Pasqulaotto AC, ed. Springer, 2009

Invasive diagnostic interventions Needle biopsy / Surgical resection Peripheral lesions Bilateral / multifocal disease BAL Focal lesions near the hilum and the great vessels in the lung, particularly in neutropenic patients, urgent thoracotomy and resection should be considered because of the risk of life-threatening haemoptysis Focal lesions near the hilum / great vessels Urgent thoracotomy and resection Lass-Florl C, Freund MC. In: Aspergillosis: from diagnosis to prevention. Pasqulaotto AC, ed. Springer, 2009

Conclusion It is mostly but not all about IA Invasive mould diseases have to be differentiated from each other

Conclusion It is mostly but not all about IA Invasive mould diseases have to be differentiated from each other Diagnosis is the most challenging step in infectious diseases

Conclusion It is mostly but not all about IA Invasive mould diseases have to be differentiated from each other Diagnosis is the most challenging step in infectious diseases We need a better understanding on the performance of the available tests

Acknowledgments Mycology team Infection Control Dept Luiz Carlos Severo Valerio R Aquino Cecilia B Severo Luciana Guazelli Melissa Xavier Infection Control Dept Teresa Sukiennik