LABORATORY HANDBOOK OF
`
`DERMATOPHYTES
`
`A CLINICAL GUIDE AND LABORATORY HANDBOOK
`
`
`
`
`OF DERMATOPHYTES AND OTHER FILAMENTOUS FUNGI
`
`
`
`FROM SKIN, HAIR, AND NAILS
`
`Julius Kane, D.Sc.,
`
`Gamma-Dynacare Medical Laboratories
`
`and St. Joseph’s Health Centre
`
`Toronto, Ontario, Canada
`
`Richard Summerbell, Ph.D.,
`Ontario Ministry of Health
`Laboratory Services Branch
`Etobicoke, Ontario, Canada
`
`Sigmund Krajden, M.D.
`St. J0seph’s Health Centre
`
`Toronto, Ontario, Canada
`
`Lynne Sigler, M.S.,
`University of Alberta
`Microfungus Collection and Herbarium
`Edmonton, Alberta, Canada
`
`Geoffrey Land, Ph.D.,
`Methodist Medical Center
`
`Dallas, Texas, U.S.A.
`
`Sfiar
`
`Publishing Company
`
`ThisR»-n‘a~tE,ria|'5‘§fa5.:!5piEd
`3“"E"‘L“"a“” I
`“E
`Subjem U.‘:1?CLa-py '
`‘Laws
`
`.
`
`CFAD V. Anacor, |PR2015-01776
`ANACOR EX. 2071 - 1/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 1/50
`
`

`
`Saar
`
`Publishing company
`1>.o. BOX 68
`
`BELMONT, CA 94002-0068
`U.S.A.
`
`phone (650) 591-3505
`fax (650) 591-3898
`
`Senior Editor: Dr. Julius Kane
`
`Assisting Editor: Dr. Richard Summerbell
`
`Publisher’s Consulting Editor: Dr. Susan Kelley
`Managing Editor: Stuart Hoffman
`Copy Editor: Roberta M. McNair
`
`Composition: Janet Hansen, Alphatype
`
`Library of Congress Cataloging-in-Publication Data
`Laboratory handbook of dermatophytes : a clinical guide and laboratory
`handbook of derrnatophytes and other filamentous fungi from skin,
`hair, and nails / Julius Kane .
`.
`. [et al.].
`p.
`cm.
`
`Includes bibliographical references and index.
`ISBN 0—89863-157-2 (hardcover)
`1. Dermatomycoses—Handbooks, manuals, etc.
`Handbooks, manuals, etc.
`1. Kane, Julius, 1924-
`[DNLM:
`l. Dermatophytes—isolation & purification.
`2. Dermatophytes—classification.
`3. Derrnatomycoses—microbiology.
`4. Dermatomycoses——diagnosis.
`QW l80.5.D3 L123 1997]
`RL765.L23
`1997
`616.5’ 7—dc21
`DNLM /DLC
`
`2. Dermatophytes—
`
`for Library of Congress
`
`97-23344
`CIP
`
`
`
`Copyright © 1997 by Star Publishing Company
`
`All rights reserved.
`No part of this publication may be reproduced, stored in an information processing and retrieval system, or transmitted, in
`any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permis-
`sion of the copyright owner and the publisher.
`
`Printed in Korea
`
`00 0102
`99
`98
`98
`97
`1234567890
`
`T,,,5,,,a,,,,.,ia.,,,a5mF.iE,d
`3“”E"'”‘*‘a”“ ma?“
`Subject USCQ-pyright: Laws
`
`CFAD v. Anacbr, |PR2015-01776
`ANACOR EX. 2071 - 2/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 2/50
`
`

`
`CONTENTS
`
`Foreword R. G. Sibbald
`
`vii
`
`Dedication and Acknowledgments
`A Personal Note
`Julius Kane
`xii
`
`xi
`
`A Historical Perspective
`
`Julius Kane
`
`xv
`
`U1.8;beN
`
`\DOG\lO\
`
`Dermatophytes and Agents of Dermatophyte-Like Infections
`
`I
`
`Dermatophytes: Epidemiology and Clinical Features
`
`5
`
`Dermatophyte Mycology: Examination of Skin, Nails and Hair
`
`33
`
`Physiological and Other Special Tests
`
`45
`
`The Biological Aspects of the Kane/Fischer System for Identification
`of Dermatophytes
`81
`
`The Genera Trichophyton and Epidermophyton
`
`131
`
`The Genus Microsporum 193
`
`Nondermatophytic Molds Causing Dermatophyte—Like Nail and Skin Infection
`
`213
`
`Chrysosporium and Molds Resembling Dermatophytes
`
`261
`
`Appendix: Media and Methods
`
`313
`
`Index
`
`333
`
`Thi5mat;er\i,a|was~mpiead
`3“'**E"‘L'°‘a”€‘ ma”?
