Candida Hyaline Molds Pigment

4/6/2016

ACCME/Disclosures

The USCAP requires that anyone in a position to influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest. Drs. Guarner and Bryan declare that they have no conflict(s) of interest to disclose.

FUNGI Case 49 years old HIV positive woman with relapsed Morphology and culture multiple myeloma presents with respiratory and Jeannette Guarner, MD renal failure. Emory University Vital signs show hypotension and on physical exam a palpable neck lymph node is noted. Molecular diagnostics The lung X‐ray showed multiple nodules. A fine needle aspirate of the neck lymph node is Andrew Bryan, MD, PhD performed. University of Washington

1 4/6/2016

Molds = hyphae Five days later the case grows Candida Hyaline molds Pigment

Septation Dematiaceous

Pauciseptated Septated

Madurella spp, Mucorales genera: Aspergillus spp., Fonsecaea spp, Rhizopus, Fusarium spp., Cladophialophora spp, Rhizomucor, Scedosporium spp., Exserohilum spp, Mucor, Trichoderma spp., Curvularia spp, Cunninghamella, Paecilomyces spp. and Bipolaris spp, and Lichtheimia (Absidia). others including others. Candida spp.

2 4/6/2016

Candida, Microorganism identification several species Matrix‐Assisted Laser Desorbtion Ionisation ‐ Time Candida albicans, C. Of Flight Mass Spectrometer glabrata, C. parapsilosis, C. tropicalis, and C. krusei. (MALDI‐TOF‐MS) Histopathologically:

Candida glabrata All others

MALDI‐TOF and Candida sp. # cases Concordance Sensitivity Candida albicans 195 187 95.9 P. aeruginosa Candida glabrata 26 22 84.6 Salmonella serotype B Candida guilliermondii 10 6 60.0 K. pneumoniae Candida krusei 86 75.0 E. coli Streptococcus Candida parapsilosis 69 65 94.2 S. aureus Candida tropicalis 32 28 87.5 total 346 316 91.3 SY Hsieh, et al. Highly efficient classification and identification of human pathogenic bacteria by MALDI‐TOF MS. Molecular & Cellular Proteomics T Spanu, et al. Mass spectrometry assay of blood culture broths for rapid 2008;7:448–456. identification of Candida species causing bloodstream infections: an observational study in two large microbiology laboratories. J Clin Microbiol 2012;50:176-179

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Candidiasis Epidemiology: Candida .Superficial: candida in Who: mouth (thrush), vaginal ◦ Immunosuppressed/ neutropenic individuals. infections, ◦ Others: Patients with central catheters, on broad onychomycosis. spectrum antibiotics, on parenteral nutrition, .Invasive hemodialysis, or after surgery. Easy to diagnose in the “usual” locations: GI tact.

Final identification Candida glabrata

JA Cortes et al. Infecciones micóticas del torrente sanguíneo en hospitales de tercer nivel en Colombia. Rev Iberoam Mycol 2011; 28: 74

4 4/6/2016

What happens if Antigenemia tests for Candida cultures aren’t and many other fungi submitted? 1,3 β‐D‐glucan in serum: ◦ Cell wall component ◦ Limited or no detection of Zygomycetes, Cryptococcus, Blastomyces ◦ Sensitivity 50–100%, specificity 44– 92% in one specimen if cut off values are high (80pg/ml) for candidemia. ◦ High rates of false positives in critically ill patients, patients with bacterial infections, medical devices with glucans

PMIDs 19375641, 26910223, 23949603

Why we speciate: Magnetic resonance Candida sp. have predictable for speciation of Candida directly from blood susceptibility patterns

Flucon‐ Itracon Voriconazole & Flucyt‐ Ampho‐ Echino‐ Speciate five Candida sp. azole ‐azole posaconazole osine tericin B candins Direct on blood without C. albicans & SS S S S S need for extraction C. tropicalis C. paropsilosis SS S S SS to R Reduce time to diagnosis compared to culture C. glabrata S‐DD to S‐DD to S‐DD to R SS to IS R R FDA‐cleared C. krusei RS‐DD to S Ito R S to IS Unclear cost‐effectiveness R C. lustaniae SS S SS to R S

