1

Monograph

on

Aspergillus and Aspergillosis in man, animals and birds

A guide for classification and identification of aspergilli, diseases

caused by them, diagnosis and treatment

By

Mohamed Refai1, Heidy Abo El-Yazid

1 and Atef Hassan

2

1. Department of Microbiology, Faculty of Veterinary Medicine, Cairo University

2. Department of Mycology and Mycotoxins, Animal Health Research Institute, Dokki

2014

2

Preface

When Micheli in 1729 discovered the Aspergillus fungus, he had not expected that this fungus would continue

for more than two and half centuries to be a subject of research, economic and medical interest. Species of the

genus Aspergillus, like Aspergillus oryzae, have long been used in the manufacture of citric acid, soy sauce and

other useful products. On the other hand, other species, like Aspergillus flavus, are known to produce highly

toxic and carcinogenic substances in the foodstuffs they infest. Moreover, other species like Aspergillus

fumigatus, cause disease in humans, animals and birds.

By the time Thom and Church published the first major monograph on the genus in 1926, Aspergillus had

become one of the best-known and most studied mould groups. Their prevalence in the natural environment,

their ease of cultivation on laboratory media and the economic importance of several of its species ensured that

many mycologists and industrial microbiologists were attracted to their study. Now we are speaking of a genus,

which is subdivided into sub-genera comprising sections and more than 250 species. The use of molecular

biology in the study of Aspergillus has resulted in the moving of species from one subhenus to the other and

from one section to the other. Some species are considered synonyms and some variants attained the status of a

species. Such changes occur in short periods, so that one needs frequent updating to his knowledge.

Since I came back from Germany in 1965 after finishing my mycology study, I depended on the 3 identification

keys of the Aspergillus species based on colours and micromorphology and we were satisfied to identify the

groups of Aspergillus. Now it becomes difficult, as many species cannot be identified on their morphological

basis, but need molecular biological techniques. This is why my post-graduate students become confused and

asked me to prepare a lecture to simplify the matter. When I started to collect data for the lecture, I was

drowned in a sea of information’s about this fungus. The enormous data which accumulated on my desk during

the last 4 months encouraged me to write a monograph and upload it on my site in the internet to be available to

all students, not only in Egypt, but in other parts of the world as well.

This monograph is dedicated to my supervisors, Prof. Dr.Dr. h.c. Kurt Wagener and Prof. Bisping who

introduced me to the eminent mycologists of Europe, when they allowed me to accompany them, few months

after arriving Germany in April 1962, to Travemuende on the Baltic Sea, to attend a meeting of mycology held

on a ship. There I met Prof. Rieth, who invited me in his laboratory for one year. Prof. Rieth was fond of

photographing fungi and writing teaching materials on all aspects of mycology.

Prof. Wagener Prof. Bisping Prof. Rieth on the ship at Travemuende, 1962

3

Contents item page Item Page

Preface 1 6. Aspergillosis in animals 91

1.Introduction 4 6.1. Aspergillosis in dogs 91

2.Historical 5 6.2. Aspergillosis in cats 94

3. Nomenclature and classification 11 6.3. Aspergillosis in horses 96

4. Description of important aspergilli 17 6.4. Aspergillosis in cattle 98

4.1. Aspergillus Section Restricti 17 6.5. Aspergillosis in Sheep and goats 100

4.2. Aspergillus Section Cervini 18 6.6. Aspergillosis in pigs 104

4.3. Aspergillus section Nigri 19 6.7. Aspergillosis in camels 104

4.4. Aspergillus section Candidi 23 6.8. Aspergillosis in American bisons 105

4.5. Aspergillus section Usti 25 6.9. Aspergillosis in monkeys 106

4.6. Aspergillus section Versicolores 26 6.10. Aspergillosis in deers 106

4.7. Aspergillus section Sparsi 30 6.11. Aspergillosis in rabbits 107

4.8.Aspergillus Section: Aspergillus 31 6.12. Aspergillosis in guinea-pigs 108

4.9. Aspergillus section Terrei 35 4.13. Aspergillosis in lesser blind mole rats 108

4. 10. Aspergillus Section Flavipedes 36 6.14. Aspergillosis in dolphins 109

4. 11. Section: Circumdati 39 7. Aspergillosis in birds 110

4. 12. Aspergillus section Flavi 40 7. 1. Aspergillosis in poultry 110

4.13. Aspergillus Section: Cremei 45 7.2. Aspergillosis in wild birds: 112

4.14. Aspergillus section Fumigati 46 7.2.1. Aspergillosis in penguins 112

4. 15. Aspergillus Section:Clavati 49 7.2.2. Aspergillosis in parrots 114

4. 16. Aspergillus Section:Nidulantes 50 7.2.3. Aspergillosis in quails 115

4. 17. Aspergillus Section:Ornati 52 7.2.4. Aspergillosis in ostrich 116

4. 18. Aspergillus sect. Aeni 52 7.2.5. Aspergillosis in red-tailed hawk 117

5. Aspergillosis in man 7.2.6. Aspergillosis in Red-billed Toucan 117

5.1. Pulmonary aspergillosis 7.2.7. Aspergillosis in Snow Owl 118

5.2. Cerebral aspergillosis 71 7.2.8. Aspergillosis in Goshawk 118

5.3. Cutaneous aspergillosis 73 7.2.9. Aspergillosis in Cormorants 119

5.4. Aspergillus Onychomycosis 75 7.2.10. Aspergillosis in swans 119

5. 5. Ocular aspergillosis 76 7.2.11. Aspergillosis in kiwi 120

5.6. Aspergillus sinusitis 78 8. Laboratory diagnosis of aspergillosis 123

5.7. Paranasal sinuses aspergillosis 79 8.1. Direct microscopic examination 123

5.8. Otoaspergillosis 81 8.2. Isolation and identification 128

5.9. Aspergillus endocarditis 82 8.3.Molecuar identification of aspergilli 132

5.10. Aspergillus thyroiditis 84 8.4. Serological techniques 135

5.11. Hepatic aspergillosis 85 9. Treatment of aspergillosis

140

5.12. Gastrointestinal aspergillosis 86 10. References

143

5.13. Urinary tract aspergillosis 87

5.13. Aspergillus osteomyelitis 88

4

1. Introduction Aspergillus is one of the oldest named genera of fungi that received its name from Micheli in

1729. In viewing the microscopic spore-bearing structure, Micheli was reminded of a device

used by the Roman Catholic clergy to sprinkle holy water during a part of the liturgy called the

asperges (Ainsworth, 1976). The asperges was described as follows in the 11th edition of The

Encyclopedia Britannica:

ASPERGES (‘thou wilt sprinkle,’ from the Latin verb aspergere), the ceremony of the Roman

Catholic Church … with which the priest begins the ceremony. The brush used for sprinkling is

an aspergill (aspergillum)… (Anonymous, 1910)

Aspergillum: a brush or small perforated container with a handle that is used for sprinkling holy

The aspergillum-like spore-bearing structure is the most important microscopic character used

in defining members of the genus Aspergillus. During mycelial differentiation certain cells

enlarge, develop a heavy cell wall and form 'T' or 'L' shaped 'foot cells', which are not separate

cells and that produce a single conidiophore perpendicular to the long axis of the cell. The erect

hyphal branch developing from the foot cell is the conidiophore, which enlarges at its apex to

form a rounded, elliptical or club shaped vesicle. The fertile area of the vesicle gives rise to a

layer of cells called phialides (sterigmata) that produce long chains of mitotic spores called

conidia or conidiospores. The size and arrangement of the conidial heads as well as the colour

of the spores they bear are important identifying characteristics.

5

2. Historical

2.1. 1729-1809

Pier Antonio Micheli was not only a Catholic Priest but also a famous botanist. His Nova

plantarum genera of 1729 was a milestone in the study of fungi. In this work, with

descriptions of 1900 plants, including new 1400, he described 900 fungi

and lichens. Micheli gave the name Aspergillus (rough head) and described Aspergillus

capitatus ochroleucus, probably some strains of Aspergillus ochraceus; Aspergillus

capilulo pulla for a black form, etc.

6

2.2. 1809-1926

Link (1809) described A. glaucus. Aspergillus candidus, Aspergillus

chevalieri, Aspergillus flavus and Eurotium herbariorum

Corda (1828) published his studies of fresh material, as seen under his microscope. He

described several species,e.g. Aspergillus phoenicis, Aspergillus repens

Desmazières (1834) described A. clavatus

DeBary (1854) noticed that an Aspergillus mycelium could produce a cleistothecium as

well as an aspergillum. Cleistothecium-producing mould had been observed before and

given its own name: Eurotium herborarium. DeBary realized that A. glaucum and E.

herborarium were different reproductive phases of the same organism, thus putting the

dual naming of the same fungus in its sexual and asexual stages.

7

Heinrich Anton de Bary Aspergillus glaucus by Anton de Bary (1854) Jean Paul Vuillemin

Fresenius (1863) described Aspergillus fumigatus and Sterigmatocystis sulphurea

Berk. & M. A. Curtis (1868) described Aspergillus erythrocephalis

van Tieghem (1967) described Aspergillus niger

Wilhelm (1877) described Aspergillus ochraceus

Ahlburg (1878) described Eurotium oryzae

Bainier (1880-1913) described A. ustus, A. flavipes, Sterigmatocystis

usta, Sterigmatocystis flavipes, Sterigmatocystis sydowii , Aspergillus gracilis and

Sterigmatocystis carbonaria

Saccardo (1882) described A. repens

Eidam (1883) described Sterigmatocystis nidulans

Winter (1884) described A. nidulans

Cohn (1884) described Aspergillus oryzae

Gasperini (1887) described Aspergillus elegans , Aspergillus violaceofuscus

Delacroix (1893) described Aspergillus brunneus , Eurotium echinulatum

Wehmer (1896-1907) devided the genus Aspergillus in Subgenus Microaspergillus and

Macroaspergillus. He described Aspergillus ostianus , Aspergillus wentii,

Aspergillus fischeri, Aspergillus giganteus, Aspergillus sulphureus , Aspergillus varians,

Aspergillus pulverulentus

Höhn (1902) described Aspergillus citrisporus

Vuillemin (1903-1908) Sterigmatocystis versicolor, Aspergillus versicolor

Saito (1904-1906) described Aspergillus caesiellus, Aspergillus japonicus

Mangin (1909-1910) described Aspergillus chevalieri , Eurotium chevalieri

Yukawa (1911) described Aspergillus melleus

Kita (1913) described Aspergillus tamarii

Blochwitz (1914) described Aspergillus conicus

Massee (1914) described Aspergillus cervinus

Thom and Church (1918) described A. terreus

8

2.3. 1926-1965

Thorn and Church (1926) revised some 350 names, but only 69 species were accepted,

which were more or less arbitrarily considered in 11 groups. Their studies resulted in the

publication in the 1926 of a monograph entitled “The Aspergilli.”

They based their taxonomy of the macroscopic colony charactes and the microscopic

features of the vesicles, conidiophores, the single or double series of the spore-bearing

phialides and the shapes and colours of conidia.

Thom and Raper (1945) used the work of Thorn and Church as a basis for subsequent

taxonomic treatments of the genus in their book entitled “A Manual of the Aspergilli”.

They classified the Aspergillus species into 14 groups and 9 series.

Charles Thom Kenneth Raper

Raper & Fennell (1965) described 150 taxa in their monograph “The Genus

Aspergillus”. They divided the species into 18 informal “groups” based on the authors”

opinions of probable relationships. These groups contained 132 species and 18 varieties.

9

Dorothy Fennell

2.4. 1965-2011

Gams et al (1985) replaced the groups by sections and divided the genus Aspergillus into

18 sections organized in six subgenera.

Pitt et al. (2000), in their the latest compilation of names in current use, listed 182.

Samson (2000) listed another 36 published between 1992 and 1999. More than 40 new

species descriptions have been published since then , bringing the total number to ∼250.

Samson, R.A., and Varga, J.(2007), in their book Aspergillus systematics in the genomic

era, they presented the activities the workshop held on 12-14 April, 2007 at

the CBS, Fungal Biodiversity Centre,Utrecht –The Netherlands covering the following

themes:

o What is the impact of Aspergillus taxonomy in terms of epidemiology, case definitions

and biological understanding of disease?

o What and how many genes should be used to delimit an Aspergillus taxon?

o How does the phylogenetic species concept translate to practical and routine diagnoses?

o What are the roles of Aspergillus databases for species identification?

o What is the value and impact of polyphasic approaches for species identification?

o What genes/methods can be used to design kits for rapid identification?

o How should new species be proposed?

11

Geiser et al. (2008) mentioned that there are approximately 250 named species of

Aspergillus and this number is likely to increase significantly in the near future because of

increasing application of the phylogenetic species concept based on DNA sequence data

rather than on visible morphological characters.

Gustavo H. Goldman and Stephen A. Osmani (2008) published their book “ The

Aspergilli Genomics, Medical Aspects, Biotechnology, and Research Methods”

M. Machida and K. Gomi (2010) published their book “Aspergillus, molecular biology

and genomics” summarized the most important aspects of Aspergillus molecular biology

and genomics. Robert A. Samson, János Varga and Jens C. Frisvad (2011) published in their book “

Taxonomic studies on the genus Aspergillus” new and revisited species in Aspergillus

section Nigri, Aspergillus section Terrei, Aspergillus section Flavi and Aspergillus section

Usti.

W. Gams John Pitt Stephen W. Peterson

Robert A. Samson János Varga Jens C. Frisvad

11

3. Nomenclature and classification

During the 20th century, as mycologists isolated and identified increasing numbers of

isolates, the number of named species of Aspergillus increased. These tended to fall into

morphologically distinct clusters. So in order to facilitate identification, the genus was

divided into intrageneric 'groups'. The Aspergillus glaucus group, for example, was

characterized by abundant, typically green conidial heads, with perithecia generally

present, while the Aspergillus ochraceous group had yellow conidia and abundant cream

to purplish-coloured sclerotia.

The term 'group' does not have nomenclatural status within the formal rules of biological

nomenclature. Therefore, the genus was reorganized into a new subgeneric taxonomic

hierarchy based on 'sections.' In this system, The genus was subdivided into subgenera.

The subgenus 'Aspergillus' consists of xerophilic species. A. glaucus is the type species,

classified in the subgenus Aspergillus and the section Aspergillus.

A new subgenus was introduced called Circumdata that encompassed seven sections, in

which 'section circumdati' was the new rubric for the old 'A. ochraceous group'. The

attempted imposition of subgeneric epithets, only one of which is called 'Aspergillus' is

extremely confusing and has not caught on. On the other hand, most taxonomists now use

12

the term 'section' rather than 'group' for Aspergillus intrageneric classifications and

identifications.

Some Aspergillus species regularly produce both sexual and asexual spores; in other

species the sexual form is rare; for still others, sexual spores have never been seen - and

perhaps never will be seen.

The names used for currently accepted sexual genera with close phylogenetic relationship

or known linkage to Aspergillus species (representative Aspergillus species given in

parenthesis) are:

1. Chaetosartorya (A. wentii);

2. Emericella (A. nidulans);

3. Eurotium (A. glaucus);

4. Fennellia (A. terreus);

5. Hemicarpenteles (A. paradoxus);

6. Neocarpenteles (A. clavatus);

7. Neosartorya (A. fumigatus);

8. Petromyces (A. flavus);

9. Sclerocleista (A. ornatus);

10. Warcupiella (A. spinnulosus).

3.1. Classification according to Thom and Raper (1945)

Thom and Raper (1945) classified the Aspergillus species into 14 groups and 9 series as

follows:

Group 1: THE ASPERGILLUS CLAVATUS GROUP

Group 2: THE ASPERGILLUS GLAUCUS GROUP 1.ASPERGILLUS REPENS SERIES

2.ASPERGILLUS RUBER SERIES

3.ASPERGILLUS CHEVALIERI SERIES

4.ASPERGILLUS AMSTELODAMI SERIES

5.ASPERGILLUS RESTRICTUS SERIES

Group 3: THE ASPERGILLUS FUMIGATUS GROUP 6.ASPERGILLUS FUMIGATUS SERIES

7.ASPERGILLUS FISCHERI SERIES

Group 4: THE ASPERGILLUS NIDULANS GROUP

Group 5: THE ASPERGILLUS USTUS GROUP

Group 6: THE ASPERGILLUS FLAVIPES GROUP

13

Group 7: THE ASPERGILLUS VERSICOLOR GROUP

Group 8: THE ASPERGILLUS TERREUS GROUP

Group 9: THE ASPERGILLUS CANDIDUS GROUP

Group 10:THE ASPERGILLUS NIGER GROUP 8.ASPERGILLUS NIGER SERIES

9.ASPERGILLUS CARBONARIUS SERIES

Group 11:THE ASPERGILLUS WENTII GROUP

Group 12:THE ASPERGILLUS TAMARII GROUP

Group 13:THE ASPERGILLUS FLAVUS-ORYZAE GROUP

Group 14:THE ASPERGILLUS OCHRACEUS GROUP

3.2. Classification according to Raper and Fennel (1965)

Raper and Fennel (1965) divided species into 18 informal “groups” , which contained 132

species and 18 varieties as follows:

3.3. Classification according to Gams, Christensen, Onions, Pitt and Samson

Gams et al (1985) replaced the groups by sections and divided the genus Aspergillus into 18

sections organized in six subgenera.