`Subject U&Ca~py‘rig.ht Laws
`
`i
`
`E
`I
`; Enacorv
`V-
`ANACOR EX. 2071 - 3/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 3/50
`
`

`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`CHAPTER 8
`
`NONDERMATOPHYTIC MOLDS
`
`CAUSING DERMATOPHYTOSIS-LIKE
`
`NAIL AND SKIN INFECTION
`by Richard C. Summerbell
`
`
`
`HOW IMPORTA NT ARE THEY?
`
`WHA T KINDS OF INFECTIONS
`
`I)O THEY CAUSE?
`
`The nondermatophytic fungi which cause tinea—like infec-
`tions of the nails and skin are often isolated by laboratories
`performing dermatologic mycology. These diverse nonder-
`matophytic molds pose two kinds of problems. Firstly, cor-
`rectly identifying which isolates are contaminants and which
`are agents of an infection may be difficult. Secondly, the
`species identification of the isolates causing infection is often
`problematic. Despite these difficulties, some surveys have
`been done in which nondermatophytic skin— and nail-infect-
`ing molds are verified as infectious by conservative standards
`and then identified to species. Such studies indicate that non-
`dermatophytic molds constitute a substantial proportion of
`isolates from nails and thickly keratinized skin areas such as
`soles and palms. A recent North American survey showed
`that 3.3% of 2,662 filamentous fungi isolated as causal agents
`of nail infections over a three—year period were nondermato-
`phytic molds.“ Such molds also caused 0.4—().8% of sole and
`palm skin infections. In a British study, 11% of nail infections
`were caused by these fungi,“ while in a Colombian study,
`these fungi caused 4.5% of the examined nail infections at
`one laboratory and 9.5% at another.33 In Gabon, Africa, Scy-
`Ialiclium climidiarum (reported under the older name Hen-
`clersonula taruloidea) alone caused approximately 20% of all
`cutaneous infections of feet and nails.” Nondermatophytic
`nail— and skin-infecting fungi comprise an important part of
`the workload of diagnostic medical mycology laboratories
`worldwide.
`
`Different nondermatophytic molds cause different kinds of
`skin and nail infections. Two once little known but now
`
`increasingly important species cause nail, sole, toe web,
`and palm infections strongly resembling those caused
`by Triclioplzyton rubrum.. These species are S. cIimidia-
`rum, better known under its old name of H. rorulaidea (tech-
`nically speaking, it was the “Scytalidimn synanamorph of
`H. Ioruloidea”), and S. liyalinum. S. r/imidimunz on rare
`occasions causes other der1natophyte—like infections such as
`tinea capitis."
`
`Other species primarily cause nail infections, especially
`toenail infections. Relatively common agents such as Scapu-
`lariopsis brevicauli.s', Aspcrgillus sydowii and other aspergilli,
`and Ifiisarimn oxysporum may cause distal—subungual and
`lateral—type onychomycoses, in which nails become discol-
`ored and thickened and may separate from the nailbed and
`become brittle? Also, F. oxyspormn, Aspergillus terreus,
`Acrenzoniiun potronii, and a small number of other species
`may cause superficial white onychomycosis (Fig. l), a disease
`in which white or yellowish, infected patches appear on the
`surface of the nail.” The nail bed is generally not affected in
`early stages of the disease. Most of the nondermatophytic
`molds that cause nail infections, excluding the two Scyta1id-
`ium. species mentioned above, are much more likely to infect
`persons over the age of 60 than younger persons.“
`
`213
`
`This material wasttsu-pied
`at: the N LM a rid may be
`Sub—jes:t‘ US {Io-pyright Laws
`
`CFAD v. Anacor, |PR2015-01776
`ANACOR EX. 2071 - 4/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 4/50
`
`

`
`CHAPTER 8
`
`NONDERMATOPHYTIC MOLDS CAUSING DERMATOPHYTOSIS-LIKE NAIL AND SKIN INFECTION
`
`Various molds are capable of causing unusual skin infec-
`tions-—a well known example is P/meoaimellomyces wer-
`neckii, the causative agent of tinea nigra. Because the
`infections caused by these molds do not resemble dermato-
`phytosis, they fall outside the scope of this book.
`
`HOW CAN INFECTIOUS MOLD
`
`ISOLATES BE DISTINGUISHED FROZVI
`CONI}<11I/IINANTS?
`
`The isolation of a dermatophyte species (i.e. a pathogenic
`Tric/toplzyton, Micrasporum, or Epidermop/zyton species)
`from a lesion is presumptive of infection; however, this is
`not true of most nondermatophytic molds. Many are com-
`mon contaminants that occasionally cause infection. The
`means of verifying that a genuine infection exists vary from
`species to species.