S‐DD = Susceptible, dose‐dependent S = Susceptible PMIDs 26371384, 24135411 R = Resistant

5 4/6/2016

Mass spectrometry (MS) for speciation of fungi In situ hybridization for speciation of fungi from both fresh specimens and FFPE from both fresh specimens and FFPE

• MALDI-TOF on blood (PMID 25636941) FISH to fungal rRNA DNA sequences has been used for identification of • PCR-electrospray ionization MS on FFPE Candida and Histoplasma from (PMID 23985922 (Pritt)) blood culture bottles, Cryptococcus from CSF, etc - m/z ratios of small PCR amplicons from several loci -> fingerprint Sensitivity 50 ‐ 95%, specificity > 90% - Good agreement with culture; detected environmental Probe intensity more limited in contaminants FFPE, but have been designed for PMID 22584138 range of yeast and molds • MALDI tissue imaging on FFPE Sequence similarity may limit (PMID 26831708, 24287632) speciation of closely related FFPE pulmonary mixed Candida and - On the horizon? organisms Aspergillus infection (PMID 26781382)

PMID 23574773 (Guarner), 21791040, 26781382 PMID 25621874

PCR followed by sequencing for speciation of Representative work‐flow of fungi from direct specimens and FFPE broad‐range fungal PCR

• Analogous to prokaryotic (bacterial) 16S rRNA sequencing • 28S rRNA DNA sequences are highly conserved, but cannot always distinguish species • Internal Transcribed Spacer (ITS) sequences are nonfunctional linkers, thus are highly polymorphic, facilitating speciation • Amplification and sequencing of ITS1 and/or ITS2, with database match for species identification

Modified from S. Salipante

6 4/6/2016

Specimen type and DNA Extraction impact Additional handling of blocks increases risk of contamination diagnostic yield of PCR (PCR true positive, biologic false positive) Fluids: centrifuged (more is typically better) Pathogen burden Tissue: 30 –50 mg 200µL elution 5µL/rxn Consider pre‐test probability: Histopath + ≠ FFPE ~17% positive rate for broad range PCR non‐FFPE ~9% positive rate Histopath ‐ Bone: Unknown Malassezia sp. Bead‐based lysis: efficient, but releases calcium (inhibitory) 6.2% of non‐FFPE cases P < 0.05 EDTA decal:  12.9% of FFPE cases Strong acids:  Shave off the top to reduce contaminants Of all positive broad‐range fungal PCRs

medibiztv.com

Balancing pre‐test probability and Initial PCR amplification of conserved risk of contamination of blocks sequences 28S, ITS1, ITS2 Duplicate patient samples Tissue permitting: Inhibition control Consider adjacent sections ITS1 –Freeze tissue (for PCR) – FFPE (histopath) BAL (Polymicrobial): Comsol.com Multiple bands

Can’t proceed with Sanger sequencing, but still can perform species‐specific assays

7 4/6/2016

Databases queried with patient sequences PCR product is sequenced by Sanger to determine species of organism method & chromatographs reviewed

Organism %ID Length Journal evalue Also search internal UW database of clinical isolates If match > 99.5% &all other species < 98.5%, report name most commonly used in clin micro otherwise generate tree

If simple database search is ambiguous, selected sequences If simple database search is ambiguous, are used to generate tree tree is generated to determine where sample clads

C. dubliniensis

C. tropicalis C. albicans

C. parapsilosis

C. glabrata

Modified from PMC4629792

8 4/6/2016

Case is reviewed and signed out Edible fungi

• Multiple independent reviewers, including director‐level sign‐out

• If alignment 97 – 99.7% with multiple species, use tree and genus +/‐ species level name most commonly used in clinical microbiology

• If no clear match, report higher level taxonomy

• Candida albicans DNA detected with ITS primer set

Case 47 year old woman with aplastic anemia treated with eculizumab, awaiting a bone marrow transplant. He presents with fever and an X‐ray showed a pneumonia. Erythematous skin lesions are also noted. Laboratory: 300 WBC/ mL, Hb 8 g/dL and 13,000 platelets/mL

9 4/6/2016

Molds = hyphae Diagnosis:

Hyaline molds Pigment Angioinvasive fungal elements.