Subgenera Sections

(Gams et al.,1985)

Groups

(Raper & Fennell,1965)

Aspergillus

Aspergillus

Restricti

A. glaucus

A. restrictus

Fumigati

Fumigati

Cervini

A. fumigatus

A. cervinus

Ornati Ornati A. ornatus

Clavati Clavati A. clavatus

Nidulantes

Nidulantes

Versicolor

Usti

Terrei

Flavipedes

A. nidulans

A. versicolor

A. ustus

A. terreus

A. flavipes

Circumdati

Wentii

Flavi

Nigri

Circumdati

Candidi

Cremei

Sparsi

A. wentii

A. flavus

A. niger

A. ochraceus

A. candidus

A. cremeus

A. sparsus

14

They then added the teleomorph names as follows:

1. Subgenus Aspergillus

1. Section: Aspergillus (Teleomorph: Eurotium)

2. Section: Restricti

2. Subgenus Circumdati

3. Section: Circumdati (Teleomorph:Neopetromyces)

4. Section: Nigri

5. Section: Flavi (Teleomorph:Petromyces)

6. Section: Cremei (Teleomorph:Chaetosartorya)

7. Section: Candidi

8. Section: Wentii

9. Section: Sparsi

3. Subgenus Clavati

10. Section: Clavati (Teleomorph:Neocarpenteles)

4. Subgenus Fumigati

11. Section: Cervini

12. Section: Fumigati (Teleomorph:Neosartorya)

5. Subgenus Nidulantes

13. Section: Nidulantes (Teleomorph:Emericella)

14. Section: Versicolores

15. Section: Flavipedes (Teleomorph:Fennellia)

16. Section: Usti

17. Section: Terrei (Teleomorph:Fennellia)

6. Subgenus Ornati

18. Section: Ornati (Teleomorph:Sclerocleista)

15

3.4. Classification according Petrson (2000)

Peterson (2000) reported that phylogenetic studies of ribosomal RNA gene sequences led to the

acceptance of 3 subgenera with a total of 16 sections and the so-called »Warcupiella group«, a

treatment currently accepted by most Aspergillus researchers:

Subgenus Aspergillus

1. Section: Aspergillus (Teleomorph: Eurotium)

2. Section: Restricti

3. Section: Cervini

4. Section: Terrei (Teleomorph:Fennellia)

5. Section: Flavipedes (Teleomorph:Fennellia)

6. Section: Nigri

7. Section: Circumdati (Teleomorph:Neopetromyces)

8. Section: Flavi (Teleomorph:Petromyces)

9. Section: Cremei (Teleomorph:Chaetosartorya)

10. Section:Candidi

Subgenus Fumigati

11. Section:Fumigati (Teleomorph:Neosartorya)

12. Section:Clavati (Teleomorph:Neocarpenteles)

Subgenus Nidulantes

13. Section:Nidulantes (Teleomorph:Emericella)

14. Section:Usti

15. Section:Versicolores

16. Section:Ornati (Teleomorph:Hemicarpenteles)

3.5. Molecular classification of Aspergillus and its Teleomorphs

Based on the phylogenetic analysis of multilocus (calmodulin, RNA polymerase 2 and rRNA)

sequence data (Samson and Varga, 2009 and Varga et al. 2010)., Aspergillus can be subdivided

into eight subgenera and 19 sections as follows:

1. Subgenus Aspergillus

1. Sections Aspergillus

2. Sections Restricti;

2. Subgenus Fumigati

3. Sections Fumigati

4. Sections Clavati

5. Sections Cervini

3. Subgenus Circumdati

6. Sections Circumdati

16

7. Sections Nigri

8. Sections Flavi

9. Sections Cremei

10. Aspergillus sect. Aeni sect. nov

4. Subgenus Candidi

11. Section Candidi;

5. Subgenus Terrei

12. Sections Terrei

13. Sections Flavipedes;

6. Subgenus Nidulantes

14. Sections Nidulantes

15. Sections Usti

16. Sections Sparsi;

7. Subgenus Warcupi

17. Sections Warcupi

18. Sections Zonati

8. Subgenus Ornati

19. Sections Ornati.

3.6. Suggested nomenclature and classification of the Genus Aspergillus (Refai, 2014)

It is suggested to divide the Genus Aspergillus into 2 Subgenera:

1. Subgenus I contains 6 Aspergillus sections that have no known teleomorphs yet,

namely Sections Restricti, Cervini, Nigri, Candidi, Usti and Versicolores.

2. Subgenus II contains 10 aspergillus sections that have teleomorphs, namely

Sections Aspergilli, Terrei, Flavipedes, Circumdati, Flavi, Cremei, Fumigati,

Clavati, Nidulantes and Ornati.

3. The name Michelius or Michelia is suggested as the genus name for all

telemorphs, which is devided into the following sections: Euroti, Fennelli,

Neopetromycei, Petromyci, Chaetosartori, Neosartori, Neocarpentelli, Emericelli

and Hemicarpentelli

Consequently, the species that have teleomorphs will be named as follows:

Aspergillus glaucus Link, 1809 Teleomorph: Michelia herbariorum

Synonym: Eurotium herbariorum Link, 1809

17

4. Description of important aspergilli

4.1. Aspergillus Section Restricti

Aspergillus Section Restricti (the Aspergillus restrictus Group) include the following species 1. Aspergillus caesiellus

2. Aspergillus penicillioides

3. Aspergillus restrictus

4. A. conicus

5. A. gracilis

6. A. itaconicus

.

4.1.1. Aspergillus restrictus G. Smith (1931)

Synonym: Penicillium fuscoflavum S. Abe (1956)

Morphology ( http://www.bcrc.firdi.org.tw/fungi/fungal) Colony diameters on Czapek’s Agar 1.3-1.5 cm in 2 weeks at 25°C, dense, rugged, floccose,

with fimbriate margins; mycelium white; conidial heads small, indistinct, covered in slime,

blackish green-gray; reverse uncolored to dark gray green. Colony diameters on M40Y 4.5-5.5

cm in 2 weeks at 25°C, velutinous, tufted or floccose; conidial heads long, narrow, columnar,

sometimes twisted, artemisia green to lily green; reverse colorless to olive brown; vesicles flask

shaped, or pyriform, 5.0-13.0 μm in diameter; stipes thin-walled, smooth or roughened, straight

or sinuous, uncolored, 78-106 × 2.0-6.0 μm. Aspergilla uniseriate, phialides 4.8-10.3 × 2.4-4.4

μm, covering the upper 2/5 of the vesicle. Conidia elongate, doliiform or ellipsoidal to

pyriform, 3.8-9.5 × 3.0-4.4 μm, rough to spinose.

18

S. S. Tzean and J. L. Chen, B Flannigan, R Samson & JD Miller , Joe Akin, Mycobank

4.2. Aspergillus Section Cervini

The section Cervini branch contains the four species placed in the group byRaper and Fennell

(1965). Full congruence of trees based on the separate loci and strong statistical support

recognize the six lineages. Isolates phenotypically identified as A. kanagawaensis and as A.

nutans each split into two separate lineages (Peterson, 1980). The following are the

species:

1. Aspergillus kanagawaensis

2. Aspergillus nutans

3. Aspergillus cervinus

4. Aspergillus parvulus

5. A. kanagawaensis

6. A. nutans

4.2.1. Aspergillus cervinus Massee, 1914

Fennell (1964) rediscovered Aspergillus cervinus as a uniseriate fawn-colored Aspergillus

obtained from Malayan forest soils. The outstanding characteristics of this sp. are a fawn to

pinkish-cinnamon surface color, thick-walled conidiophores up to 9.5[mu] wide, a single series

of sterigmata borne radiately on the vesicle, and globose smooth conidia. The Malayan cultures

and an obtained Wisconsin isolate matched in essential details Massee's original diagnosis and

Neill's later description of A. cervinus. They appear to be the only extant representatives of the

sp. The mold Aspergillus cervinus is considered to be rare, as it has only been isolated from the

tropical rainforest soils of Malaya, Puerto Rico, Wisconsin, New Zealand and India.

19

4.3. Aspergillus section Nigri

Aspergillus section Nigri are industrially one of the most important taxa of filamentous fungi.

Several strains belonging to this section are used in the fermentation industry for the production

of different organic acids and hydrolytic enzymes.

The observation that black aspergilli including A. niger and A. carbonarius are able to

produce ochratoxins is economically important since A. niger is extensively used in the

food industry.

Taxonomic evaluation of this species complex was carried out using different methods

(Varga et al,. 2011). Among the genotypic approaches, nuclear and mtDNA

polymorphisms and PCR based techniques led to the recognition of four species within

this species complex (A. niger, A. tubingensis, A. brasiliensis, A. foetidus).

Several well known species names such as A. awamorii, A. usamii, A. phoenicis and A.

ficuum have been reduced to synonymy.

Regarding other black Aspergillus species, phylogenetic analysis of ITS sequence data

indicates that at least 9 species belong to this section: 1. Aspergillus heteromorphus,

2. Aspergillus ellipticus,

3. Aspergillus carbonarius,

4. Aspergillus japonicus,

5. Aspergillus aculeatus,

6. Aspergillus. niger,

7. Aspergillus. tubingensis,

8. Aspergillus foetidus and

9. Aspergillus brasiliensis.

Ochratoxin production has been observed only in A. niger and A. carbonarius isolates.

These species are now considered as major sources of ochratoxin contamination in

tropical and subtropical foods including dried vine fruits, wines and coffee

The identification of species of Aspergillus section Nigri depends on:

a. Colony morphology, conidial size and ornamentation of the cultures

b. The temperature range of all species

c. Growth characteristics on creatine agar and boscalid agar.

d. The extrolites produced by each species

e. The response of the Ehrlich reaction

f. Molecular characterization (β-tubulin or calmodulin sequence data)

21

4.3.1. Aspergillus niger van Tieghem 1867

Synonyms: Sterigmatocystis nigra (Tiegh.) Tieghem, (1877)

Aspergillopsis nigra (Tiegh.) Speg., (1910)

Rhopalocystis nigra (Tiegh.) Grove (1911)

Aspergillus pyri W.H. English

Aspergillus fuliginosus Peck, (1873)

Aspergillus cinnamomeus E. Schiemann (1912)

Aspergillus fuscus E. Schiemann (1912)

Aspergillus niger var. altipes E. Schiemann, (1912)

Aspergillus schiemanni Thom (1916)

Morphology:

On Czapek dox agar, colonies consist of a compact white or yellow basal felt covered by a

dense layer of dark-brown to black conidial heads. Conidial heads are large (up to 3 mm x 15-

20 um in diameter), globose, dark brown, becoming radiate and tending to split into several

loose columns with age. Conidiophores are smooth-walled, hyaline or turning dark towards the

vesicle. Conidial heads are biseriate with the phialides borne on brown, often septate metulae.

Conidia are globose to subglobose (3.5-5.0 um in diameter), dark brown to black and rough-

walled.

Varga et al., 2011 Mycobank

21

A. niger is one of the most common species of the genus Aspergillus. It causes a disease called

black mold on certain fruits and vegetables such as grapes, onions, and peanuts, and is a

common contaminant of food. It is ubiquitous in soil and is commonly reported from indoor

environments,

1. Aspergillus niger can cause aspergillosis in man, in particular, frequent

among horticultural workers that inhale peat dust, which can be rich in Aspergillus

spores. It is one of the most common causes of otomycosis (fungal ear infections), which

can cause pain, temporary hearing loss, and, in severe cases, damage to theear

canal and tympanic membrane.

2. Various strains of Aspergillus niger are used in the industrial preparation of citric

acid and gluconic acid and have been assessed as acceptable for daily intake by the

World Health Organisation. A. niger fermentation is "generally recognized as safe"

(GRAS) by the United States Food and Drug Administration Many useful enzymes are

produced using industrial fermentation of Aspergillus niger, e,g, , A.

niger glucoamylase , pectinases , Alpha-galactosidase and proteases.

3. Aspergillus niger growing from gold mining solution contained cyano metal complexes;

such as gold, silver, copper iron and zinc. The fungus also plays a role in the

solubilization of heavy metal sulfides. Alkali treated Aspergillus niger binds to silver to

10% of dry weight. Silver biosorbtion occurs via stoichiometric exchange with Ca(II) and

Mg(II) of the sorbent.

4. In 2006, it was reported that a secreted RNase produced by Aspergillus

niger called actibind has antiangiogenic and anticarcinogenic characteristics.

4.3.2. Aspergillus carbonarius (Bainier) Thom, (1916) Synonyms: Sterigmatocystis carbonaria Bainier, (1880)

Rhopalocystis carbonaria (Bainier) (1911)

Aspergillopsis puchella Speg. (1910)

Aspergillopsis pulchella Speg., (1910)

Aspergillus atropurpureus (1902)

Sterigmatocystis acini-uvae Caball., (1928)

Aspergillus fonsecaeus Thom & Raper, (1965)

Morphology ( http://www.bcrc.firdi.org.tw/fungi/fungal)_

Colony diameters on Czapek’s Agar 4.0-4.5 cm in 7 days at 25°C, basal mycelium white,

velvety, marginal mycelium low; reverse white to dark grey; exudate clear to slight greyish

yellow; conidial heads globose to radiate or rarely splitting into loose columns, deep olive or

black ; stipes 300-5720 × 6.4-31.5 μm, smooth to rough, thick-walled, uncolored to brown or

22

reddish brown towards the vesicle; vesicles globose to subglobose 20-96 μm in diameter.

Aspergilla biseriate, metulae covering the upper 1/2 to the entire sur-face of the vesicle, 14.0-

50.0 × 6.0-14.0 μm; phialides 8.0-12.0 × 5.0-8.0 μm. Conidia spherical, 5.6-12.0 μm in

diameter, spinulose when young, tuberculate at maturity. Colony diameters on Malt Extract

Agar 5.0-6.0 cm in 7 days at 25°C; conidial heads radiate to globose, or splitting into more or

less well defined columns, blackish brown; reverse uncolored.

Aspergillus carbonarius resembles A. niger in many features, and indeed the two species are

very closely related. A. carbonarius differs from A. niger most notably in the production of

larger spores, although other minor morphological differences exist. The available information

on its physiology indicates a broad similarity toA. niger. Recently, black aspergilli,

mainly Aspergillus carbonarius and members of the Aspergillus nigeraggregate, have been

described as a main possible sources of ochratoxin (OTA) contamination in grapes from

Argentina and Brazil, France, Italy, Spain, Portugal, Greece and Australia as well as in dried

vine fruits from different origins. Strong evidence of the contribution of A. carbonarius to the

OTA contamination of wine has been also reported. Ochratoxin A is a highly harmful

metabolite classified in 1993 by the International Agency for Research on Cancer (IARC, 1993)

as a possible human carcinogenic toxin (group 2B).

www.bustmold.com. www.cirad.frm, James Baker (cepecity), Mycobank

23

4.4. Aspergillus section Candidi

Aspergillus section Candidi historically included a single white-spored species, A. candidus.

Later studies clarified that other species may also belong to this section. The revised

sectionCandidi by Varga et al. (2007) includes 4 species: 1. candidus,

2. campestris,

3. taichungensis

4. tritici.

Morphological characteristics of section Candidi:

Slow growing colonies with globose conidial heads having white to yellowish conidia,

Conidiophores smooth, small conidiophores common, metulae present and covering the

entire vesicle,

Some large Aspergillus heads with large metulae,

Presence of diminutive heads in all species,

Conidia smooth or nearly so with a subglobose to ovoid shape,

Presence of sclerotia in three species (A. candidus, A. taichungensis and A. tritici).

All species produce terphenyllins and candidusins and three species (A. candidus, A.

campestris and A. tritici) produce chlorflavonins. Xanthoascins have only been found

in A. candidus.

Each of the species in section Candidi produce several other species specific extrolites,

and none of these have been found in any other Aspergillus species.A. candidus has often

been listed as a human pathogenic species, but this is unlikely as this species cannot grow

at 37 °C.

4.4.1.Aspergillus candidus Link, (1809) Synonyms: Aspergillus albus K. Wilh.

Aspergillus okazakii Saito, (1907)

Aspergillus albus var. thermophilus Nakaz., Takeda & Suematsu, (1932)

Aspergillus tritici B.S. Mehrotra & M. Basu, (1976)

Aspergillus triticus B.S. Mehrotra & M. Basu (1976)

Morphology ( http://www.bcrc.firdi.org.tw/fungi/fungal

Colony diameters on Czapek’s Agar 1.5-1.7 cm in 14 days at 25°C, dense, plane; conidial heads

radiate, white to ivory yellow; mycelium white; reverse white to cream color or warm buff to

light ochraceous-buff, stipes 64-800 × 4.0-8.7 μm, hyaline, smooth; vesicles subglobose,

globose, ellipsoidal or obovoid, 5.6-26.0 μm wide. Aspergilla biseriate, occasionally unseriate;

metulae 4.4-11.1 × 2.1-3.8 μm, usually swollen, covering the whole surface of the vesicle;

phialides 5.8-10.6 × 2.5-3.6 μm. Conidia subglobose or globose to ellipsoidal, smooth, 2.2-3.7

μm wide. Colony diameters on Malt Extract Agar 1.8-2.2 cm in 14 days at 25°C, dense,

24

velutinous; conidial heads radiate, white to pale ivory; mycelium white; reverse ivory yellow to

cream color.