`
`Two nondermatophytes can be treated as if they were der-
`matophytes from the point of view of verifying infection.
`These are S. diniicliatimz and S. hyalinimi. These species are
`not isolated as contaminants in temperate areas, and rarely
`are so isolated in tropical/subtropical areas. Isolation from
`skin or nails should be considered presumptive of an infec-
`tion, and this can be verified by showing thick-walled but
`otherwise dermatophyte-like (or rarely, brown) filaments in
`direct mounts of the affected areas.
`
`Photos of the infecting filaments of Scytalicliimz and other
`nondermatophytic agents of onychomycosis are depicted
`under the individual species. For comparison with dermato-
`phyte filaments, refer to Chapter 3.
`
`With nonderinatophytic onychomycosis agents other than
`S. clinizicliatznn and S. /iyalimmz, the possibility of isolating
`the fungus as a contaminant rather than as an infectious
`agent must be considered. Fungi such as S. brevicaulis and
`A. terreus may cause nail infection, but they are also com-
`mon soil and indoor air fungi. Furthermore, their kerati-
`nolytic abilities probably give them the ability to live on
`moist leather, thus possibly making them a part of the nor-
`mal llora of shoes.
`
`Certainly, for any non—ScyIali‘dium mold from nails or skin,
`finding fungal filaments in the direct mount of the patient’s
`specimen is an absolute prerequisite for considering the pos-
`
`
`
`
`
`Figure 1. Superficial white onychomycosis attributed to
`Scopu/ariopsis brevicau/is.
`
`sibility of a mold infection. It must be stressed that any true
`infection by mold fungi will be signalled by the presence of
`filaments or other fungal structures in tissue. One must, how-
`ever, always consider the possibility that the filaments seen
`in direct mounts may be dermatophyte filaments regardless
`of what kind of fungus is isolated. Sometimes a physician
`will take a sample from an older portion of the infected area
`where nonviable dermatophyte filaments are found. In this
`case, a contaminant growing from the sample may be mis-
`taken for an infectious agent. Potentially misleading, dead
`dermatophyte filaments may also be found in cases where
`the patient has already used antifungal agents on the infected
`area. In other cases, the dermatophyte filaments may be
`viable; however, an aggressive mold contaminant may over-
`grow the isolation medium. In these cases, a mistake might
`easily be made and a mold contaminant reported as an infec-
`tious mold.
`
`This problem can be resolved in two ways. Firstly, with
`some mold species, this problem can be resolved the EASY
`WAY. Some molds causing nail infections produce highly
`distinctive microscopic structures within the infected tissue.
`These structures can easily be recognized as not having been
`produced by a dermatophyte. For example, some, but not all,
`S. brevicaulis infections are accompanied by the production
`of masses of golden-brown, leinon—shaped Scopulariopsis
`conidia in the nail tissue. /lspergillus syclowii and A. versi-
`colar infections may have whole Aspergillus conidial heads
`associated with the nail tissue along with many interwoven
`irregular filaments. Wangiella zlernzatitidis infections will
`show masses of deep brown filaments and yeast cells. Molds
`
`
`
`214
`
`
`
`Th is material wastes-pied
`at the N LM and may be
`5U‘IJjEsZI'. US Copyright Laws
`
`CFAD v. Anacor, |PR20‘|5-01776
`ANACOR EX. 2071 - 5/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 5/50
`
`

`
`NONDERMATOPHYTIC MOLDS CAUSING DERMATOPHYTOSIS-LIKE NAIL AND SKIN INFECTION
`
`CHAPTER 8
`
`;,-
`n
`«_;
`'j‘ .._,....._.__.,.J
`'
`c
`
`.
`
`
`
`
`
`Figure 3. Irregular filament of frondose morphology from
`an infected nail. Fusarium solani grew, but it could not be
`confirmed as an infectious agent because no repeat
`specimen was received.
`
`A
`
`,
`
`..
`
`
`
`-
`
`.,_ .«
`
`
`«-
`
`lsirect NaOH mount from nail scrapings showing
`Figure 2.
`dark filaments and C/adosporium-like conidia. Long spirals
`of lignin reveal the material as plant material from subun-
`gual debris. Trichophyton mentagrophytes grew from
`the specimen.
`
`in these situations may be-considered infectious. The labora-
`torian must, however, be careful to ensure the distinctive
`
`microscopic structures were actually seen in nail tissue and
`not in debris of environmental origin from underneath the
`nail or dust trapped in crevices of the nail surface. Penetra-
`tion among the cells of actual nail tissue by fungal filaments
`must always be ascertained. Deceptive subungual debris is
`often seen (Figure 2).