Septation Dematiaceous

Pauciseptated Septated

Pathologic misclassifications Misclassifications occur when: Retrospective studies that correlate Frequent misclassifications when: few, folded, culture results with histopathology and fragmented or necrotic fungal elements. cytology show: ◦ accuracy ranges from 20 to 80%. There is a false sense of ability to name the fungal genus based on morphology alone (hyaline septated The lowest correlation: invasive septate hyphae rather than Aspergillus spp.). molds. Lack of knowledge of morphologic mimics of yeasts Special stains do not improve and hyphal forms. pathologists’ diagnostic capabilities. Patel AJ, et al. Am J Surg Pathol 2010;34:256-261. Sangoi AR, et al. Am J Clin Pathol 2009;131:364-375.

10 4/6/2016

Case continues Identification 5days after the skin biopsy was obtained the blood culture bottles showed:

What happens Direct tests for Aspergillus if it all goes antigen in patient fluids into formalin? Galactomannan in serum or other fluid: ◦ ELISA for Aspergillus galactomannan ◦ Sensitivity 40‐100%, specificity 56‐100% ◦ More specific than 1,3 β‐D‐glucan, but still not entirely specific to Aspergillus ‐ May also detect Penicillium, Alternaria, Paecilomyces, Geotrichum and Histoplasma ‐ False‐positives in 50% of patients taking antibiotics (β‐lactam)

LJ Wheat. Clin Chest Med 2009;30:367–377

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How to choose a PCR Assay: Nested PCR & probes can Broad‐range vs. species‐specific increase sensitivity and specificity

Want to detect the broadest number of organisms? ◦ ‐> Broad‐range 1st round PCR Polymicrobial? ◦ ‐> Species‐specific 2nd round PCR You have a decent guess on what it is? (nested) ◦ ‐> Species‐specific with reflex to broad‐range (or vice a versa) Final product Want the highest sensitivity [for a specific organism]? ◦ ‐> Species‐specific (usually)

Additional considerations: ◦ Highly sensitive assays may detect organisms that are not clinical significant

◦ Broad‐range primers may amplify contaminants or normal microbiota dyes.gene‐quantification.info

Broad‐range PCR consists of 2 PCR reactions: Broad‐range fungal PCR 1. 1‐step (simple) PCR‐sequencing of 28S/ITS 2. Sensitive nested PCR screening test Broad‐range Broad‐range

Beta‐globin control Beta‐globin control ITS2 1st round of nested PCR ITS, 28S primer sets ITS, 28S primer sets

Sequence Sequence SYBR Screen

28S: 1,000 genomes/rxn 28S: 1,000 genomes/rxn Duplicate ITS1: 100 genomes/rxn ITS1: 100 genomes/rxn specimens with Cts < 28 A. fumigatus control

Negative Ct = cycle threshold, Low Ct = more template DNA

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Broad‐range PCR consists of 2 PCR reactions: 1. 1‐step (simple) PCR‐sequencing of 28S/ITS Highly sensitive (nested PCR) 2. Sensitive nested PCR (if passes screening test) requires clinical correlation Broad‐range Reagent contamination from manufacturer:

Beta‐globin control Cladosporium, Phoma, Nectria, Aureobasidum 1st round of nested PCR ITS, 28S/26S Not reported if only detected by nested assay primer sets SYBR screen Consider specimen source, grossing, histology, and any Sequence other source of contamination when interpreting 28S: 1,000 genomes/rxn ITS1: 100 genomes/rxn Malassezia sp. Nested PCR primers Internal Transcribed Spacer 2 (ITS2) Not reported if only detected by nested assay Sequence Pan 2 A. fumigatus 0.2 genomes/rxn Pan 1 A. flavus 0.92 genomes/rxn Consider anatomic site (e.g. is it Candida from a BAL), Pan 1 Mold 2 Pan 2 Mold 2 H. capsulatum 3.77 genomes/rxn is that clinical significant?