Aspergillus candidus. Samson et al., 2011

Aspergillus candidus is a common contaminant of grain dust and which causes

respiratory disease in humans. The species is widely distributed in nature and develops

upon vegetation in the later stages of decay. İt has been reported

from grain, flour, hay, compost and a fur processing facility.

Growth of A. candidus on barley grain occurs at the substrate water content 20-25% and

maximal temperature 30-40°C.

A. candidus may produce citrinin and other mycotoxins. Also this species produces p-

terphenyl metabolites and, which are potent cytotoxic substances.

Some strains of A. candidus produce kojic acid and / or citrinin, molecules that can cause

renal disease in swine.

A. candidus is involved in various human infections: invasive aspergillosis, otomycosis,

onychomycosis.

25

4.5. Aspergillus section Usti

Based on phylogenetic analysis of sequence data (Samson et al.,2011), Aspergillus section

Usti includes the following species: 1. Aspergillu ustus,

2. Aspergillu. puniceus,

3. Aspergillus granulosus,

4. Aspergillu pseudodeflectus,

5. Aspergillu calidoustus,

6. Aspergillus insuetus

7. A. compatibilis (Emericella heterothallica )

8. Aspergillus heterothallicus (Fennellia monodii)

9. A. keveii sp. Nov.

10. Aspergillus germanicus sp. nov. was isolated from indoor air in Germany. This

species is unable to grow at 37 °C, similarly to A. keveii and A. insuetus.

11. Aspergillus carlsbadensis sp. nov. was isolated from the Carlsbad Caverns

National Park in New Mexico. This species is also unable to grow at 37 °C, and

acid production was not observed on CREA.

12. Aspergillus californicus sp. nov. is proposed for an isolate from chamise chaparral

(Adenostoma fasciculatum) in California. This species grew well at 37 °C, and

acid production was not observed on CREA.

13. Aspergillus turkensis sp. nov. was isolated from soil in Turkey. This species grew,

although rather restrictedly at 37 °C, and acid production was not observed on

CREA.

14. Aspergillus pseudoustus sp. nov. was isolated from stored maize, South Africa

15. Aspergillus monodii comb. Nov

4.5.1.Aspergillus ustus (Bainier) Thom & Church, 1924.

Synonyms: Sterigmatocystis usta Bainier (1881)

Aspergillus humus Abbott (1926)

A. ustus is a variable species. A. ustus isolates may vary in their colony colour from mud brown

to slate grey, with colony reverse colours from uncoloured through yellow to dark brown.

Colony diam, 7 d, in mm: CYA 36-43; CYA37 no growth; MEA25 39-46; YES 42-50. Colony

texture: floccose, plane, sulcate or umbonate. Conidial head: radiate to hemispherical. Stipe:

400 × 3-6 μm, aerially borne stipes up to 125 × 2-5 μm, smooth, brownish. Vesicle diam/shape:

7-15 μm, hemispherical to subglobose. Conidium size/shape/surface texture: 3.2-4.5 μm,

globose, roughened, greenish to dark yellow brown. Hülle cells: irregularly ovoid or elongate,

usually scattered. Ehrlich reaction: no reaction. Growth on creatine: good growth with faint

yellow mycelium, no acid production.

26

Diagnostic features: No growth at 37 °C; good growth on creatine with faint yellow pigmented

mycelium; Hülle cells typically scattered or form irregular masses and not associated with

pigmented mycelium.

Aspergillus ustus, Samson et al., 2011

4.6. Aspergillus section Versicolores

Aspergillus section Versicolores was originally erected as the Aspergillus versicolor group

by Thom & Church (1926) and was subsequently revised by Thom & Raper (1945) to contain

four species. Raper & Fennell (1965) revised the genus Aspergillus and accepted 18 species in

the A. versicolor group. Gams et al. (1985) formalized the sectional taxonomy of Raper &

Fennell’s (1965) groups. Peterson (2008) accepted four phylogenetically distinct species in the

section based on multilocus DNA sequence analysis.

Aspergillus versicolor is the most widely reported and studied species in section Versicolores. It

has been isolated from, indoor environments, various foods and feeds and hypersaline water,

and is associated with many health issues of humans and animals. It is a producer of the

mycotoxin sterigmatocystin that is a precursor of aflatoxin B1.

The section Versicolores was revised by Jurjevic et al. (2012) and contains the following

species::

1. Aspergillus sydowii,

2. Aspergillus creber,

3. Aspergillus venenatus,

4. Aspergillus tennesseensis,

5. Aspergillus cvjetkovicii,

6. Aspergillus jensenii and

7. Aspergillus puulaauensis;

8. Aspergillus versicolor,

9. Aspergillus tabacinus,

10. Aspergillus fructus,

27

11. Aspergillus protuberus,

12. Aspergillus amoenus and

13. Aspergillus. Austroafricanus

14. Aspergillus subversicolor.

4.6.1. Aspergillus sydowii Thom & Church (1926)

Synonyms: Sterigmatocystis tunetana Langeron, (1924)

Aspergillus sydowii var. achlamydosporus Nakaz. et al. (1934)

When grown in pure culture on agar plates, A. sydowii produces blue-green colonies with

reddish-brown shades. Colony on Czapek’s Agar is plane to floccose; conidial heads radiate to

loosely columnar, light grayish olive or green; mycelium white; reverse ivory yellow or maroon

; stipes hyaline to pale brown, smooth; vesicles clavate to subglobose. Aspergilla biseriate,

metulae covering 1/2 to 4/5 of the vesicle; Conidia globose, conspicuously roughened to

spinose. Small aspergilla often present resembling the fruiting structures of Penicillium. Hulle

cells occasionally present, globose to subglobose.

28

Jos A.M.P. Houbraken, Ronald P. de Vries, Robert A. Samson, A. sydowii Mycobank

Aspergillus sydowii is a saprophytic fungus found in soil that can contaminate food and is

occasionally pathogenic to humans. It is the predominant fungus found on wheat Qu, the most

widely used source of raw microorganisms and crude enzymes for Chinese rice

wine brewing.[5]

Since the 1990s it has been found to be present in sea water in the Caribbean

region and has been shown to be the cause of aspergillosis in sea fans

Aspergillus sydowii has been implicated in the pathogenesis of several human diseases,

including aspergillosis, onychomycosis, and keratomycosis.

4.6.2. Aspergillus versicolor (Vuill.) Tirab., (1908).

Synonyms:Aspergillus amoenus Roberg, 1931

Aspergillus versicolor var. fulvus Nakaz. et al., 1932

Aspergillus versicolor var. rutilobrunneus J.N. Rai, S.C. Agarwal & J.P. Tewari, 1971

Sterigmatocystis versicolor Vuill., 1903

Morphology

Colonies on CYA 16-25 mm diam, plane or lightly sulcate, low to moderately deep, dense;

mycelium white to buff or orange; conidial heads sparse to quite densely packed, greyish green;

pink to wine red exudate sometimes produced; reverse orange or reddish brown. Colonies on MEA

12-25 mm diam, low, plane, and dense, usually velutinous; mycelium white to buff; conidial heads

numerous, radiate, dull or grey green; reverse yellow brown to orange brown. Colonies on G25N

10-18 mm diam, plane or umbonate, dense, of white, buff or yellow mycelium; reverse pale,

29

yellow brown or orange brown. No growth at 5°C. Usually no growth at 37°C, occasionally

colonies up to 10 mm diam formed.

Conidiophores borne from surface or aerial hyphae, stipes 300-600µm long, with heavy yellow

walls, vesicles variable, the largest nearly spherical, 12-16µm diam, fertile over the upper half to

two-thirds, the smallest scarcely swollen at all and fertile only at the tips, bearing closely packed

metulae and phialides, both 5-8µm long; conidia mostly spherical, very small, 2.0-2.5µm diam,

with walls finely to distinctly roughened or spinose, borne in radiate heads.

Aspergillus versicolor isolates produce the aflatoxin precursor sterigmatocystin, a compound that is

mutagenic and tumorigenic. Animal feed infested with three morphotypes of A. versicolor, all of

which produce sterigmatocystin, have been implicated in dairy animal toxicosis, but it is unknown

whether sterigmatocystin caused the toxicosis.

Aspergillus versicolor has been implicated as the causitive agent of disseminated aspergillosis in

dogs has probably caused aspergillosis in transplant recipients and has been isolated from the

infected eye of a patient suffering from HIV.

A. versicolor, Mycota, Mold-pro.com, fungi myospecies inf.

www.tamagawa.ac.j

31

Jurjevic Z, Peterson SW, Horn BW - IMA Fungus (2012)

Aspergillus versicolor, www.tamagawa.ac.jp

4.7. Aspergillus section Sparsi

The Aspergillus sparsus species group (Aspergillus section Sparsi; Gams et al. 1985) was

established by Raper & Fennell (1965) to accommodate four species isolated from

tropical or subtropical soils. Species assigned to this group have large globose conidial

heads, which irregularly split with age, with colours ranging from light grey to olive-

buff.

The data obtained by Varga et al. (2010) indicate that the revised section Sparsi includes

10 species:

1. Aspergillus anthodesmis,

2. Aspergillus biplanus,

3. Aspergillus conjunctus,

4. Aspergillus diversus,

5. Aspergillus funiculosus,

6. Aspergillus implicatus,

7. Aspergillus panamensis,

8. Aspergillus quitensis,

9. Aspergillus sparsus,

10. Aspergillus haitiensis ( recently described)

31

Aspergillus quitensis and Aspergillus ecuadorensis are synonyms of Aspergillus

amazonicus based on both molecular and physiological data.

The white-spored species A. implicatus has also been found to belong to this section.

Aspergillus haitiensis sp. nov. is characterised by whitish colonies becoming reddish

brown due to the production of conidial heads, and dark coloured smooth stipes.

The taxon produces gregatins, siderin and several unknown but characteristic metabolites.

4.8. Aspergillus Section: Aspergillus (Teleomorph: Eurotium)

Aspergillus section Aspergillus contains economically important, xerophilic fungi that

are widely distributed in nature and the human environment and are known for their

ability to grow on substrates with low water activity.

Eurotium species are the sexual states of Aspergillus species, notably the Aspergillus

glaucus group among others. Eurotium is common and is most closely related to

Emericella, another genus with Aspergillus anamorphs.

Health effects, allergenicity, and toxicity of Eurotium are closely related to the

Aspergillus anamorph and, for the most part, have not been studied apart from that

primary phase. The Aspergillus anamorph is likely to be the identifiable result, at least

with primary growth within one week.

The taxa were revised based on sequence data from four loci, PCR fingerprinting, micro-

and macromorphology, and physiology. The number of taxa was reduced to the following

species:

1. Aspergillus proliferans (The only anamorphic species)

2. Aspergillus niveoglaucus (≡Eurotium niveoglaucum)

3. Aspergillus brunneus [Eurotium echinulatum.].

4. Aspergillus neocarnoyi [Eurotium carnoyi.].

5. Aspergillus glaucus [Eurotium herbariorum].

6. Aspergillus repens (Eurotium repens)

7. Aspergillus rubrobrunneus [Eurotium rubrum]

8. Aspergillus tonophilusi [Eurotium tonophilum].

9. Aspergillus hollandicus (Eurotium amstelodami)

10. Aspergillus chevalieri var. intermedius (≡ Eurotium intermedium)

11. Aspergillus equitis (Eurotium chevalieri)

12. Aspergillus cristatus (≡Eurotium cristatum)

13. Aspergillus xerophilus. [Eurotium xerophilum.].

14. Aspergillus leucocarpus [Eurotium leucocarpum].

32

4.8.1. Aspergillus glaucus Link, 1809

(Teleomorph: Eurotium herbariorum (Wiggers) Link, 1809.

A. glaucus is not very invasive and is rarely encountered in the clinical laboratory. It has been

implicated as a cause of ocular (eye) infections, particularly after some traumatic

injury. Cerebral, orofacial, cardiovascular and pulmonary infections are rare but have been

reported. A. glaucus may also cause sinusitis (nasal) and otitis (ear) infections. A.

glaucus may be considered an opportunistic fungus particularly with immunocompromised

patients.

Morphology:

Growth is slow to moderate, maturing in about 7 to 21 days. Colony size expands rather

slowly. Colony colouration is media dependent but is described as a dull to deep green to a

greyish turquoise, with yellow to orange areas where cleistothecia are being produced. The

reverse is pale yellow to yellow. Hyphae are septate and hyaline.

Teleomorph – Sexual state is seen with the production of cleistothecia (ascomata). These

structures are globose to subglobose, about 60 µm to 150 µm in diameter. In their natural state

they appear yellow to golden in colour and their presence may be seen macroscopically as

distinctly yellowish areas within the maturing colony. Within the cleistothecia/ascomata, 8-

celled asci are produced which are released at maturity or when ruptured. The 8-celled asci (10

µm - 12µm diameter) are dehiscent (dissolve) and release individual ascospores on

maturity). The Ascospores themselves mature in about two weeks’ time and are lenticular (lens

shaped) with a noticeable longitudinal furrow. They range between 5 µm to 7 µm by 3 µm to 5

µm in size).

Eurotium herbariorum , B.Flannigan, R Samson & JD Miller Anamorph – smooth walled conidiophores extend between 300 µm – 700 µm in length and are

between 7 µm – 12 µm in width. Vesicles are globose (spherical) to subglobose (subspherical)

to pyriform (tear-drop) in shape and roughly 18-30 µm in diameter. A.glaucus is uniseriate with

phialides 7 – 11 µm to 3 – 7 µm in size and generally covers most of the vesicle. The conidia (4

µm to 8 µm diameter) are spherical to ellipsoidal in shape and are echinulate to spinose (finely

roughened/fine sp

33

GEFOR, Galería de imágenes: Aspergillus Glaucus

4.8.2. Aspergillus equitis Samson & Gams, 1985

Teleomprph: Eurotium chevalieri Mangin, 1909.

Morphology

Colony diameters on Czapek’s Agar 2.8-3.2 cm in 14 days at 25°C; conidial heads radiate, deep greenish glaucous to pistachio green; mycelium yellow. Cleistothecia yellow to buffy citrine, or deep colonial buff to olive; soluble pigment yellow; exudate clear; reverse yellow to Saccardo’s umber. Colony diameters on Czapek’s Agar with 20% added sucrose 6.0-6.5 cm in 14 days at 25°C; conidial heads radiate, gnaphalium green to near dark sage green; mycelium yellow to greyish yellow orange shades; exudate clear; reverse near asphodel green to apricot yellow, or mahogany red to bay; stipes 62-680 × 7.0-20.0 μm, smooth, colorless to pale brown; vesicles obovoid to globose 10.0-46.0 μm wide. Aspergilla uniseriate, phialides covering the entire surface of the vesicle, 4.0-13.1 × 2.8-6.2 μm. Conidia ellipsoidal to doliiform, less commonly subglobose, rough to irregularly roughened, 3.2-7.1 × 2.4-5.0 μm. Cleistothecia greyish yellow, subglobose to globose, up to 178 μm wide, asci 8-spored, spherical to subspherical, ascospores lenticular, with wall smooth to finely roughened, with 2 distinct longitudinal trough flanges, 4.2-5.4 × 3.3-4.0 μm. Colony diameters on M40Y 7.5-8.5 cm in 14 days at 25°C; conidial heads abundant, light hellebore green. Cleistothecia abundant as on C20S enmeshed in or-ange-red hyphae; reverse orange-brown.

34

Conidia Ascospores Mycobank

Aspergillus equitis Samson & W. Gams 1985. , S. S. Tzean and J. L. Chen

4.8.3. Aspergillus hollandicus Samson & W. Gams 1985. Teleomorph: Eurotium amstelodami Mangin, 1909.

Colony diameters on Czapek’s Agar 2.0-2.5 cm in 14 days at 25°C; conidial heads radiate to

columnar, dark terre verte to dusky green; mycelium white to yellow; reverse uncolored, amber

yellow to olive lake; cleistothecia yellow. Colony diameters on Malt Extract Agar 1.8-2.2 cm in

14 days at 25°C; conidial heads radiate, dark grass green to ivy green; mycelium white to

yellow; reverse colorless or dull wax yellow to dull primuline yellow; cleistothecia yellow.

Colony diameters on Czapek’s Agar with 20％ added sucrose 3.5-4.5 cm in 14 days at 25°C;

conidial heads radiate to columnar, ivy green to dark grass green; myce-lium white to yellow;

reverse uncolored, yellow to pale green brown; stipes 60-360 × 4.0-16.0 μm, smooth, colorless,

or pale brown to middle brown; vesicles globose or subglobose to obovoid or pyriform, 6.8-

32.5 μm in diameter. Aspergilla uniseriate, phialides covering 1/2 to 4/5 of the vesicle, 3.2-9.3 ×

3.0-5.3 μm. Conidia globose or subglobose to ovoid, 3.3-6.4 × 3.0-5.2 μm, rough to irregularly

roughened. Cleistothecia globose to subglobose, yellow 60.0-168.0 μm in diameter. Asci 8-

spored, subglobose to globose, 8.7-12.7 × 8.0-10.3 μm. Ascospores lenticular 3.8-5.2 × 3.3-4.8

μm, longitudinal furrow with two irregular ridges, convex surfaces irregularly roughened.