`
`Laboratorians who have considerable experience with recog-
`nition of dermatophyte filaments in tissue may also recog-
`nize that some irregular—shaped filaments in nails are outside
`the range of variability of dermatophyte filaments. This kind
`of decision may be difficult even for the experienced person,
`because dermatophytes, especially T nzentagropltyres, may
`produce aberrant frond-like hyphae in nails.” Nonetheless, if
`an experienced mycological laboratorian isolates 21 known
`nail—infecting mold from a nail sample which clearly con-
`tains nondermatophytic filaments, this is strongly suggestive
`of a mold infection. Some typical irregular filaments of the
`frondose type are shown in Figure 3. For final confirmation,
`the mold should be shown to have been isolated from several
`
`(at least four) separate inoculum pieces. Also, the possibil-
`ity that the unusual filaments are yeast filaments must be
`ruled out. If yeast-like blastoconidia are seen being produced
`on the filaments in the direct mount, there is a high proba-
`bility that the infection is caused by a yeast and merely over-
`grown by a mold. If filaments do not bear such structures,
`but both a filament-producing, potentially nail—infecting
`
`yeast (e.g., Candida albicans, C. parapsilosis, C. krusei,
`Trichosporon beigelii) and a mold grow from the inoculum
`pieces, a repeat sample should be done to determine which is
`the infectious agent.
`
`Many nail—infecting molds produce filaments in tissue
`that are essentially indistinguishable from dermatophyte
`filaments. These molds present a problem that can only
`be resolved by verifying infections the HARD WAY. If a
`nail sample shows dermatophyte-like or ambiguous fila-
`ments in the direct mount and yields only a potentially nail-
`infecting mold in culture, this is “suggestive” of a mold
`infection. If the mold grows from a large proportion of the
`inoculum pieces (eg, from many separate nail pieces on
`the culture medium), this is strongly suggestive of a mold in-
`fection. Growth from only a small number of inoculum
`pieces is less strongly suggestive, and some workers dis-
`count molds that do not grow from at least four separate
`inoculum pieces.
`
`Once one has recorded a suggestive infection, one must
`advise the physician to consider obtaining a repeat sample of
`the patient’s putatively infected lesion. If the repeat sample
`again shows similar filaments, grows the same mold, and
`fails to yield a dermatophyte, then and only then can the
`infection be considered confirmed.
`
`A protocol for investigating possible nondermatophyte ony-
`chomycoses is outlined in Fig. 4.
`
`215
`
`This material W‘aS-Estlplflrd
`at the N LM 5! rid may be
`Subject‘ U355-pyright Laws
`
`CFAD v. Anacor, |PR2015-01776
`ANACOR EX. 2071 - 6/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 6/50
`
`

`
`NONDERMATOPHYTIC MOLDS CAUSING DERMATOPHYTOSlS—LlKE NAIL AND SKIN INFECTION
`CHAPTER 8
`
`
`Table 1 Checklist of Nondermatophytic Molds Substantiated as Agents of Nail Infection
`
`Species
`
`/lcremonimn palronii
`Alter/mria altcrnala
`
`Aplzanmzscusfitlve.rccn.r
`As/zergillus camliz/us
`Aspergillus flavus
`A.vpcrgi1lus finnigatux
`Axpcrgillus “glaucu.r series”
`Aspergi/lu.\* nirlulunr
`Aspergilluy niger
`Aspergillus xydowii
`A5/wrgi//(IS terreus
`/type!‘/,'iI/us u,rIu.s'
`Aspcrgillux varirms
`Aspergi/lu.s' versicolor
`"Ccphalosporiuin," various namcsv
`sec Acremonium and Fusarium
`Clmemmimn glolzoszmi
`C/c/(lop/Iiulop/mm currionii
`Cl!l'VH/"""(’ /”’"”“
`Fu.s'ar1'mn oxyspormn
`
`Fz1sm'imn salani
`GyII1Il(l.§'(‘(’//(I z/mzka/iwI.ri.s'
`Lasiaz/iploz/ia I/zen/zramuc
`/Wit/'r)r1.s'c'ir.s' L'iIIereu.s'
`Mi(.'mu.s'('zr.\‘ ('iI'I”().Yll.\‘
`()nyc/mcn/a cm1udcI1.s'i.\'
`l’t'I1!'<‘!'/”""” >"I’-
`])_wm/elirolizmz avale
`5(,‘()[)1Ii(lI‘l()/7,YfS l2revic'(mli.s'
`560/’"/"’i"/"ml I’’‘“’’'/’’’'’.