Species‐specific PCR: Species‐specific assays for polymicrobial site, Lightcycler with FRET probes sensitivity, or specific pathogen of interest A. fumigatus complex probe

Same specimen extraction Positives confirmed with Beta‐globin control second reaction, Broad‐range Specific‐specific: more specific 1st round of (semi) nested PCR A. fumigatus probe

A. fumigatus Clinical species‐specific fungal Primers & Zygomycetes assays also offered at UW: probes primer mix Cryptococcus Sequence Histoplasma if amplification Coccidiodes without probe match Sequence Pneumocystis A. fumigatus 1 genome/rxn Zygomycetes 1 genome/rxn

13 4/6/2016

Fungi that cause death Case 68 years old female with painful, pruritic, 1 cm nodule in right ear for the past 2 months. A lung CT scan shows multiple cavities. Renal transplant 3 years ago on tacrolimus. No fever, chills, or weight loss.

PAP BAL

Mucin

14 4/6/2016

Histoplasma* Small Talaromyces* Small yeasts Size/ budding (2 ‐10 µ) Sporothrix* Grouping Cryptococcus Yeasts Epidemiology Candida glabrata Pneuumocystis

Large Blastomyces* (>10 µ) Coccidioides* Paracoccidioides* Chrysosporium*

*Thermally dimorphic fungi: Parasites: “Mold in the cold, Toxoplasma, Yeast in the beast” Leishmania, Trypanosome

Positive Negative In microbiology: Case continues Cryptococcal antigen in serum 1:16 Cryptococcal antigen in CSF 1:8 Culture of tissue: Crytococcus neoformans Urease Susceptibility testing is not standardized Caffeic acid test

Cryptococal antigen India ink staining L‐Canavanine, glycine, 2 bromthymol blue (CGB) agar

15 4/6/2016

1,000,000 new cases of When to use melanin stains: cryptococcosis each year Use of Fontana‐Masson Bishop JA, et al. Hum Pathol 2012;43:898-903 silver stain for detecting Positive Negative melanin in the diagnosis of Cryptococcus 9/9 Histoplasma 0/10 Cryptococcous and dematiaceous fungi. Coccidioides 7/7 Sporothix 0/1

However Aspergillus Blastomyces 4/10 Prototheca 0/2 (particularly A. niger) and others will stain. Paracoccidioides 2/2

Few studies with limited Lacazia 1/1 numbers of organisms. Rhinosporidium 1/1

What happens if culture is negative AND stains are ambiguous?

http://www.cdc.gov/fungal/diseases/cryptococcosis‐neoformans/screening.html

16 4/6/2016

Why is my PCR negative, there’s tons of organisms in my block??? Formalin

Hypothesis: Distribution of Formalin inhibits PCR Fresh Small biopsy + β‐globin Ct values DNA extraction alone is insufficient to remove inhibitors tissue time in formalin = from formalin fixed tissue‐‐full tissue processing needed low DNA yield FFPE (usually to the FFPE stage) Formalin causes strand breakage and base changes DNA damage is time dependent Fresh tissue (longer time in formalin = decreased amplification) has lower Cts = Shorter amplicons may amplify FFPE better, more amplifiable DNA but can result in loss in discriminatory power

20 25 30 PMID 25421801, 20392915 Ct value

Balancing pre‐test probability, risk of contamination of blocks, Additional molecular reduced yield from formalin challenges of Cryptococcus • Strain/serogroup differentiation important Tissue permitting: • Cryptococcal Antigen Tests (CrAg): sensitivity of different Consider adjacent sections serotypes may vary (improved with lateral flow assays) –Freeze tissue (for PCR) • PCR – FFPE (histopath) ‐ Capsule may reduce extraction efficiency Comsol.com ‐ Cryptococcus sequences are too similar always differentiate by 28S and ITS methods ‐ Hemi‐nested PCR of the ribosomal intergenic regions (IGS), followed by sequencing, allows species‐level determination for Cryptococcus

17 4/6/2016

28S and ITS cannot always speciate Sequencing of IGS Cryptococcus sequences allow speciation

Cryptococcus, non‐ neoformans or gattii & other C. gatti related organisms Patient isolate

C. gattii C. neoformans C. neoformans Patient isolate

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18 4/6/2016