35

Vesicle Yellow cleistothecia Conidia Ascospores

Aspergillus hollandicus (Eurotium amstelodami)

4.9. Aspergillus section Terrei (Teleomorph:Fennellia)

Aspergillus section Terrei (Gams et al. 1985; A. terreus species group according to Raper &

Fennell 1965) includes species with columnar conidial heads in shades of buff to brown. The

most important species of this section is A. terreus, which is an ubiquitous fungus in our

environment. Strains of this cosmopolitan species are frequently isolated from desert and

grassland soils and compost heaps, and as contaminants of plant products like stored corn,

barley and peanuts.

Molecular studies have since indicated that this section should be expanded to include the

following species (Peterson 2000, 2008, Varga et al. 2005):

1. Aspergillus terreus

2. A. terreus var. africanus

3. A. terreus var. aureus

4. Aspergillus niveus

5. Aspergillus carneus,

6. Aspergillus niveus var. indicus,

7. Aspergillus allahabadii,

8. Aspergillus ambiguus

9. Aspergillus microcysticus

36

10. Aspergillus. alabamensis

Aspergillus terreus is an economically important species from a number of aspects. Aspergillus

terreus isolates are used in the fermentation industry for the production of itaconic acid and

itatartaric acid and for enzyme production.

Aspergillus terreus isolates produce a range of secondary metabolites, some of which

have properties valuable for mankind, including lovastatin, a cholesterol lowering drug,

the antitumor metabolites terrein, quadrone and asterriquinone, acetylcholinesterase

inhibitors like territrem B and terreulactone, butyrolactones, and cyclosporine A.

Antiviral compounds such as acetylaranotin has also been reported from Aspergillus

terreus.

Other secondary metabolites reported to be produced by A. terreus isolates are considered

as mycotoxins, including citreoviridin, patulin, citrinin, terretonin, geodin, territrems,

gliotoxin, and cytochalasin E.

Aspergillus terreus is also an important human pathogen, and often causes disseminated

infection with increased lethality compared to other Aspergillus spp.

4.9.1. Aspergillus terreus Thom, (1918) Synonym: Aspergillus terrestris

Morphology( http://www.bcrc.firdi.org.tw/fungi/fungal)

Colonies on potato dextrose agar at 25°C are beige to buff to cinnamon. Reverse is yellow and

yellow soluble pigments are frequently present. Moderate to rapid growth rate. Colonies

become finely granular with conidial production. Hyphae are septate and hyaline. Conidial

heads are biseriate (containing metula that support phialides) and columnar (conidia form in

long columns from the upper portion of the vesicle). Conidiophores are smooth-walled and

hyaline, 70 to 300µm long, terminating in mostly globose vesicles. Conidia are small (2-2.5

µm), globose, and smooth. Globose, sessile, hyaline accessory conidia (2-6 µm) frequently

produced on submerged hyphae.

On Malt-Agar growth medium (MA) (initial pH 5) – Moderately fast growing colonies

(reaching 78 cm in 21 days), velvet-like, white at first and then becoming cinnamon to brown-

orange. The reverse is cream to slightly orangey. Emission of a yellowish pigment in the

medium. The species slightly acidifies the medium (final pH 4).

This fungus is readily distinguished from the other species of Aspergillus by its cinnamon-

brown colony colouration and its production of aleurioconidia. Aspergillus terreus is a

thermotolerant species since it has optimal growth in temperatures between 35–40 °C, and

maximum growth within 45–48 °C.

37

mycology.adelaide.edu.au W. New Mexico Univ

Mycobank S. S. Tzean and J. L. Chen

4. 10. Aspergillus Section Flavipedes (Teleomorph:Fennellia) Aspergillus flavipes

Aspergillus niveus

Aspergillus iizukae

Aspergillus carneus

Aspergillus aureofulgens

Aspergillus janus

Aspergillus brevijanus

4.10.1.Aspergillus flavipes (Bain. & Sart.) Thom & Church, 1926.

Colony diameters on Czapek’s Agar 1.3-1.5 cm in 14 days at 25°C, dense, raised; conidial

heads radiate to loosely columnar, white to cartidge buff ; mycelium white to pale capucine

buff; exudate abundant, light yellow; soluble pigment buff-yellow; reverse deep chrome or

38

capucine orange to amber brown; stipes 140-1510 × 2.8-11.1 μm, colorless or light yellow to

pale brown, smooth to slightly roughened; vesicles obovoid, subglobose or pyriform, 6.0-29.4

μm wide. Aspergilla biseriate, metulae covering 1/2 to 4/5 of the vesicle, 4.0-9.4 × 2.5-5.7 μm;

phialides 4.8-8.7 × 2.2-3.0 μm. Conidia globose to subglobose, 2.0-4.0 μm in diameter, smooth

walled. Colony diameters on Malt Extract Agar 3.0-3.5 cm in 14 days at 25°C, plane to

velutinous; conidial heads radiate to columnar, white to light buff; mycelium white to pinkish

buff; soluble pigment red brown; reverse light pinkish cinnamon or vinaceous-cinnamon to

mikado brown.

Gardner, D. E. flavipes, Wiley & Simmons,

4. 11. Section: Circumdati (Teleomorph:Neopetromyces)

Aspergillus section Circumdati historically includes species with biseriate conidial heads in

shades from yellow to ochre. Species of Aspergillus section Circumdati are economically

important as ochratoxin A (OA) producing spoilage organisms.

Aspergillus section Circumdati formed two main clades, which could also be distinguished

based on phenotypic methods. A sexually reproducing ochratoxin producing species and

ochratoxin non-producing. (Frisvad et al., 2004). The section contains the following species:

1. Aspergillus auricomus

2. Aspergillus bridgeri

3. Aspergillus cretensis

4. Aspergillus elegans

5. Aspergillus flocculosus

39

6. Aspergillus insulicola

7. Aspergillus melleus

8. Aspergillus neobridgeri

9. Aspergillus ochraceus

10. Aspergillus ostianus

11. Aspergillus persii

12. Aspergillus petrakii

13. Aspergillus pseudoelegans

14. Aspergillus robustus

15. Aspergillus roseoglobulosus

16. Aspergillus sclerotiorum

17. Aspergillus steynii

18. Aspergillus sulphurous

19. Aspergillus westerdijkiae

20. Neopetromyces muricatus

4.11.1. Aspergillus ochraceus K. Wilh., (1877) Synonyms: Sterigmatocystis ochracea (G. Wilh.) Tiegh. (1877)

Aspergillus alutaceus Berk. & M.A. Curtis, (1875)

Aspergillus ochraceus var. microsporus Tirab., (1908)

Sterigmatocystis japonica Aoki et al. (1951)

Morphology:

Colony diameters on Czapek’s Agar 3.0-3.5 cm in 10 days at 25°C, wrinkled; conidial heads

spherical, or splitting into compact divergent columns, cream color, pinkish buff or near dark

olive-buff; mycelium white, inconspicuously floccose to floccose; exudate uncolored; reverse

dull yellow brown to victoria lake; soluble pigment, pale capucine buff ; stipes 360-1390 × 4.0-

16.0 μm, pale yellow to light yellow brown, slightly to coarsely roughened; vesicles spherical to

subspherical, 8.8-46.0 μm in diameter. Aspergilla biseriate, metulae covering the entire surface

of the vesicle, 4.8-33.3 × 2.4-10.3 μm; phialides 5.6-143.0 × 1.8-4.8 μm. Conidia spherical to

subspherical, smooth to irregular rough, 2.0-3.8 . Sclerotia produced by the same isolate, purple,

up 1000 μm in diameter. Colony diameters on Malt Extract Agar 5.0-5.5 cm in 10 days at 25°C,

more or less floccose; conidial heads globose or splitting into a few columns, near antimony

yellow to ochraceous-buff; mycelium white, reverse dull yellow brown to pale auburn.

lookfordiagnosis.com Show.wnmu.edu1155 × 1148 www.drthrasher.org Mold Library

41

S. S. Tzean and J. L. Chen Mycobank

4. 12. Aspergillus section Flavi (Teleomorph:Petromyces)

Aspergillus section Flavi includes species with conidial heads in shades from yellow-

green to brown, and dark sclerotia

Several species assigned to this section are either important mycotoxin producers

including aflatoxins, cyclopiazonic acid, ochratoxins and kojic acid, or are used in

oriental food fermentation processes and as hosts for heterologous gene expression.

The data obtained by Varga et al. (2011) using morphological characters, extrolite

production and partial calmodulin, β-tubulin and ITS sequences indicated that

Aspergillus section Flavi involves 22 species:

1. Aspergillus albertensis

2. Aspergillus alliaceus

3. Aspergillus arachidicola

4. Aspergillus avenaceous

5. Aspergillus bombycis

6. Aspergillus coremiformis

7. Aspergillus caelatus

8. Aspergillus coremiformis

9. Aspergillus flavus

10. Aspergillus lanosus

11. Aspergillus leporis

12. Aspergillus minisclerotium

13. Aspergillus nomius

14. Aspergillus oryzae

15. Aspergillus parasiticus,

41

16. Aspergillus parvisclerotigenus

17. Aspergillus pseudocaelatus

18. Aspergillus pseudonomius

19. Aspergillus pseudotamarii

20. Aspergillus sojae

21. Aspergillus tamari

22. Aspergillus togoensis

The A. flavus« clade includes species characterised with Q-10(H2) as their main

ubiquinone, conidial colours in shades of green and dark sclerotia.

1. Aspergillus flavus Group I includes isolates producing only aflatoxin B and having large

or small sclerotia. This group also includes isolates of A. oryzae, which has previously

been described as having a recombining population species of this clade, P. alliaceus and

P. albertensis, produce high amounts of OA (50–300 mg/mL), and are considered to be

responsible for OA contamination of figs.

2. Aspergillus flavus Group II includes isolates that may produce aflatoxins B or G, and

have large or small sclerotia.

3. Aspergillus Group III includes isolates able to produce both aflatoxins B and G

and have small sclerotia,

4. Aspergillus flavus is the most common species associated with aflatoxin contamination of

agricultural crops.

5. flavus soil populations also contain isolates from two morphologically distinct sclerotial

size variants, termed the L-strain for isolates with average sclerotial size greater than 400

μm and the S-strain for isolates with sclerotial size less that 400 μm. On typical

laboratory growth media S-strain isolates produce higher levels of aflatoxins, more

abundant sclerotia, and generally fewer conidia. Atoxigenic S-strain isolates are very

rarely found in natural environments.

42

4.12.1. Aspergillus flavus Link, 1809 Synonyms: Monilia flava (Link) Pers., (1822)

Sterigmatocystis lutea Tiegh., (1877)

Aspergillus flavus var. proliferans Anguli, Rajam, Thirum., Rangiah & Ramamurthi, (1965)

Morphology

A. flavus is known as a velvety, yellow to green or brown mould with a goldish to red-brown

reverse. On Czapek dox agar, colonies are granular, flat, often with radial grooves, yellow at

first but quickly becoming bright to dark yellow-green with age. Conidial heads are typically

radiate, mostly 300-400 um in diameter, later splitting to form loose columns .The

conidiophores are variable in length, rough, pitted and spiny. They may be either uniseriate or

biseriate. They cover the entire vesicle, and phialides point out in all directions. Conidia are

globose to subglobose, conspicuously echinulate, varying from 3.5 to 4.5 mm in diameter.

Based on the characteristics of the sclerotia produced, A. flavus isolates can be divided into two

phenotypic types. The S strain produces numerous small sclerotia (average diameter ,400 mm).

The L strain produces fewer, larger sclerotia (Cotty, 1989). Within theS strain, some isolates,

termed SB, produce only B aflatoxins, whilst others, named SBG, produce both B and G

aflatoxins.

Aspergillus flavus: human pathogen, allergen and mycotoxin producer

Fungi mycospecies info www.drjacksonkungu.com William McDonald

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Rahayu WP Mycobank

4.12.2. Aspergillus parasiticus Speare, 1912 Synonyms: Aspergillus flavus subsp. parasiticus (Speare) Kurtzman, Smiley, Robnett & Wicklow,1986

Aspergillus chungii Y.K. Shih, (1936)

Morphology

Colony diameters on Czapek’s Agar larger than 9 cm in 10 days at 25°C, distinctly

floccose, sporulation abundant at margin; mycelium fimbriate, white; reverse uncolored;

conidial heads mostly radiate or splitting into fine columns or rarely globose, small,

primuline yellow, or wax yellow to yellowish citrine; stipes smooth to roughened,

colorless, 86-2140 × 6.8-24.0 μm; vesicles globose to pyriform, 19.0-94.0 μm wide.

Aspergilla mostly biseriate, occasionally uniseriate; metulae covering 1/2 to the whole

surface of the vesicle, 9.5-21.4 × 4.8-12.7 μm; phialides 8.3-15.1 × 3.8-6.0 μm; conidia

globose to subglobose, 5.5-8.3 × 4.4-7.1 μm, irregularly roughened to very roughened.

Colony diameters on Malt Extract Agar 6.5-7.0 cm in 10 days at 25°C, floccose to plane;

mycelium white; conidial heads distinctly radiate, occasionally loosely columnar,

yellowish oil green, serpentine green to grass green, or cedar green; reverse colorless;

44

Mycobank S. S. Tzean and J. L. Chen

4.12.3. Aspergillus oryzae (Ahlb.) Cohn, 1884 Synonyms:Eurotium oryzae Ahlb., 1878

Aspergillus flavus var. oryzae (Ahlb.) Kurtzman, M.J. Smiley, Robnett & Wicklow1986

Morphology

Colony diameters on Czapek’s Agar 4.5-5.5 cm in 10 days at 25°C, floccose; conidial heads

radiate, or splitting into several loose columns, Kronberg’s green to citron green; mycelium

white; reverse cream color to mustard yellow and pale isabella color; soluble pigment light

yellow; stipes smooth to rough, hyaline, 56-1160 × 6.4-20.6 μm; vesicles globose, subglobose,

pyriform to somewhat elongate, 15.8-50.0 μm wide. Aspergilla uniseriate, biseriate, or both

coexisting on the same vesicle, metulae cov-ering the entire vesicle, 5.2-36.5 × 2.8-9.5 μm;

phialides 4.0-14.3 × 2.8-7.1 μm, hyaline to light yellow; phialides of uniseriate aspergilla

covering 1/2 to the entire surface of the vesicle. Conidia subglobose, rarely ellipsoidal or ovoid,

2.8-6.0 μm wide, with walls smooth to irregularly roughened. Colony diameters on Malt Extract

Agar larger than 9 cm, floccose, in 10 days at 25°C; co-nidial heads enmeshed within the

loosely aerial mycelium, ivy green to citron green, and olive-ocher to olive-yellow; mycelium

white; reverse uncolored to pale buffy olive in center.

45

Aspergillus oryzae (ex-type , A–C. Colonies incubated at 25 °C for 7 d, A. CYA, B. MEA, C. YES,

S. S. Tzean and J. L. Chen Mycobank

4.13. Aspergillus Section: Cremei (Teleomorph:Chaetosartorya)

1. Aspergillus stromatoides

2. Aspergillus stromatoides

3. A. itaconicus

4. Aspergillus cremea

5. Aspergillus chrysella,

6. Aspergillus wentii,

7. Aspergillus dimorphicus,

8. Aspergillus pulvinus

9. Aspergillus flaschentraegeri.

10. Aspergillus gorakhopurensis

11. Aspergillus brunneouniseriatis

46

4.13.1. Aspergillus wentii Wehmer (1896)

Synonyms: Aspergillus archaeoflavus Blochwitz, (1933)

Aspergillus wentii var. minimus Nakaz. et al., (1934)

Aspergillus wentii. R.A. Samson, CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht,

4.14. Aspergillus section Fumigati (Teleomorph:Neosartorya)

This section involves the anamorphs of at least 20 sexual Neosartorya species, and 5 asexual

aspergilli. The most important species among them is Aspergillus fumigatus, which is an

ubiquitous filamentous fungus in the environment, and also an important human pathogen.

Several Neosartorya species have also been described as causative agents of human diseases

including invasive aspergillosis, osteomyelitis, endocarditis and mycotic keratitis. Many of the

Neosartorya species produce several mycotoxins, many of which may cause serious health

hazards.