`S00/21(Ia/‘iup.ri.s' camlirla
`_§'¢:y1c/li(/izmi (/inzidiulzmi
`Scylrl/it/I‘III77 /W"/"'l“"1
`W11/zgiel/ct zlernmririz/i_s'
`
`Norrzs
`
`Status as Agent of Onychomycosis
`
`Status as Medical Contaminant
`
`uncommon
`rare
`
`rare
`rare to uncommon but regular‘
`rare but regular’
`rare
`rare
`rare
`rare
`uncommon but regular‘
`uncommon but regular"
`rare
`rare
`uncommon but regular‘
`
`rare
`rare
`FGTC
`rare but regular‘
`mm
`nu-c
`rare, regular in some areas‘-3
`rm-c
`rare
`rare, but regular‘
`Talc"
`rare
`common
`“W
`rare
`mm [0 common?
`rare to uncommon but regular'<3
`rm-e
`
`uncommon
`abundant
`
`uncommon
`uncommon
`abundant
`abundant
`common
`common
`abundant‘
`abundant
`common
`uncommon to common
`rare
`abundant
`
`abundant
`mrci
`uncommon to common
`abundant
`
`abundams
`mm
`mrefi
`mm
`mm
`mm-I
`abundant
`mm
`abundant
`uncommon to common‘
`Common
`mrcz
`ya,-62.4
`Common
`
`Species listed as “regular” agents of infection never cause more tl1an 5% of total nail infections and often cause less than 1% of these infections. None-
`theless, they cause infections with predictable frequency. They will be infrequently but regularly reported by laboratories dealing with large numbers
`of specimens. Smaller laboratories Wlll see such an infection from time to time but not with any noticeable regularity.
`More common in tropical and subtropical areas or in patients who have lived in such areas. Occurrence as a contaminant only in tropical or subtrop-
`ical areas, not in temperate areas.
`Infectious status not well demonstrated; possibly not an agent of onychomycosis.
`No environmental source yet known; to date, never reported as a contaminant.
`Species add to this table based upon new information while this book was in production, and therefore not included in the descriptions.
`
`]_
`
`7 H
`
`.
`3.
`4,
`5'
`
`
`
`216
`
`This material was-*:>u-pied
`at the N LM 5! rid may be
`Su.bjse».=:t: US %3«t:»i:e;‘right Laws
`
`CFAD v. Anacor, |PR20‘|5-01776
`ANACOR EX. 2071 - 7/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 7/50
`
`

`
`NONDERMATOPHYTIC MOLDS CAUSING DERMATOPHYTOSIS-LIKE NAIL AND SKIN INFECTION
`
`CHAPTER 8
`
`Figure 4. A flow chart for the interpretation of isolations of nondermatophytic fungi from nails.
`
`Protocol for Dealing with Possible Cases of Non-dermatophytic Onyohomycosis
`
`Step l. Microscopic examination: Classify structures seen.
`Note type of structttre seen (e.g., “Sc0pttIari0p.ris
`A. Distinctive non-dermatophytic structures (e.g., masses of Sc()[1uIari0pSi.s' conidia,
`C0T1idii1")
`or Aspcrgilltts heads and eonidia)
`Note that “irregular” filaments seen
`B. Unusual or irregular filaments (e.g., with irregular nodules)
`Note that fungal filaments seen
`C. Typical dertnatophytic filaments (cylindrical filaments or regular chains
`of spherical artltroconidia)
`[)_ Fungal structures not 3-gen
`Note as microscopic negative
`
`.
`
`Step 2. Culture Result
`
`B.
`S(;y(()[i[]f(un
`(limiditizum or
`S, hyalinum
`
`C.
`Known agent of
`onychomycosts other than
`dermatopliyte or Scylaliditmt
`
`D.
`Fungus itot known as an
`agent of non-dermatophytic
`onychomyeosis
`
`A.
`B
`an
`-E I)erm;1u)phy1c
`E
`E
`
`:U
`
`
` E
`
`‘%
`
`55)‘
`3
`E
`E
`
`T Report as
`infectious
`agent,
`regardless of
`E microscopic
`:13;
`strttctures
`E
`seen
`E’
`E
`§
`
`Report as
`infectious
`agent,
`provided
`fungal
`filaments
`seen in direct
`microscopy;
`if not, notify
`physician of
`presence of
`fungus and
`solicit repeat
`sample for
`
`Matches
`distinctive
`non-
`dertnatophytic
`structttres in
`direct
`microscopy
`
`Report as
`infectious
`agent
`
`Found in the
`presence of
`“irregular"
`lilatnents
`
`Found in the
`presence of
`regular fungal
`filaments
`
`Found in the
`absence of
`fungal
`structures
`
`Found itt the
`presence of
`“irregulat"‘
`filaments
`
`Found in the
`presence of
`regular fungal
`filaments
`
`Found in the
`absence of
`fungal
`structures
`
`Found in the
`presence of
`distinctive
`tton-
`d°r"“1l0Ph)’liC
`structures in
`[M1105
`
`Disregard
`
`Likely a
`contatninant:
`note in your
`records but
`do not report
`or solicit
`repeat
`satnple
`
`Note as
`possible
`etiologic
`agent;
`attetnpt to
`conlirtn by
`repetition
`
`Note
`presence of
`fttngus but do
`ttot suggest
`etiology;
`solicit repeat
`satuple for
`further
`investigation
`
`Likely a
`contatninant;
`note in your
`records but
`do not report
`or solicit
`repeat
`sample
`
`Note as
`possible
`etiologic
`agent;
`attempt to
`confirm by
`repetition
`
`Note
`presence of
`fungus bttt do
`not suggest
`etiology; if
`fungus is a
`regularly
`occurring
`agcm of
`onychomycosis,
`solicit repeat
`Silmplc TOT
`flII‘IhCr
`evaluation
`
`confirmation
`l__
`
`
`217
`
`T,;i;,,,a..;;c,i;.w.;.5,m.,;t;d
`"*L*~*a”dmaWe
`Su btjzect US {>t:-pejrighr.‘ Laws
`
`CFAD v. Anacor, |PR201 5-01 776
`ANACOR EX. 2071 — 8/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 8/50
`
`

`
`CHAPTER 8
`
`NONDERMATOPHYTIC MOLDS CAUSING DERMATOPHYTOSIS-LIKE NAIL AND SKIN INFECTION
`
`Mixed infections of dermatophytes and nail-infecting molds
`may occur. Such cases must show consistent and profuse
`growth of the mold from a number of repeat samples; how-
`ever, this is difficult to obtain, since treatment aimed at the
`
`dermatophyte may also suppress the mold. In some mixed
`infections, the dermatophyte may be relatively difficult to
`detect, and overgrown by the mold. In such situations, the
`isolation of the dermatophyte may be facilitated by scatter-
`ing small fragments of nail specimen across a plate of
`Littman’s oxgall agar (see Appendix for details about
`media).
`
`A/IE THODS OF IS OLA TION
`
`Because many nondermatophytic agents of skin and
`nail
`infection are partially or completely inhibited by
`cycloheximide, they cannot be reliably isolated on the
`cycloheximide—containing media (e.g., Sabouraud agar
`+ cycloheximide, Mycosel, Dermatophyte Test Medium)
`usually used to isolate dermatophytes. The recommended
`procedure for isolating these fungi is to plate appropriate
`dermatological samples (especially nail scrapings and
`drillings, but also,
`in places of tropical/subtropical climates
`or with immigration from such areas, sole, palm, and intert-
`riginous scrapings) on Littman oxgall agar (Difco Labora-
`tories, Detroit) or cycloheximide-free Sabouraud agar.
`Littman oxgall agar is mycologically preferable because it
`restricts colony growth, thus allowing the user to determine
`the number of separate inoculum pieces from which the sus-
`pected disease agent has grown. It also restricts rapidly
`growing contaminants. Sporulation on Littman oxgall agar is
`usually poor or aberrant, so subculturing is necessary as out-
`lined below. Alternaria allermira spontaneously dies after
`seven to 14 days on Littman oxgall agar and should be sub-
`cultured sooner.
`
`
`
`W!E THODS OF IDENTIFICA TION
`
`Many of the fungi causing nondermatophytic onychomyco—
`sis and tinea will sporulate poorly, aberrantly, or not at
`all on Sabouraud agars, Littman oxgall agar, Dermatophyte
`Test Medium, brain heart infusion agar, and most bacteri-
`
`ological media such as blood agar and chocolate agar.
`Cycloheximide—containing media (e.g., Mycoscl) will inhibit
`any growth in some species and will discourage sporulation
`
`in others. The use of general—purpose mycological sporula-
`tion media is recommended (_e.g., potato dextrose agar or
`modified Leonian’s agar) (see Appendix). Other sporulation
`media such as potato flake agar and Bandoni’s MYP are
`equally effective.
`
`In addition, many species are best examined in a classic
`Riddell-type slide culture” with a sporulation medium used
`to compose the agar block. For those without the glass petri
`plates and bent glass rods required for the Riddell technique,
`an approximation can be made by partially embedding a
`sterile, glass coverslip into growth medium on a slant in the
`path of a growing colony, so that the fungus grows onto the
`slanting aerial portions of the coverslip. Finally, an excellent
`facsimile of the Riddell method is provided by the Harris
`method, in which the fungus is inoculated into four points
`on a block'of sporulation medium placed onto a surface
`of nutrient-free water agar (see Appendix, Part E). A
`sterile coverslip is then placed over the block of sporula-
`tion medium.