47

4.14.1. Aspergillus fumigatus Fresenius, 1863.

Synonyms:

Aspergillus fumigatus var. acolumnaris Rai, Agarwal & Tewari (1971)

Aspergillus fumigatus var. albus Rai, Tewari & Agarwal (1974)

Aspergillus fumigatus var. cellulosae Sartory, Sartory & Mey. (1935)

Aspergillus fumigatus var. coeruleus Malchevsk. (1939)

Aspergillus fumigatus var. ellipticus Raper & Fennell ( 1965)

Aspergillus fumigatus var. fulviruber Rai, Tewari & Agarwal (1974)

Aspergillus fumigatus var. fumigatus Fresen. (1863)

Aspergillus fumigatus griseibrunneus var. Rai & Singh (1974)

Aspergillus fumigatus var. helvolus Yuill (1937)

Aspergillus fumigatus var. lunzinense Svilv. (1941)

Aspergillus fumigatus var. minimus Sartory (1919)

Aspergillus neoellipticus Kozak. (1989)

Aspergillus phialoseptus Kwon-Chung (1975)

Aspergillus bronchialis Blumentritt (1901)

Aspergillus septatus Sartory & Sartory (1943)

Aspergillus arvii Aho, Horie, Nishimura & Miyaji (1994)

Description

Colony diam (7 d): CYA25: 21-67 mm; MEA25: 25-69 mm; YES25: 48-74 mm; OA25: 34-62

mm, CYA37: 60-75 mm, CREA: poor growth, no or very weak acid production

Colour: greyish turquoise or dark turquoise to dark green to dull green. Reverse colour (CYA):

creamy, yellow to orange

Colony texture: velutinous, st. floccose .Conidial head: columnar. Conidiation: abundant, rarely

less abundant. Stipe: 50-350 × 3.5-10 μm. Vesicle diam, shape: 10-26 μm, pyriform to

subclavate, sometimes subglobose, but rarely globose. Conidia length, shape, surface texture: 2-

3.5(-6) μm, globose to ellipsoidal, smooth to finely rough

48

Aspergillus fumigatus, Mycobank

4.14.2. Aspergillus felis

Colonies grow rapidly on CYA agar attaining a diameter of 5.0 to 5.5 cm in 7 days at 25°C and

on MEA reach 5.5 cm in diameter in 7 days at 25°C. On CYA the colony texture is mostly

floccose; colonies are usually white and often sporulate poorly. On MEA colonies are more or

less velvety with abundant greenish sporulation occurring after 5 to 7 days. In reverse, colonies

are cream to light green. Conidiophores are uniseriate with greenish stipes and subclavate,

“nodding” heads. Vesicles are subclavate with a diameter of 15–16.5 µm. Conidia are green,

globose to subglobose, finely roughened and 1.5–2.5 µm in dimensions. Cleistothecia are white

to creamish, 100–230 µm. Asci are globose, 8-spored, 12–16 µm in diameter. Ascospores are

lenticular with two prominent equatorial crests and with short echinulate convex surfaces 5.0–

7.0×3.5–5.0 µm

This species had been found in cats with chronic invasive FRS and retrobulbar masses (SOA),

IPA or with sinonasal cavity infection only (sinonasal aspergillosis, SNA), in a dog with

disseminated IA, in a human with chronic IPA and in an indoor air sample in Germany.

Fungi like Aspergillus felis can be easily misidentified as the closely related fungus Aspergillus

fumigatus. However, Aspergillus felis is intrinsically more resistant to antifungal drugs

than Aspergillus fumigatus and this has important implications for therapy and prognosis.

Aspergillus felis colonies growing seven days at 25 C on Czapek Yeast Extract Agar, left, and Malt

Extract Agar, right (Barrs)

49

4. 15. Aspergillus Section:Clavati (Teleomorph:Neocarpenteles)

Aspergillus section Clavati has been revised by Varga et al. (2007) using morphology,

secondary metabolites, physiological characters and DNA sequences. Phylogenetic analysis of

beta-tubulin, ITS and calmodulin sequence data indicated that Aspergillus section Clavati

includes 6 species:

1. Aspergillus clavatus

1. giganteus

2. longavesica

3. clavatonanicus

4. rhizopodus

5. Neocarpenteles acanthosporus

4. 15. 1. Aspergillus clavatus Desmazières (1834)

Synonyms: A. apicalis Mehrotra & Basu,1976

A. pallidus Kamyschko,1963

Colony diameters on Czapek’s Agar 4.7-5.0 cm in 14 days at 25°C, zonation conspicuous to

inconspicuous; conidial heads radiate or splitting into well defined columns in age, niagara

green to bice green, or artemisia green to slate-olive (R., Plate XVII, XXXIII, XLVII); my-

celium white; exudate clear; reverse colorless, or ivory yellow to cartridge buff (R., Plate

XXX); stipes 250-2300 × 4.8-40.0 μm, uncolored, smooth; vesicles clavate, 8.7-80.0 μm wide.

Aspergilla uniseriate, phialides covering the entire surface of the vesicle, 5.3-21.4 × 2.4-5.6 μm.

Conidia subspherical, ellipsoidal, occasionally cylindrical, 3.3-7.1 × 2.4-4.4 μm, smooth.

Colony diameters on Malt Extract Agar 5.0-5.5 cm in 14 days at 25°C, zonation conspicuous;

conidial heads radiate or splitting into well defined columns, bluish gray-green to artemisia

green (R., Plate XLII, XLVII); mycelium white; reverse uncolored.

51

labmed.ucsf.edu Varga, Frisvad , Samson RA S. S. Tzean and J. L. Chen Mycobank

4. 16. Aspergillus Section:Nidulantes (Teleomorph:Emericella)

1. A. nidulans

2. A. quadrilineata

3. E. rugulosa

4. E. nidulans var. echinulata

4.16.1. Aspergillus nidulans (Eidam) G. Winter (1884) Teleomorphic state: Emericella nidulans

Synonyms: Sterigmatocystis nidulans Eidam (1883)

Diplostephanus nidulans (Eidam) Neveu-Lem. (1921)

Sterigmatocystis nidulans var. nicollei Pinoy,(1906)

Aspergillus nidulans var. cesarii Pinoy (1915)

Aspergillus nidulellus Samson & W. Gams (1986)

51

Morphology Colonies on potato dextrose agar at 25°C are dark green with orange to yellow in areas of

cleistothecial production. Reverse is purplish to olive. Exudate is usually present and may be

brown to purplish. Growth rate is slow to moderate in comparison with other clinically

significant Aspergillus species.

Hyphae are septate and hyaline. Conidial heads are columnar. Conidiophores are brown, short

(60-150 µm in length), and smooth-walled. Vesicles are hemispherical, small (8-12 µm in

diameter), with metulae and phialides occurring on the upper portion. Conidia are globose (3-4

µm) and rough. A. nidulans is a homothallic species capable of producing the teleomorph

(sexual stage) without mating studies. The ascomycetous telemorph (Emericella nidulans)

produces brown to black globose cleistothecia (100-250 µm) that are engulfed with globose

Hülle cells. Ascospores are reddish brown, lenticular (4 x 5 µm), with two longitudinal crests

visualphotos Aspergillus nidulans, Wikipedia Mycota

Mycobank www.mycology.adelaide.edu SEM Micrograph Gallery, public.gettysburg.edu

52

4. 17. Aspergillus Section:Ornati (Teleomorph:Hemicarpenteles)

4.17.1. Aspergillus ornatus Raper, Fennell & Tresner (1953) Synonyms: Sclerocleista ornata (Raper, Fennell & Tresner) Subram., (1972)

Neosartorya ornata (Raper, Fennell & Tresner) Malloch & Cain, (1973)

Hemicarpenteles ornatus (Raper, Fennell & Tresner) Arx, (1974)

Hemicarpenteles ornata (Subram.) Arx (1974)

Chaetosartorya ornata (Raper, Fennell & Tresner) Bilai & Koval, (1988) [

4. 18. Aspergillus sect. Aeni

Aspergillus sect. Aeni is a new section that includes the following species 1. Aspergillus karnatakaensis

2. Aspergillus aeneus,

3. Aspergillus crustosus,

4. Aspergillus eburneocremeus,

5. Aspergillus heyangensis,

6. Emericella bicolor,

7. Emericella discophora,

8. Emericella spectabilis,

9. E. foeniculicola.

Aspergillus karnatakaensis isolates were found to produce karnatakafurans A and B, terrein,

gregatins, asteltoxin and the partially characterised metabolite NIDU. Both gregatins and NIDU

are also produced by A. granulosus, while karnatakafurans are produced in common with A.

aeneus and A. multicolor. Emericella bicolor produces sterigmatocystin, versicolorins, some

anthraquinones, and a polar extrolite with end-absorption; E. foeniculicola produces

sterigmatocystin (and many other sterigmatocystin and versicolorin-related compounds),

xanthocillin derivatives, and the partially characterized (but common) metabolite DRI; E.

spectabilis produces two members of the shamixanthone biosynthetic family (both more polar

than shamixanthone itself) and a member of the sterigmatocystin biosynthetic family; A.

heyangensis produces a decaturin in common with A. aeneus and A. karnatakaensis and NIDU,

while E. discophora produces sterigmatocystin and versicolorins (Varga et al., 2010)

53

4.18.1. Aspergillus granulosus Raper and Thom (1944)

Morphology

Colonies are cream to white and floccose at the periphery and buff to yellowish tan centrally,

with a very slight clear exudate. Colonies on CZA are mostly cream, more irregularly furrowed,

and exhibited reduced conidiation. The central granular areas on PFA consisted of colorless

masses of thick-walled (6 to 8 μm), predominately globose but also oval to elongate to

irregularly shaped Hülle cells with individual, mature cells ranging from 20 to 40 μm.

Robust Aspergillus fruiting heads are sparse and are borne on long (200 to 480 μm), subhyaline

to brown, smooth, thick-walled conidiophores terminating in small (12 to 18 μm wide by 15 to

25 μm long), oval to elliptical vesicles. Metulae and bottle-shaped phialides of almost equal

length (3.5 to 5.5 μm) covered most of the surface of the vesicle. Conidia pale green in mass,

globose, and finely echinulate, measuring 3.5 to 5.5 μm in diameter. More commonly, fruiting

structures are reduced in size with small, Penicillium-like vesicles or single chains of conidia

are borne from solitary phialides

A. granulosus, Sutton et al., J. Clin. Microbiol. October 2009vol. 47 no. 10 3386-3390

A. granulosus, Houbraken J, Due M, Varga J, Meijer M, Frisvad JC, Samson RA - Stud. Mycol. (2007), Mycobank

54

5. Aspergillosis in man

5.1. Pulmonary aspergillosis

5.1.1. Aspergilloma (Saprophytic aspergillosis, fungus ball)

Aspergilloma is an Aspergillus infection without tissue invasion.

The aspergilloma is a mass composed of fungal hyphae, inflammatory cells, fibrin,

mucus and tissue debris that develops in a pre-existing cavity in the lung, without invade

the surrounding lung parenchyma or blood vessels.

The most common underlying causes are tuberculosis and sarcoidosis. Other conditions

that occasionally may be associated with aspergilloma include bronchogenic cyst,

pulmonary sequestration, and pneumatoceles secondary to Pneumocystis

carinii pneumonia in patients with acquired immunodeficiency syndrome (AIDS).

At radiography, aspergilloma is characterized by the presence of a solid, round or oval

mass with soft-tissue opacity within a lung cavity. Typically, the mass is separated from

the wall of the cavity by an airspace of variable size and shape, resulting in the “air

crescent” sign. A change in the position of the fungus ball after moving the patient,

demonstrating that this mass is mobile because does not usually invade the surrounding

lung parenchyma.

Aspergillomas are often associated with thickening of the cavity wall and adjacent pleura.

In such cases, pleural thickening may be the earliest radiographic sign before any visible

changes are seen within the cavity. Approximately 10% of mycetomas resolve

spontaneously. Reversibility of the pleural thickening corresponding to the resolution of

intracavitary fungal material has been demonstrated at follow-up radiography

Surgical resection of the cavity and removal of the fungus ball is usually indicated in

patients with recurrent haemoptysis, if their pulmonary function is sufficient to allow

surgery or bronchial artery embolization in patients with poor lung function.

55

Aspergilloma www.brown.edu radiopaedia.org pathhsw5m54.ucsf.edu

Aspergilloma - “Chest x-ray of a man with extensive bullous lung

pathologyimages.wordpress.com, de.wikipedia.org Aspergilloma,

www.histopathology-india.net www.flickriver.com

56

Recently reported cases: 1. Aspergilloma and massive haemoptysis.

45

2. A rare case of calcified pulmonary aspergilloma46

.

3. Adjuvant antifungal therapy after pulmonary surgery foraspergilloma: is it useful?47

4. Aspergilloma and the surgeon.48

5. Pulmonary Diseases with Imaging Findings MimickingAspergilloma. Lung.49

6. Is video-assisted thoracic surgery a versatile treatment for both simple and

complex pulmonary aspergilloma? 50

7. Pulmonary Aspergilloma: An Unexpected Complication of Radiofrequency Ablation in the Management

of Targeted Therapy for a Patient With Metastatic Renal Cell Carcinoma.51

8. Treatment of pulmonary aspergilloma in Srinagarind Hospital52

.

9. Surgical therapy of pulmonary aspergillomas: a 30-year North American experience.53

10. Intrapulmonary aspergilloma in an old tuberculous cavity with access to the bronchial system.54

11. . Case 3: aspergilloma. 55

12. Pulmonary aspergilloma: a treatment challenge in sub-Saharan Africa. 56

13. [Gougerot-Sjögren syndrome complicated by pulmonaryaspergilloma].57

14. A case of pulmonary aspergilloma treated with radiofrequency ablation.58

15. [Surgical treatment of pulmomary tuberculosis complicated withaspergilloma].59

16. Always expect the unexpected: lung abscess due to pseudomonas aeruginosa

mimicking pulmonary aspergillomain acute B-cell leukemia.60

17. Video-assisted thoracic surgery for pulmonary aspergilloma: a safe and effective procedure. 61

18. 'Monod' and 'air crescent' sign in aspergilloma.62

19. Calcium oxalate crystal deposition in a patient withAspergilloma due to Aspergillus niger. 63

20. Surgical treatment of aspergilloma grafted in hydatid cyst cavity.64

21. Haemoptysis after four years of lobectomy for aspergilloma.65

22. Aspergilloma coexisting with idiopathic pulmonary fibrosis: a rare occurrence. 66

23. Aspergilloma mimicking a lung cancer. 67

24. A Case of Endobronchial Aspergilloma Associated with Foreign Body in Immunocompetent Patient

without Underlying LungDisease.68

25. Pulmonary aspergilloma in a cavity formed after percutaneous radiofrequency ablation.69

26. Co-existence of HIV, active tuberculosis and aspergilloma in a single individual--a case report.70

.

57

5.1.2. Allergic bronchopulmonary aspergillosis (Hypersensitivity reaction)

Allergic bronchopulmonary aspergillosis (ABPA) is seen most commonly in patients

with long-standing bronchial asthma.

It is characterized by the presence of plugs of inspissated mucus

containing Aspergillus organisms and eosinophils.

This results in bronchial dilatation typically involving the segmental and subsegmental

bronchi.

It is caused by a complex hypersensitivity reaction to Aspergillus organisms.

The fungi proliferate in the airway lumen, resulting in the production of a

constant supply of antigen.

A type I hypersensitivity reaction with immunoglobin E and immunoglobin

G release occurs.

Immune complexes and inflammatory cells are then deposited in the

bronchial mucosa, producing necrosis and eosinophilic infiltrates (type III

reaction) with bronchial wall damage and bronchiectasis.

Excessive mucus production and abnormal ciliary function lead to mucoid

impaction.

Many patients cough up thick mucous plugs in which hyphal fragments can be

demonstrated at culture or histologic analysis.

Acute clinical symptoms include recurrent wheezing, malaise with low-grade fever,

cough, sputum production, and chest pain. Patients with chronic allergic

bronchopulmonary aspergillosis may also have a history of recurrent pneumonia.

Radiologic manifestations include homogeneous, tubular, finger-in-glove areas of

increased opacity in a bronchial distribution, usually predominantly or exclusively

involving the upper lobes. Occasionally, isolated lobar or segmental atelectasis may

occur. CT findings in allergic bronchopulmonary aspergillosis consist primarily of

mucoid impaction and bronchiectasis involving predominantly the segmental and

subsegmental bronchi of the upper lobes. In approximately 30% of patients, the impacted

mucus has high attenuation or demonstrates frank calcification at CT.

58

Areas of tubular (Panel A, arrows) and cystic (Panel A, arrowhead) bronchiectasis, predominantly in the upper lobes, and bilateral mucous plugging (Panel B, arrows)

101

The CXR shows bronchial wall thickening and impressive central bronchiectasis.101

Recently reported cases 1. Antifungal treatment in allergic bronchopulmonary aspergillosis with and without cystic fibrosis: a

systematic review. Clin Exp Allergy. 71

2. Omalizumab therapy for allergic bronchopulmonary aspergillosis in children with cystic fibrosis: a

synthesis of published evidence72

.

3. Fungal allergy in asthma-state of the art and research needs.73

4. Excellent outcome of Aspergillous endophthalmitis in a case ofallergic bronchopulmonary aspergillosis. 74

5. A retrospective study of patients with a delayed diagnosis ofallergic bronchopulmonary

aspergillosis/allergicbronchopulmonary mycosis. 75

6. Allergic bronchopulmonary aspergillosis coexists with hereditary bisalbuminemia. 76

7. Role of inhaled amphotericin in allergic bronchopulmonary aspergillosis. 77

8. Pulse methylprednisolone in allergic bronchopulmonary aspergillosis exacerbations. 78

.