`
`At this time there are no biochemical or serological tests
`relevant to the diagnosis of nondermatophytic dermatologi-
`cal pathogens, nor do any commercial kits exist.
`
`NONtWYCOLOGIST’S KEY TO AUTHENTIC
`
`NAIL-INFECTING MOLDS
`
`After keying to a final key choice, check out the full descrip-
`tion of the fungus to ensure that there is a good match
`between your isolate and the described species.
`
`I. Colonies uniformly black, dark gray or drab greenish-
`gray at the end of seven days at 25°C, or at least
`80% dark but with a pale fringe around the colony
`border........................................................................ .. 2
`
`(Follow choice la for colonies basically pale-colored
`or medium brown but developing a grayish to khaki
`overlay of sexual or asexual fruiting bodies. Such
`structures will be over 90 pm in diameter and will pro-
`duce spores or conidia in their interiors. Follow choice
`2 for purely black or deep gray colonies producing
`fruiting bodies. Choice 2 is again appropriate for
`colonies beginning pale but turning predominantly
`deep gray to black by seven days and producing fruit-
`ing bodies after seven or more days.)
`
`218
`
`This material was smpied
`at the N LM a rid may be
`Su.bjet:t US {lo-wright‘ Laws
`
`CFAD v. Anacor, |PR2015-01776
`ANACOR EX. 2071 - 9/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 9/50
`
`

`
`NONDERMATOPHYTIC MOLDS CAUSING DERMATOPHYTOSIS-LIKE NAIL AND SKIN INFECTION
`
`CHAPTER 8
`
`la. Colonies other colors at seven days .............. .. ll
`(Difficult choices are keyed out in both branches of
`the key.)
`
`DARK GRAY OR BLACK FUNGI
`
`2.(l)
`
`3.(2)
`
`3a.
`
`4.(3)
`
`4a.
`
`5.(2)
`
`5a.
`
`6.(5)
`6a.
`
`7.(_6)
`
`Colony fast—growing, diameter over 4 cm after
`seven days or, if slower growing, then producing
`conidia in chains from specialized flask-shaped
`fertile cells (phialides or annelides) ................... .. 5
`Colonies slow growing, under 4 cm in diameter in
`seven days, and never with phialides or annelides
`producing conidia in chains ................................ .. 3
`Colonies at center covered with a conspicuous
`
`like
`state, often shiny black,
`black yeast
`melted asphalt; fertile cells in slide cultures
`are flask— or candle—shaped “phialides” bearing
`conidia in slimy balls; colony able to grow at
`40°C — ............................... .. VI/czlzgiella dermaliriclis
`Colony without a black yeast state ...................... .. 4
`Colony in slide culture showing occasionally
`branching chains of dark conidia tapering down to
`narrow necks at the points where they connect to
`one another (blastoconidia) ................ .. Clac/0-
`phialop/wra carrimzii (For differentiation from
`normally nonpathogenic Cladosparium species, see
`comments under the description of this species.)
`Colony in slide culture showing occasionally
`branching chains of dark conidia connected end
`to end without any narrowing, giving a rectan-
`gular or finger joint—like appearance (arthroco-
`nidia) .......................... .. Scytaliclizmz (limidiatum,
`slow-growing Asian form.
`Colonies nonsporulating for at least two weeks,
`eventually (in fresh isolates) forming macroscopi-
`cally visible, blaek, irregular, hard fruiting bodies
`containing large (over 22 um long) clear or brown-
`striped conidia ........... .. Lasiodiplodici I/ieobrolncte
`(fungi keying out here which remain persistently
`nonsporulating pose an identification problem
`beyond the scope of this book.)
`Colonies sporulating earlier or in other ways .... .. 6
`Conidia multicelled ............................................. .. 7
`
`Conidia single celled or 2—eelled .......................... .. 8
`Conidia with cross walls only in one plane (divid-
`ing the eonidium into cells of shorter length, but
`
`7a.
`
`8.(6)
`
`8a.
`
`9.(8)
`
`9a.
`
`l0.(9)
`
`I021.
`
`not dividing the cell into components of smaller
`width)
`Curvularia lzmara
`At least the larger conidia with cross walls in
`two planes, crosswise and lengthwise;
`larger
`conidia grenade shaped or with a “beak” at the
`tip ........................................ .. Alternaria alternara
`Colony producing long, upright fertile stalks with
`a swollen, bulbous vesicle at the top, bearing
`masses of dry conidia in chains from fertile struc-
`tures mounted on the vesicle (i.e., Aspergillus
`heads); colonies usually with brownish fertile
`stalks and bizarre, squashed-banana—shaped, thick-
`walled “hiille cells” within the colony; colony
`drab grayish brown ................... .. Aspergillus u.s‘m.s'
`(If black colony with tall stalks and no hiille
`cells,
`the likely identity is Aspergillus niger,
`a species not yet substantiated as an agent of
`authentic nail infections.) Note added in proof:
`Rare nail infection by A. niger has recently been
`established.