9. Unexpected decrease in total IgE in a patient with allergic bronchopulmonary aspergillosis treated with

omalizumab. 79

59

10. ABPA Concomitantly Occurring with Invasive SinusAspergillosis: A Short Report on Two Patients. 80

11. Atypical Presentation of Allergic Bronchopulmonary Aspergillosis: An Unusual Cause of Difficult-to-

Treat Asthma. 81

12. [Allergic bronchopulmonary aspergillosis in patients with chronic obstructive pulmonary disease: report

of 3 cases].82

13. Evaluation of serum levels of carcinoembryonic antigen inallergic bronchopulmonary aspergillosis. 83

14. Allergic bronchopulmonary aspergillosis mimicking relapsing chronic eosinophilic pneumonia in non-

asthma patient. 84

15. Treatment options in severe fungal asthma and allergic bronchopulmonary aspergillosis.85

16. Anti-IgE therapy for allergic bronchopulmonary aspergillosis. 86

17. [Analysis of clinical features and allergic status of asthmatic patients with positive serum mycosis-

specific IgE]. 87

18. Allergic bronchopulmonary aspergillosis in asthma: epidemiological, clinical and therapeutic issues.88

19. Bronchial asthma with ABPA presenting as PTE. 89

20. Anti-IgE therapy for allergic bronchopulmonary aspergillosisin people with cystic fibrosis. 90

21. . Development of allergic bronchopulmonary aspergillosiswith central bronchiectasis over a 10-year

period: the need to recheck allergen sensitization. 91

22. Dissociation between sensitizing and colonizing fungi in patients with allergic bronchopulmonary

aspergillosis.92

23. . Allergic bronchopulmonary aspergillosis in garden waste (compost) collectors--occupational

implications.93

24. Allergic bronchopulmonary aspergillosis with repeated isolation of nontuberculous mycobacteria.94

25. Allergic bronchopulmonary aspergillosis: review of literature and proposal of new diagnostic and

classification criteria. ABPA complicating asthma ISHAM working group. 95

26. Uncertain areas in the diagnosis of allergic bronchopulmonary aspergillosis in patients with asthma.96

27. When to suspect and work up allergic bronchopulmonary aspergillosis.97

28. Allergic bronchopulmonary aspergillosis: a clinico-serological correlation with radiologic profile.98

29. Allergic bronchopulmonary aspergillosis presenting as chronic cough in an elderly woman without

previously documented asthma.99

30. Coexistence of allergic bronchopulmonary aspergillosis andallergic aspergillus sinusitis in a patient

without clinical asthma.100

31. Ting-hsu Chen,., and Helen Hollingsworth, Allergic Bronchopulmonary Aspergillosis.101

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5.1.3. Chronic necrotizing aspergillosis (Semi-invasive aspergillosis)

Semi-invasive aspergillosis, also known as chronic necrotizing aspergillosis, is an

indolent, destructive process of the lung due to local invasion by Aspergillus species,

with tissue necrosis and granulomatous inflammation.

Factors associated with the development of this form of aspergillosis include chronic

debilitating illness, diabetes mellitus, malnutrition, alcoholism, advanced age, prolonged

corticosteroid therapy, and chronic obstructive pulmonary disease.

Clinical symptoms include chronic cough, sputum production, fever, and constitutional

symptoms. In patients with chronic obstructive pulmonary disease, semi-invasive

aspergillosis may manifest with a variety of nonspecific clinical symptoms such as

cough, sputum production, and fever lasting more than 6 months. Hemoptysis has been

reported in 15% of affected patients.

Radiologic manifestations of semi-invasive aspergillosis include unilateral or bilateral

segmental areas of consolidation with or without cavitation or adjacent pleural

thickening, and multiple nodular areas of increased opacity. Aspergillus necrotizing

bronchitis may be seen at CT as an endobronchial mass, obstructive pneumonitis or

collapse, or a hilar mass. Only a few reports have described CT findings

in Aspergillus necrotizing bronchitis involving the central airways; reported

abnormalities include circumferential bronchial wall thickening and bronchial

obstruction.

In clinical practice, the diagnosis of Aspergillus necrotizing bronchitis is usually based on

the presence of abnormal findings at chest radiography and bronchoscopic biopsy, which

are consistent with tissue invasion.

Aspergilloma/chronic necrotising pulmonary aspergillosis

122

61

Recently reported cases:

1. Chronic necrotizing pulmonary aspergillosis in a renal transplant recipient.122

2. Prognostic factors in 194 patients with chronic necrotizingpulmonary aspergillosis. 123

3. [Three autopsy cases of chronic necrotizing pulmonaryaspergillosis]. 124

4. Systemic dissemination of chronic necrotizing pulmonaryaspergillosis in an elderly woman without

comorbidity: a case report.125

5. Systemic biomarkers of inflammation and haemostasis in patients with

chronic necrotizing pulmonary aspergillosis.126

6. Efficacy and safety of short- and long-term treatment of itraconazole

on chronic necrotizing pulmonary aspergillosisin multicenter study.127

7. [Chronic necrotizing pulmonary aspergillosis following an infection by Mycobacterium malmoense].128

8. [Retrospective analysis of the safety of four hours administration of liposomal amphotericin B in

patients with chronicnecrotizing pulmonary aspergillosis]. 129

9. A surgically treated case of chronic necrotizing aspergillosiswith pleural invasion. 130

10. Chronic necrotizing pulmonary aspergillosis presenting as bilateral pleural effusion: a case report.131

11. Chronic necrotizing pulmonary aspergillosis. 132

12. Resolution of galactomannan antigenemia in a patient receiving oral voriconazole

for chronic necrotizing pulmonaryaspergillosis. 133

13. [Chronic necrotizing pulmonary aspergillosis as a complication of silicosis].134

14. [Case of chronic necrotizing pulmonary aspergillosiscomplicated by elevated eosinophils and serum

IgE].135

15. Chronic necrotizing pulmonary aspergillosis in a patient treated with a tumor necrosis factor-alpha

inhibitor. 136

16. [Lethal case of chronic necrotizing pulmonary aspergillosis(CNPA)].137

17. Progressive increase in cavitation with the evolution of fungus ball: A clue to the diagnosis

of chronic necrotizing pulmonaryaspergillosis.138

18. [A case of pulmonary actinomycosis mimicking chronicnecrotizing pulmonary aspergillosis].139

19. Clinical characteristics and treatment outcomes of chronicnecrotizing pulmonary aspergillosis: a review

of 43 cases.140

20. Chronic necrotizing pulmonary aspergillosis or invasive pulmonary aspergillosis. 141

62

5.1.4. Invasive pulmonary aspergillosis

Invasive pulmonary aspergillosis occurs almost exclusively in immunocompromised

patients with severe neutropenia.

The reasons of the substantial increase in the number of patients at risk for developing

invasive aspergillosis include the development of new intensive chemotherapy regimens

for solid tumors, difficult-to-treat lymphoma, myeloma, and resistant leukemia as well as

an increase in the number of solid organ transplantations and increased use of

immunosuppressive regimens for other autoimmune diseases.

Invasive pulmonary aspergillosis is characterized by the presence of Aspergillus

organisms deep to basement membrane of bronchi or bronchioles, usually with a

neutrophil reaction and presence of hyphae in the involved airway. Surrounding the

airway is often found a variably sized zone of hemorrhage and/or organizing pneumonia.

Invasive pulmonary aspergillosis is characterized at histologic analysis by the invasion

and occlusion of small to medium-sized pulmonary arteries by fungal hyphae, which

leads to the formation of necrotic hemorrhagic nodules or pleura-based, wedge-shaped.

Characteristic CT findings consist of nodules surrounded by a halo of ground-glass

attenuation (“halo sign”) or pleura-based, wedge-shaped areas of consolidation. These

findings correspond to hemorrhagic infarcts. In severely neutropenic patients, the halo

sign is highly suggestive of invasive pulmonary aspergillosis.

63

Acute airway invasive aspergillosis, manifested as a bronchopneumonia. posterng.netkey.at. HRCT.

Recently reported cases: 1. Prevalence of Invasive Aspergillosis Among (PTB) Patients in Kanchipuram,

142

2. Clinical utility of Aspergillus galactomannan and PCR in bronchoalveolar lavage fluid for the diagnosis

of invasive pulmonary aspergillosis in patients with haematological malignancies.143

3. Evaluation of galactomannan enzyme immunoassay and quantitative real-time PCR for the diagnosis

of invasive pulmonary aspergillosis in a rat model.144

4. Independent contribution of bronchoalveolar lavage and serum galactomannan in the diagnosis

of invasive pulmonary aspergillosis.145

5. A neonatal case of chronic granulomatous disease, initially presented with invasive pulmonary

aspergillosis.146

6. Host biomarkers of invasive pulmonary aspergillosis to monitor therapeutic response. 147

7. Performance of Galactomannan, Beta-D-Glucan, Aspergillus Lateral-Flow Device, Conventional

Culture and PCR tests for Diagnosis of Invasive Pulmonary Aspergillosis in Bronchoalveolar Lavage

Fluid. 148

8. What's new in invasive pulmonary aspergillosis in the critically ill. 149

9. Galactomannan antigen assay from bronchoalveolar lavage fluid in diagnosis of invasive pulmonary

aspergillosis in intensive care units patients.150

10. Invasive pulmonary aspergillosis post extracorporeal membrane oxygenation support and literature

review.151

11. Screening of the central nervous system in children withinvasive pulmonary aspergillosis.152

12. Intrapulmonary posaconazole penetration at the infection site in an immunosuppressed murine model

of invasive pulmonary aspergillosis receiving oral prophylactic regimens.153

64

13. Diagnosis of invasive pulmonary aspergillosis: updates and recommendations. 154

14. Approach to invasive pulmonary aspergillosis in critically ill patients.155

15. Invasive pulmonary aspergillosis after influenza a infection in an immunocompetent patient.156

.

16. Clinical findings in 19 cases of invasive pulmonary aspergillosis with liver cirrhosis. 157

17. Altered CD8(+) T-cell counts as an early predictor of prognosis in critically ill immunocompromised

patients with invasive pulmonary aspergillosis. 158

18. Prognostic value of serum galactomannan index in critically ill patients with chronic

obstructive pulmonary disease at risk ofinvasive pulmonary aspergillosis. 159

19. Real-life diagnosis of invasive pulmonary aspergillosis in a non neutropenic critically ill patient. 160

20. Hypothermic endpoint for an intranasal invasive pulmonary aspergillosis mouse model. 161

21. Early diagnosis and treatment of invasive pulmonary aspergillosis in a patient with cystic fibrosis.162

22. Early diagnosis of invasive pulmonary aspergillosis in hematologic patients: an opportunity to improve

the outcome. 163

23. Risk factors for invasive pulmonary aspergillosis and hospital mortality in acute-on-chronic liver failure

patients: a retrospective-cohort study. 164

24. Invasive pulmonary aspergillosis in patients with chronic obstructive pulmonary disease. 165

25. Coinfection of Pneumocystis jirovecii and invasive pulmonary aspergillosis in an immunocompromised

patient: a diagnostic challenge. 166

26. Isavuconazole (BAL4815) pharmacodynamic target determination in an in vivo murine model

of invasive pulmonary aspergillosis against wild-type and cyp51 mutant isolates of Aspergillus

fumigatus. 167

27. Diagnosis of airway-invasive pulmonary aspergillosis by tree-in-bud sign in an immunocompetent

patient: case report and literature review.168

28. The strategy for the diagnosis of invasive pulmonary aspergillosis should depend on both the underlying

condition and the leukocyte count of patients with hematologic malignancies. 169

29. Invasive pulmonary aspergillosis: prediction at thin-section CT in patients with neutropenia--a

prospective study.170

30. Invasive aspergillosis in the immunocompromised host: utility of computed tomography and

bronchoalveolar lavage. 171

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5.1.5. Angioinvasive aspergillosis

Angioinvasive aspergillosis is most aggressive form of aspergillosis, with a mortality rate

that may exceed 50%.

It occurs when the hyphae invade bronchial wall and subsequently the accompanying

arterioles, with consequent thrombosis and the formation of necrotic hemorrhagic

nodules or subpleural wedge-shaped hemorrhagic infarcts.

It is seen almost exclusively in severely immunocompromised. Neutropenia is the most

important risk factor, following by prolonged Corticosteroid therapy, transplantation,

hematologic malignancy, Cytotoxic therapy and AIDS.

Aspergillus may disseminate to another organs, most commonly to the brain (seizures,

ring-enhancing lesions, cerebral infarctions, intracranial haemorrhage, meningitis and

epidural abscess), skin, kidneys, heart, esophagus and liver.

Radiographic abnormalities include single or multiple nodular infiltrates; segmental or

subsegmental consolidation; diffuse ground-glass pattern (often progressing to

consolidation) and cavitation (air-crescent sign); pleural effusions are uncommon.

Chest CT scan may shows multiple nodules surrounded by areas of ground-glass

attenuation known as halo sign and areas of segmental and non-segmental consolidation,

which are often bilateral, with or without a halo.“The halo sign” represents haemorrhage

around a pulmonary nodule and is highly suggestive of angioinvasive aspergillosis.

Another finding are wedge-shaped areas of consolidation with a broad base abutting a

peripheral pleural surface by hemorrhagic infarcts.

The cavitation and “air crescent sign" are viewed when area of necrotic tissue is

reabsorbed from the periphery, which causing retraction of the infarct from viable lung

parenchyma and leaves a space of air surrounding. This air-space doesn´t change with

position of patient and it´s different to monod sign of aspergilloma.

A typical image of Angioinvasive aspergillosis in a 45 year-old neutropenic patient after hematopoietic stem cell transplantation

(HSCT) , Tomas Franquet Casas, Hospital de Sant Pau in Barcelona

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Angioinvasive aspergillosis ww.flickr.com

5.1.6. Aspergillus bronchitis and tracheobronchitis

Aspergillus tracheobronchitis is defined as an Aspergillus infection which is limited

entirely or predominantly confined to the tracheobronchial tree. In addition, Denning

suggested the term be applied to patients in whom there is evidence of bronchial and/or

tracheal inflammation, excess mucus production, with Aspergillus as the only pathogen

and without invasion of bronchial mucosa on biopsy, but full criteria for this diagnosis

have not been validated.

Aspergillus infection of the large airways has several manifestations. Many authors try to

propose a classification to describe the disease, but to date; there is still no ideal

classification. Overlapping clinical features can make cases difficult to classify.

Interestingly Young et al. described a non-invasive form of infection, with the bronchial

mucosa is usually preserved, but this contrasts with many reported invasive cases in

patients with immunosuppression and poor immunological host responses

Aspergillus fumigatus is the most common species being isolated (74-84%), followed

withAspergillus flavus (8-21%). Other Aspergillus species (3-5%) have also been

isolated, such as Aspergillus niger, Aspergillus terreus, Aspergillus

nidulans and Aspergillus versicolor

Wu et al.(2009) proposed a classification based on bronchoscopic morphology of the

intraluminal lesions into four types: superficial infiltration type (Type I), full-layer

involvement type (Type II), occlusion type (Type III) and mixed type (Type IV), then a

mixed type which an overlapping bronchoscopic features seen in all morphological types.

1. Superficial infiltration type: Inflammatory infiltration, mucosa

hyperaemia, mucosa oedematous and superficial ulcer which is confined to

the mucosa and submucosa; mild plaques of pseudomembrane formation

without obvious airway obstruction or deeper tissue invasion

2. Full-layer involvement type: Tracheobronchial lesions infiltrating through

the matrix layer of bronchi, often with substantial and deep ulceration,

67

extensive tissue necrosis with cartilage invasion and destruction of normal

airway structures

3. Occlusion type: Airway obstruction or constriction > 50% of the original

caliber of involved bronchi caused by extensive pseudomembrane formation,

polypoid granulation or necrotic tissues as a result of Aspergillus infection,

without definite proof of full-layer invasion.

4. Mixed type:Two or more different forms of typical bronchoscopic features

coexisting at the time of diagnosis

Bronchoscopic manifestations of Aspergillus tracheobronchitis. (a) Type I. Inflammatory infiltration, mucosa hyperaemia and plaques

of pseudomembrane formation in the lumen without obvious airway occlusion. (b) Type II. Deep ulceration of the bronchial wall. (c)

Type III. Significant airway occlusion by thick mucous plugs full of Aspergillus without definite deeper tissue invasion. (d) Type IV.

Extensive tissue necrosis and pseudomembrane formation in the lumen with airway structures and severe airway occlusion (Wu 2009).

Recently reported cases:

1. An unusual case of invasive Aspergillus ulcerative tracheobronchitis without involvement of lung

parenchyma in a post-renal transplant patient.172

2. Ulcerative tracheobronchitis due to Aspergillus.173

3. Colonization with small conidia Aspergillus species is associated with bronchiolitis obliterans

syndrome: a two-center validation study.174

4. [The aspergillus laryngotracheobronchitis. A case report and literature review]. 175

5. Aspergillus bronchitis without significant immunocompromise. 175

6. Aspergillus tracheobronchitis in allogeneic stem cell transplanted recipient. 176

7. [Aspergillus tracheobronchitis in a lung transplant recipient]. 177

8. Pseudomembranous Aspergillus tracheobronchitis in a heart transplant recipient. 178

9. Isolated invasive Aspergillus tracheobronchitis: a clinical study of 19 cases.179

68

10. Ulcerative and pseudomembranous Aspergillus tracheobronchitis in a patient with acute myeloid

leukemia. 180

11. Aspergillus niger causing tracheobronchitis and invasive pulmonary aspergillosis in a lung transplant

recipient: case report. 181

12. Ulcerative tracheobronchitis due to Aspergillus.199

13. Tracheal oxalosis associated with Aspergillus niger tracheobronchitis. 200

14. Colonization with small conidia Aspergillus species is associated with bronchiolitis obliterans

syndrome: a two-center validation study. 201

15. [The aspergillus laryngotracheobronchitis. A case report and literature review].202

16. Aspergillus bronchitis without significant immunocompromise.203

17. Tracheobronchial Manifestations of Aspergillus Infections 204

.

18. Pseudomembranous Aspergillus tracheobronchitis in a heart transplant recipient.205

19. Isolated invasive Aspergillus tracheobronchitis: a clinical study of 19 cases. 206

5.1.7. Chronic cavitary pulmonary aspergillosis (CCPA)

The commonest symptom complex consists of weight loss, chronic cough, hemoptysis,

fatigue and shortness of breath.

While the clinical signs and symptoms of CCPA are non-specific, their presence and

severity is an important point of distinction from simple aspergilloma.

Radiological examination usually reveals one or more cavities, typically within the upper

lobes which may or may not contain fungus balls. New cavity formation or expansion of

one or more existing cavities over time is highly characteristic.

Peri-cavitary infiltrates and adjacent pleural thickening are frequently observed and

appear to be indicative of disease activity; these radiological abnormalities may remit

with effective antifungal therapy leaving residual thin-walled empty cavities.

Severe bilateral chronic pulmonary aspergillosis with the left upper lobe replaced by one large and several smaller

cavities. w ww.life-worldwide.org

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Chronic cavitary pulmonary aspergillosis is a multicavitary disease in immunocompetent patients and progresses over time. Previous mycobacterium infection w ww.life-worldwide.org

Recently reported cases:

1. A prominent role for the IL1 pathway and IL15 in susceptibility tochronic cavitary pulmonary

aspergillosis. 182

2. Pulmonary aspergillosis in an immunocompetent patient. 183

3. Itraconazole in chronic cavitary pulmonary aspergillosis: a randomised controlled trial and systematic

review of literature.184

4. Cavitary pulmonary lesion associated with Aspergillus fumigatus infection in a German shepherd dog. 185

5. Distinct alleles of mannose-binding lectin (MBL) and surfactant proteins A (SP-A) in patients

with chronic cavitary pulmonary aspergillosis and allergic bronchopulmonary aspergillosis. 186

6. The efficacy and tolerability of voriconazole in the treatment ofchronic cavitary pulmonary

aspergillosis. 187

5.1.8. Disseminated aspergillosis (Invasive aspergillosis)

Invasive aspergillosis is generally seen in severely immunocompromised individuals and

carries a high mortality rate.

Risk factors for invasive aspergillosis include neutropenia, immunosuppressive therapy,

high-dose systemic corticosteroids, AIDS, solid organtransplant and haematopoietic stem

cell transplant.

The lungs are the most common site of primary invasive disease. The CNS is the most

common secondary site of invasive disease.

Invasive infections due to Aspergillus fumigatus are increasing, and despite new

therapies, are associated with greater than 50% mortality.

Invasion of blood vessels is a hallmark pathologic feature of invasive aspergillosis and

plays a critical role in the development of local and disseminated disease.

This process of angioinvasion and subsequent dissemination is unique to A. fumigatus

and a handful of other pathogenic molds, and its mechanism is not well understood.

Histopathologic studies suggest that there are several key steps by which the organism

gains entry to and subsequently exits from the vascular system.

Hyphae from an infectious focus must invade the abluminal surface of the blood vessel,

and penetrate endothelial cells to gain access to the blood vessel lumen.

Hyphal fragments are borne by the bloodstream to distal sites where they adhere to and

penetrate the luminal surface of the endothelial cells lining the blood vessel and

thereby invade the deep organs.

It is hypothesed that the interactions of A. fumigatus with the vascular endothelium are

critical to the pathogenesis of invasive aspergillosis.

71

Recently reported cases:

1. Primary gut aspergillosis in a patient with acute myeloid leukaemia: the importance of

early suspicion and definitive treatment.188

2. Disseminated aspergillosis as the herald manifestation of chronic granulomatous disease

in an adult patient. 189

3. Immune reconstitution syndrome-like entity in lung transplant recipients with

invasive aspergillosis.190

4. Aspergillus felis sp. nov., an emerging agent of invasiveas pergillosis in humans, cats,

and dogs. 191

5. [Mixed invasive fungal infection due to Rhizomucor pusillus and Aspergillus niger in an

immunocompetent patient.]192

6. [An autopsy case of ulcerative colitis with disseminated aspergillosis, complicated by

brain hemorrhage]. 193

7. Invasive aspergillosis masquerading as catastrophic antiphospholipid syndrome.194

8. Disseminated Aspergillosis due to Aspergillus niger in Immunocompetent Patient: A

Case Report. 195

9. Clinical experience in invasive fungal infections: multiple fungal infection as the first

presentation of HIV.196

10. Pharmacodynamics and dose-response relationships of liposomal amphotericin B against

different azole-resistant Aspergillus fumigatus isolates in a murine model of

disseminated aspergillosis. 197

11. Systemic dissemination of chronic necrotizing pulmonary aspergillosis in an elderly

woman without comorbidity: a case report. 198

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5.2. Cerebral aspergillosis

Cerebral Aspergillosis is the most lethal manifestation of infection due to Aspergillus

species arising most commonly as hematogenous dissemination from a pulmonary focus,

direct extension from paranasal sinus infection or direct inoculation through trauma and

surgery of the central nervous system (CNS).

Meningitis is rare and spinal cord involvement is barely reported.

It has non-specific clinical presentation and non-specific radiological abnormalities

Cerebral aspergillosis in the immunocompromised host is often not diagnosed until

autopsy, and carries a mortality of about 99%.

In the non-immunocompromised patient cerebral aspergillosis is rare.

Cerebral aspergillosis. Aspergillus/Aspergillosis Website

Abscesses in the brain caused by Aspergillus spp, ww.life-worldwide.org

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Cerebral aspergillosis, brain biopsy, Braz J Infect Dis vol.12 no.4 Salvador Aug. 2008

Recently freported cases:

1. Improving Outcomes in Cerebral Aspergillosis. World Neurosurg. 207

2. High weekly doses of liposomal amphotericin B as secondary prophylaxis

after cerebral aspergillosis in a paediatric patient. 208

3. Striated enhancement sign in invasive cerebral fungal granuloma by Aspergillus: a case

report. 209

4. Cerebral aspergillosis in adult critically ill patients: a descriptive report of 10 patients

from the AspICU cohort. 210

5. A Case of Acute Cerebral Aspergillosis Complicating Influenza A/H1N1pdm 2009. 211

6. Cerebral aspergillosis and acute myeloid leukemia.212

7. Pituitary aspergillosis in a kidney transplant recipient and review of the literature. 213

8. Isolated Cerebral Aspergillosis in Immunocompetent Patients.214

9. Aspergillus brain abscess. 215

10. Cerebral aspergillosis with multiple enhancing nodules in the right cerebral hemisphere

in the immune-competent patient. 216

11. Unusual case of cerebral aspergillosis with clinical and imaging findings mimicking

lymphoma. 217

73

12. Early diagnosis of cerebral aspergillosis with various methods: a case report.218

13. Cerebral aspergillus infection in pediatric acute lymphoblastic leukemia induction

therapy. 219

14. Aspergillus galactomannan antigen for diagnosis and treatment monitoring

in cerebral aspergillosis.229

15. Cranial aspergilloma masquerading as meningioma.221

16. Antifungal therapy of aspergillosis of the central nervous system and aspergillus

endophthalmitis.222

17. Fatal case of cerebral aspergillosis : a case report and literature review. 223

18. Central nervous system aspergillosis: a series of 14 cases from a general hospital and

review of 123 cases from the literature.224

19. Relapsing cerebral aspergilloma associated with pregnancy.225

20. Treatment of refractory cerebral aspergillosis in a liver transplant recipient with

voriconazole: case report and review of the literature. 226

5.3. Cutaneous aspergillosis

Cutaneous aspergillosis has been described as primary or secondary to haematogenous

dissemination.

Primary cutaneous aspergillosis usually follows local trauma and appears as a purplish

oedematous lesion at the site of inoculation. Primary cutaneous aspergillosis is a rare

disease usually caused by Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus

and Aspergillus ustus.

It is usually seen in immunocompromised hosts, though some cases are also reported in

immunocompetent hosts.

It usually develops in relation to local trauma (intravenous injection site, injury or burns).

Clinically, it is characterized by the presence of violaceous macules, papules,

haemorrhagic bullae, ulcerations with central necrosis, and pustules or subcutaneous

abscess.

The occurrence of varying clinical manifestations of fungal diseases has been

demonstrated in patients with altered host defences. Immunocompromised patients with

primary cutaneous aspergillosis occasionally present with solitary non-ulcerated nodules.

74

Neonatal Primary Cutaneous Aspergillosis – Buttocks Lesion. Multiple cutaneous lesions on the leg in a case of

Woodruff CA, et al. Pediatr Dermatol 2002;5:439-44. disseminated aspergillosis.due to A. fumigatys

Punch biopsied specimen of skin shows acute and chronic inflammation with hyaline, septated, and acute-angle branched hyphae invading vessels in dermis and subcutis (A) Periodic acid Schiff (H&E stain, ×400), (B) Grocott-Gomori methenamine silver (H&E stain, ×400).Jin-Hee Noet et al., Infect Chemother. 2010 Aug;42(4):244-248.

Recently reported cases: 1. Aspergillus fumigatus infection on a home-made tattoo.

227

2. What is your diagnosis? primary cutaneous aspergillosis.228

3. Fatal-mixed cutaneous zygomycosis-aspergillosis: a case report. 229

4. SPrimary cutaneous aspergillosis due to Aspergillus tamarii in an immunocompetent host. 230

5. A case of dual infection in a paediatric trauma victim of primarycutaneous aspergillosis caused by

Aspergillus flavus and Aspergillus terreus.231

6. [Primary cutaneous aspergillosis in an extremely low birth weight preterm infant].232

7. Primary cutaneous aspergillosis complicating tumor necrosis factor-α blockade therapy in a patient with

psoriasis. 233

8. Post-filarial cutaneous aspergillosis. 234

9. Cutaneous aspergillosis associated with HIV infection.235

10. Primary Cutaneous Aspergillosis Complicating Tumor Necrosis Factor-α Blockade Therapy in a Patient

With Psoriasis. 236

11. Primary cutaneous aspergillosis in two pediatric trauma patients.

237

12. Primary cutaneous aspergillosis from Tamilnadu diagnosed by fine needle aspiration cytology. 238

. 13. Primary cutaneous aspergillosis in a patient with systemic lupus erythematosus.

239

14. [Case of primary cutaneous Aspergillus caldioustus infection caused by nerve block therapy]. 240

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15. Severe cutaneous aspergillosis in a premature neonate linked to nonsterile disposable glove

contamination? 241

16. Investigation of a cluster of cutaneous aspergillosis in a neonatal intensive care unit.

242

17. Nonhealing scalp wound infected with Aspergillus niger in an elderly patient. 243

18. Tracheobronchial aspergillosis following primary cutaneous aspergillosis in a lung-transplant recipient. 244

19. [Posttraumatic primary cutaneous aspergillosis with Candida guilliermondii infection in a healthy host]. 245

20. Primary cutaneous aspergillosis. 246

21. A Case of Disseminated Aspergillosis Presenting Solely as Multiple Cutaneous Lesions in an Acute

Leukemia Patient, 247

5.4. Aspergillus Onychomycosis

Many different species of Aspergillus may cause onychomycosis including A. niger, A.

fumigatus, A. versicolor, A. terreus and some rare species.

Among non-dermatophyte mould onychomycosis, proportional rates

of Aspergillus onychomycosis vary from 5% to as high as 30%

There are 2 common patterns of disease, destructive and superficial white

onychomycosis, but lateral and distal onychomycosis may also be seen.

Particular features suggestive of Aspergillus infection are a chalky, deep white nail with

early involvement of the lamina and painful perionyxis without pus.

Trauma is the common predisposing factor. The affected nail is most often a toenail;

peripheral vascular disease is occasionally implicated.

Onychomycosis :A. fumigatus aspergillus.org.uk Onychomycosis :A. terreus,subclin.org.

A. fumigatus onychomycosiswww.studyblue.com, A. ochraceus onychomycosis ,aspergillus.org.uk

76

5. 5. Ocular aspergillosis

Presence of fungus in the conjunctiva is a constant threat to the eyes, because these

microorganisms, defined as opportunistic, may provoke severe ocular infections, in

situations as low organic resistance, use of immunosuppressants, antibiotics and epithelial

alteration

Aspergillus species have been implicated in a wide variety of primary ocular conditions,

characterized by either slow and asymptomatic infection, or rapid, uncontrollable

progression and sometimes death.

Endogenous Aspergillus endophthalmitis is most commonly reported in

immunosuppressed patients with organ transplants or after valve replacement and in

individuals suffering from hematological malignancies. It may also be the presenting

feature of disseminated aspergillosis

Aspergillus keratitis is an important ophthalmological problem worldwide, but especially

in outdoor workers in agricultural communities in the developing world and in tropical

and subtropical areas, where it frequently occurs following traumatic inoculation

of Aspergillus conidia into the cornea through injury.

In recent years, Aspergillus keratitis has been reported to occur following ocular surgical

procedures, such as radial keratotomy, laser-assisted in situ keratomileusis and

keratoplasty

Aspergillus keratitis is a medical emergency, since the patient frequently presents with

extreme pain and loss of vision, and needs to be recognized and treated promptly.

fumigatus is the commonest cause of Aspergillus keratomycosis In recent years, other

species, particularly Aspergillus flavus, have been reported to predominate

in Aspergillus keratitis.

Aspergillus keratitis in patient from Pakistan Shrunken eye as a consequence of this infection

Dr Andrew Tullo, Royal Eye Hospital, Manchester

77

Severe aspergillus infection with large area of corneal ulceration and deep stromal involvement Severe central aspergillus infection with a “cheesey” looking area of the lesion and hypopyon (fluid level of

inflammatory cells in the anterior chamber)

Bilateral A. fumigatus endophthalmitis in association with pulmonary and cerebral aspergillosis, complicating severe autoimmune

disease treated with intense immunosuppression. Dr Richard Wintle and Dr Steven Charles, Royal Eye Hospital, Manchester, UK

Recently reported cases:

1. Aspergillus terreus endogenous endophthalmitis in a nonimmunocompromised patient with a history of

bronchiectasis. 248

2. Epidemiology of Aspergillus keratitis at a tertiary care eye hospital in South India and antifungal

susceptibilities of the causative agents.249

3. Aspergillus endophthalmitis in one eye subsequent to bilateral anterior chamber phakic intraocular lens

implantation. 250

4. Ocular aspergillosis: Obtaining a specimen is crucial for diagnosis. A report of three cases. 251

5. Detection of Candida and Aspergillus species DNA using broad-range real-time PCR for fungal

endophthalmitis. 252

6. Molecular characterization of drug-resistant and drug-sensitive Aspergillus isolates causing infectious

keratitis. 253

7. Aspergillus keratitis in vernal shield ulcer--a case report and review.

254

8. Mycotic keratitis due to Aspergillus nomius. 255

. 9. Infectious keratitis caused by Aspergillus tubingensis.

256

10. A. flavus keratomycosis in a cat treated with topical 1% voriconazole solution.257

.

78

5.6. Aspergillus sinusitis

Aspergillus disease can happen in the sinuses leading to Aspergillus sinusitis. Just as in

the lungs, Aspergillus can cause the three diseases - allergic sinusitis, a fungal ball or

invasive aspergillosis.

Allergic disease is associated with long standing symptoms of a runny blocked up nose,

and may lead to nasal polyps.

The fungal ball caused by Aspergillus happens in a similar way to an aspergilloma. In

those with normal immune systems, stuffiness of the nose, chronic headache or

discomfort in the face is common.

When patients have damaged immune systems - if, for example they have had leukaemia

or have had a bone marrow transplant - Aspergillus sinusitis is more serious. In these

cases the sinusitis is a form of invasive aspergillosis. The symptoms include fever, facial

pain, nasal discharge and headaches.

Base of skull at autopsy showing pus in the sphenoid sinus and destruction of the lateral wall of the sinus, as demonstrated on the CT

scan. Cultures from the sinus and brain abscess grew Aspergillus fumigatus, ww.njmoldinspection.com

Recently reported cases:

1. [Allergic fungal sinusitis--new aspects of clinical features, laboratory diagnosis and therapy]. 258

.

2. Aspergillosis sinusitis with cranio-orbital spread in an apparently immunocompetent patient. 259

3. Mycotic aneurysm due to Aspergillus sinusitis.

260

4. Coexistence of allergic bronchopulmonary aspergillosis and allergic aspergillus sinusitis in a patient

without clinical asthma. 261

5. [Clinicopathologic study of invasive fungal rhinosinusitis caused by Aspergillus and Mucorales] 262

6. [Cellular and tissue reactions of the mucous membrane of the maxillary sinus in the patients presenting

with odontogenic aspergillous maxillary sinusitis]. 263

7. [Application of PCR combined with reverse line blot assay in detection and identification of common

pathogenic Aspergillusin fungal sinusitis]. 264

79

5.7. Paranasal sinuses aspergillosis

Paranasal sinus Aspergillosis is now classified into invasive (acute fulminant, chronic

invasive, granulomatous invasive) and non-invasive (fungus ball and allergic fungal

rhinosinusitis) forms with their own pathophysiology and clinical presentation.

Any type of paranasal Aspergillosis may progress to more aggressive disease illustrating

the importance of early recognition of this increasingly encountered disease.

Prerequisites for diagnosis are sinonasal polyps, infiltrative or non-infiltrative fungal

hyphae on microscopy with Potassium Hydroxide (KOH) and histopathological

examination of the resected polyps and positive fungal culture of the tissue following

surgery.