`'
`
`an Aspergillus; no long fertile stalks
`Not
`present ................................................................ .. 9
`Colony in slide culture showing occasionally
`branching chains of dark conidia (some pale coni-
`dia may also be present) connected end to end with-
`out any narrowing, giving a rectangular or finger
`joint-like appearance (_arthroconidia) .... .. ScyIalizI—
`ium climiclianun, fast-growing forms
`Colony in slide culture showing unbranched chains
`of dark conidia arising from llask— or candle—shaped
`“annellides” (Scopulariopsis state); these fertile
`cells arise singly or in small, vaguely broom—like
`clusters near the end of a branch; the large, 1naero—
`scopically visible, rounded, dark structures of the
`sexual state may or may not be present ........... .. l()
`(Note added in proof: Scopulariopsis brumjltii,
`recently established as an agent of nail infection,
`will key out here.)
`(Successful formation of sexual state necessary for
`separation of the species in key eouplet I0.) Spores
`formed in macroscopically visible, dark, rounded
`sexual fruiting bodies are lens shaped, sometimes
`concave on one side, and usually at least twice as
`long as broad ........................ .. Micr0ascu.s‘ cinereus
`Spores in sexual fruiting bodies almost heart
`shaped, one side strongly indented, less than twice
`as long as broad .................... .. Microascus cirmsus.
`
`219
`This material was -:5-piad
`at‘ the NLls.r1an.»:l may be
`SL2 Eject US {aspyright Laws
`
`CFAD v. Anacor, |PR20‘l5-01776
`ANACOR EX. 2071 - 10/50
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2071 - 10/50
`
`

`
`CHAPTER 8
`
`NONDERMATOPHYTIC MOLDS CAUSING DERM/\TOPHYTOS|S—L|KE NAIL AND SKIN INFECTION
`
`PALE, BRIGHT COLOURED, AND
`PREDOMINANTLY BROWN FUNGI
`
`l1.(l)
`
`NOTE:
`
`Colonies producing long, upright, unbranched fer-
`tile stalks with a swollen, bulbous vesicle at the top,
`bearing masses of dry conidia in chains from fertile
`structures mounted on the vesicle (Aspergillus
`species) ............................................................. .. 12
`If the largest vesicles seen are small (under 10 um
`diameter in a seven-day-old colony), bear fewer
`than 20 short fertile cells, and are attached to thin-
`walled stalks, isolate is a member of Pem'cillium
`subgenus Aspergilloides, not
`further keyed
`out here.
`
`lla.
`
`Not Aspergillus: colonies lack long fertile stalks
`topped by swollen vesicles ............................... .. 20
`
`(ASPERGILLUS SPECIES)
`
`12.(ll)
`
`l2a.
`
`l3.(l2)
`
`l3a.
`
`l4.(l3)
`
`l4a.
`
`15.04)
`
`1521.
`
`l6.(l5)
`
`Colony blue-green; fertile stalks distinctly blue-
`greenish; fertile cells “uniseriate” (i.e., fertile cells
`mounted directly on the vesicle surface, without
`intervening short branches); colony growing at
`45"C ......... .; ....................... .. Aspergillusfimligatus
`Not as above ...................................................... .. 13
`
`Colony yellow—green; fertile stalk walls roughened
`with minute granules conspicuous at 400X magni-
`fication ...................................... .. Aspergillusflavus
`Not as above ..................................................... .. l4
`
`fertile heads
`Colony pale cinnamon-brown;
`biseriate (fertile cells mounted on short branches
`arising from the vesicle surface); chains of dry
`conidia in undisturbed heads amassing in cylindri-
`cal vertical columns rising up from the fertile
`stalk ........................................ .. Aspergillus terreus
`Not as above ..................................................... .. l5
`
`Colony surface navy-blue; fertile heads biseriate
`(fertile cells mounted on short branches aris-
`ing from the vesicle surface); fertile stalks clear
`in color and smooth walled; colony under-
`surface usually a pale or deep hue of reddish-
`brown ...................................... .. Aspergz'llu.s' syclowii
`Not as above ..................................................... .. l6
`
`Colony at seven days on sporulation medium usu-
`ally with patches of grey—green sporulation, later
`becoming increasingly bright yellow or red as the
`small cottony puffs of sexual state form on the
`
`colony surface; conidia mostly barrel shaped with
`broad scars where they were attached to their
`neighbours in conidial chains; sexual spores origi-
`nally seen held together i