Intracranial spread of the infection occurs due to close proximity of the sinuses with

cranial cavity. It is a dreaded complication, as it is usually fatal if not treated promptly.

Orbital involvement occurs by contiguous spread of the disease from paranasal sinuses,

by expansion or bone erosion due to pressure effect of the polyps or fungal tissue

invasion.

It is considered to worsen the prognosis of sinonasal aspergillosis. Moreover, the superior

orbital fissure and optic canal directly open into the middle cranial fossa, and are ready

pathways for further intracranial spread of the infection.

Acute inflammatory polyp obstructing the sinuses in an operational patient Allergic polyps with fungal infection, Vijay K. Andad

Left:Double density sign (white arrows) of non-invasive aspergillus infection of paranasal sinuses on coronal

contrast enhanced CT scan.Right:Extensive sino-nasal polyposis (white arrow) involving all sinus cavities and

nose on coronal contrast enhanced CT scan.

81

Sections of the maxillary sinus show (a) Aspergillus in necrotic background (H and E, ×250), (b) fungal hyphae

in giant cell (H and E, ×400), (c) many eosinophils and hyphae (H and E, ×250), special stains highlighting the

fungal morphology (d; PAS, ×250) and (e; silver methanamine, ×250). Divya Sharma, Nidhi Mahajan, Seema Rao, Nita

Khurana, Shyama Jain, Department of Pathology, Maulana Azad Medical College, New Delhi, India

Recently reported cases 1. Diagnosis and treatment of paranasal sinus fungus ball of odontogenic origin: case report.

265

2. Computed tomographic features of feline sino-nasal and sino-orbital aspergillosis. 266

3. Granulomatous invasive aspergillosis of paranasal sinusesmasquerading as actinomycosis and review of

published literature. 267

4. ABPA Concomitantly Occurring with Invasive SinusAspergillosis: A Short Report on Two Patients. 268

5. [Allergic fungal sinusitis--new aspects of clinical features, laboratory diagnosis and therapy]. 269

6. [Invasive aspergillosis of sphenoidal sinus in a patient in Djibouti, revealed by palsy of cranial nerves: a

case report]. 270

7. Invasive fungal rhinosinusitis.271

8. [Unilateral frontal sinus aspergillosis: the combined endoscopic and mini-trephination approach]. 272

9. Isolated primary frontal sinus aspergillosis: role of endonasal endoscopic approach.

273

10. Chronic invasive aspergillosis of paranasal sinuses: A case report with review of literature. 274

11. Highly effective unconventional management of aspergillosis of the left maxillary sinus in an 11-year-

old girl with rhabdomyosarcoma embryonale of the frontal sinus. 275

12. [Chronic diseases of the nose and nasal sinuses in cats: a retrospective study]. 276

13. Simultaneous actinomycosis with aspergillosis in maxillary sinus. 277

14. Clotrimazole and enilconazole distribution within the frontalsinuses and nasal cavity of nine dogs with

sinonasalaspergillosis. 278

81

15. [Invasive aspergillosis of the maxillary sinus in an immunocompetent patient].279

16. Aspergillus Infection of Paranasal Sinuses.

280

5.8. Otoaspergillosis (Otomycosis)

Otomycosis is subacute or chronic superficial fungal infection of the external auditory

canal and auricle. It is the most frequently encountered fungal infection in ear, nose and

throat clinics. The fungi are usually secondary invaders of tissue already rendered

susceptible by bacterial infections, physical injury or excessive accumulation of cerumen

in the external auditory canal. Sometimes it is merely non-pathogenic fungal colonization

of the external auditory canal.

The disease is worldwide in distribution. Otomycosis is more prevalent in warm, humid

climates, particularly in the rainy season as compared to arid or cold climates. It is more

frequent in individuals of lower socio-economic status with poor hygienic conditions.

There are many local predisposing factors of otomycosis such as chronic infection of the

ear, use of oils, ear-drops, steroids, swimming and evidence of fungal infection

elsewhere. Local lesions observed in bacterial otitis create favourable conditions for the

growth of fungi in the external and middle ear, as well as in post-operative cavities,

especially in cases of open-type surgery. Persistent wetness of the external auditory canal

predisposes to fungal

In about 75-92% cases of otomycosis, Aspergillus genus alone is the causative

agent. A. niger is the most common cause, with occasional cases caused by A.

flavus and A. fumigatus .

Otomycosis usually presents with a history of itching, irritation, discomfort, pain and

scanty discharge from the affected ear. There is also a feeling of blockage in the ear due

to collection of debris material in external auditory canal. Irritation is more marked in

fungal as compared to bacterial otitis externa. Pruritus and discharge are the most

common symptoms, with reddened epidermis and lining of the tympanic cavity being

common.

Otoscopic examination reveals infection confined to the ear canal. There is greenish or

black fuzzy growth on cerumen or debris resembling wet ‘blotting paper’, which may fill

up the entire meatus. There may be slight conduction deafness also due to mechanical

obstruction of the external auditory canal. The local area may be hyperemic and

sometimes bleeding may be observed. In immunocompromised patients especially

diabetics, Aspergillus may invade locally to adjacent anatomical sites like mastoid bone

or even brain.

Aspergillus may cause invasive external otitis (necrotizing or malignant otitis externa)

with local spread to bone and cartilage, which is a severe and potentially life-threatening

disease. This may be associated with underlying immunocompromised situation, diabetes

mellitus or patient receiving haemodialysis entailing high mortality. Invasive otitis

externa is more frequently caused by A. fumigatus than A. niger . In addition,

82

invasive Aspergillus tympanomastoiditis may be encountered in immunocompetent

patients as well.

Otomycosis (Aspergillus niger) , Dr. T. Balasubramanian

Recently reported cases

1. Otomycosis in the north of Iran: common pathogens and resistance to antifungal agents. Eur Arch

Otorhinolaryngol. 281

2. Prevalence of otomycosis in Khouzestan Province, south-west Iran.

282

3. Otomycosis in central iran: a clinical and mycological study. 283

4. Otomycosis in immunocompetent and immunocompromised patients: comparative study and literature

review. 284

5. Molecular identification and antifungal susceptibilities of blackAspergillus isolates

from otomycosis cases in Hungary. 285

6. Species assignment and antifungal susceptibilities of black aspergilli recovered from otomycosis cases

in Iran. 286

7. Otomycosis due to filamentous fungi. 287

8. Prevalence of otomycosis in Ibadan: a review of laboratory reports.

288

9. Otomycosis: Diagnosis and treatment. 289

5.9. Aspergillus endocarditis

Most cases of Aspergillus endocarditis (AE) involve adults.

Pre-requisites for developing AE. are valvular anatomic abnormalities, rheumatic heart

disease, previous bacterial endocarditis. previous surgery.

Conditions that predispose to invasive fungal infections (eg malignancy, antimicrobial

use, neutropenia, injecting drug use and immunosuppressive states and therapy) are also

commonly found in AE.

83

Diagnosis of AE is difficult and frequently delayed: the diagnosis is made post-mortem.

Blood cultures are almost always negative. Mycological examination of vegetations or

emboli is the most common means of diagnosis.

Crude mortality from FE in general is around 72% although in recent years this has

dropped to around 59%. For AE the mortality rate was 68% (McCormack, 2010). The

cause of death is directly attributable to Aspergillus in 90% of cases and is usually due to

catastrophic valvular failure or complications secondary to major embolic phenomena:

usually cerebral.

Aspergillus endocarditis, www.aspergillus.org.uk Aspergillus pericarditis

Recently reported cases:

1. Aspergillus endocarditis in a paediatric patient after a cardiac surgery, associated with septic pulmonary

embolism and pulmonary hypertension. 290

2. Isolated aspergillosis myocardial abscesses in a liver-transplant patient. 291

3. Acute myocardial infarction caused by coronary embolism from Aspergillus endocarditis.292

4. Aspergillus pacemaker lead endocarditis. 293

5. Fungal endocarditis. 294

6. Successful surgical intervention in an unusual case of Aspergillus endocarditis with acute myeloid

leukemia. 295

7. . [Aspergillus prosthetic aortic valve endocarditis; report of a case]. 296

8. Aspergillus endocarditis in lung transplant recipient: successful surgical treatment. 297

9. The tell-tale heart: Aspergillus fumigatus endocarditis in an immunocompetent patient. 298

10. Aspergillus endocarditis in the era of new antifungals: major role for antigen detection. 299

11. A Report of 2 Cases of Disseminated Invasive Aspergillosis with Myocarditis in Immunocompromised

Patients . 300

12. Aspergillus endocarditis in a native valve without prior cardiac surgery. 301

13. Aspergillus endocarditis: a case of near complete left ventricular outflow obstruction.302

14. NikoTwo cases of aspergillus endocarditis in non neutropenic children on chemotherapy for acute

lymphoblastic leukaemia. 303

15. Infective endocarditis due to Aspergillus following kidney transplantation. 304

16. Molecular diagnosis of Aspergillus fumigatus endocarditis. 305

17. . Cardiac aspergillosis: endocardial or endomyocardial? 306

18. [Cardiac invasive aspergillosis in a heart transplant recipient]. 307

84

19. Aspergillus endocarditis in lung transplant recipients: case report and literature review. 308

20. Aspergillus endocarditis: a review of the literature. 309

5.10. Aspergillus thyroiditis

Invasion of the thyroid gland is common in disseminated invasive aspergillosis.

Patients typically present with fever and tender thyroid enlargement, involving one or

both lobes.

Clinically the thyroid may be diffusely enlarged or a nodule may be palpable.Marked

thyroid enlargement leading to fatal airway obstruction has been described, as has

compression of the oesophagus leading to dysphagia.

Histology of thyroid tissue usually demonstrates abscesses containing necrotic debris,

haemorrhage and Aspergillus hyphae. Vascular invasion with thrombosis and infarction

is sometimes prominent.

Aspergillus hyphae with septae, branching at 45° (PAS) Aspergillus hyphae (silver methenamine, Erdem H, Uzunlar AK, Yildirim Ü, Yildirim M, Geyik M F.. Indian J Pathol Microbiol 2011;54:814-6

Recently reported cases:

1. Aspergillus thyroiditis: a review of the literature to highlight clinical challenges.

310

2. Rapidly growing thyroid mass in an immunocompromised young male adult. 311

3. Aspergillus thyroiditis: a complication of respiratory tract infection in an immunocompromised patient. 312

4. Successful treatment of acute thyroiditis due to Aspergillus spp. in the context of disseminated invasive

aspergillosis in a kidney transplant patient. 313

5. Aspergillus thyroiditis in a renal transplant recipient mimicking subacute thyroiditis. 314

6. Aspergillus thyroiditis in a living donor liver transplant recipient.

315

7. Aspergillus thyroiditis. 316

85

5.11. Hepatic aspergillosis

Liver transplant recipients are at high risk of infection with Aspergillus species, which

may cause invasive disease and severe complications in this patient population.

There is a high incidence of invasive aspergillosis (IA) in liver transplant recipients

The mortality of IA in liver transplant recipients is approximately 90%.

Invasive pulmonary aspergillosis is most common but disseminated disease occurs in

~50% of liver transplant recipients affected.

A 52-year-old woman; status after bone marrow transplantation for acute myelogenous leukemia with ringenhancing lesions

in the liver and spleen caused by aspergillosis fungal infection. Marchelle J. Bean, MD, Karen M. Horton, MD, and Elliot

K. Fishman, MD, J Comput Assist Tomogr 2004;28

Left: chronic hepatic aspergilloma with central necrosis surrounded by few Giant cells (arrows) and fibrotic wall (HE ×125) Right: active hepatic abscess showing giant cells with negative profiles of fungal hyphae (arrows) surrounded by inflammatory cells ( PAS × 325)., Gupta KL, Rajaram K G, Joshi K, Sakhuja V. Indian J Pathol Microbiol 2012;55:580-2

86

Recently reported cases:

1. Primary hepatic aspergillosis following induction chemotherapy for acute leukemia. 317

2. Progression of hepatic aspergillosis following second renal transplantation in a patient with recurrent

glomerulonephritis. 318

3. Invasive aspergillosis in patients with severe alcoholic hepatitis. 319

4. Aspergillosis after liver transplantation in the context of common variable immunodeficiency: case

report. 320

5.12. Gastrointestinal aspergillosis

There are few reports in the literature of gastrointestinal aspergillosis without

pulmonary involvement as the initial presentation of disseminated disease.

Tumour-like masses (aspergillomas) in the gastrointestinal tract have been very rarely

reported.

The port of entry is usually the lungs and wide dissemination is possible after vascular

invasion.

The gastrointestinal tract may be involved via oesophageal or bowel ulceration,

especially in the immunocompromised host.

Necrotizing enterocolitis may precede aspergillosis and disruption of mucosal barriers

may provide a port of entry for germinating spores.

The features of gastrointestinal tract involvement are mild anaemia, weight loss,

diarrhoea with malabsorption, in the absence of any other cause for the diarrhoea,

gastrointestinal haemorrhage and a swinging fever not responding to antibiotics.

Occasionally, there may by signs of peritonitis, especially if there is bowel perforation

or infarction.

Gastrointestinal haemorrhage is a known complication of aspergillus infection of the gut.

This may be due to gut infarction, colonic or duodenal ulceration.

Uncontrollable upper gastrointestinal bleeding due to erosion into the descending aorta,

resulting in a rapidly fatal outcome, has been reported in oesophageal aspergilloma.

Recently reported cases:

1. Primary gastrointestinal aspergillosis 6 months after allogeneic hematopoietic cell transplantation: a case

report.321

2. Invasive Aspergillus infection localized to the gastric wall: report of a case. 322

3. A retrospective series of gut aspergillosis in haematology patients.323

4. A case of isolated invasive Aspergillus colitis presenting with hematochezia in a nonneutropenic patient

with colon cancer. 324

5. Necrotizing colitis caused by systemic aspergillosis in a burn patient.325

6. Successful treatment of acute thyroiditis due to Aspergillus spp. in the context

of disseminated invasive aspergillosis in a kidney transplant patient. 326

87

5.13. Urinary tract aspergillosis

Aspergillosis limited to the urinary tract is a rare disease, often occurring in

immunocompromised patients.

Three different patterns of renal Aspergillus infection have been: disseminated

aspergillosis with hematogenous renal involvement, aspergillosis of the renal pelvis with

bezoars formation, and ascending panurothelial aspergillosis. Extrarenal aspergillosis of

the urinary tract is sparse, with few cases of testicular and adrenal involvement being

described.

The kidney is the most frequently involved part of the urinary tract in invasive

aspergillosis.

Both bladder and prostate aspergillosis are also described.

Outflow obstructive symptoms, similar to those due to benign prostate hypertrophy

(BPH) are predominant. Other related symptoms could be dysuria, perineal or suprapubic

discomfort and hematuria.

The prostate fungal infection is indistinguishable from BPH, bacterial and tuberculus

prostatitis or malignancy of the gland. Prolonged courses of antibiotics, steroid intake and

an indwelling bladder catheter have been identified as presumed predisposing factors in

these cases.

Renal involvement is usually silent if the disease is localised to the cortex of the kidney

and is a relatively frequent finding at autopsy in the context of disseminated disease.

Despite the increased incidence of invasive aspergillosis in transplant recipients, urinary

tract infections caused by Aspergillus species are uncommon and usually as a

consequence of a systemic haematogenous dissemination, characterized by multiple

parenchymal microabscesses.

More common, although still rare, is renal pelvis involvement. Involvement may be

unilateral or bilateral. Typical host groups include diabetics and intravenous drug

abusers. Fungal masses (bezoars) fill the pelvis of the kidney causing hydronephrosis.

These masses may be passed in the urine as `balls' and may cause renal colic.

Anuria due to bilateral ureteral obstruction with mycelial clumps, although rare, has been

recently reported. Aspergillus is usually cultured from the urine and can be cultured and

visualised in the fungal balls.

Recently reported cases:

1. An unusual case of non-disseminated bladder aspergillosis in a setting of transitional cell carcinoma. 327

2. Nosocomial urinary tract aspergilloma in an immunocompetent host: an unusual occurrence. 328

3. Localized primary renal aspergillosis in a diabetic patient following lithotripsy--a case report. 329

4. An unusual cause of ureteral obstruction in a renal transplant recipient: ureteric aspergilloma. 330

5. Aspergillus in a cervico-vaginal smear of an adult postmenopausal female: An unusual case. 331

.

6. Isolated bladder aspergillosis as the primary presentation of non-oliguric acute renal failure. 332

88

7. Aspergillus "fungus ball" of the bladder after hematopoietic transplantation in a pediatric patient:

successful treatment with intravesical voriconazole and surgery. 333

8. Emphysematous renal tract disease due to Aspergillusfumigatus. 334

9. Renal aspergillosis secondary to renal intrumentation in immunocompetent patient.

335

10. Aspergillosis prostatitis post-renal transplantation. 336

11. Prostatitis and epididymo-orchitis due to Aspergillus fumigatus in a patient with AIDS. 337

12. [Aspergillus prostatitis and prolonged corticotherapy. Apropos of a case report].

338

13. [Renal aspergilloma: first manifestation of an occult sarcomatoid carcinoma]. 339

14. Aspergillus vertebral osteomyelitis and ureteral obstruction after liver transplantation. 340

5.13. Aspergillus osteomyelitis

Aspergillus osteomyelitis is an uncommon manifestation of invasive aspergillosis. It may

involve bones of the axial or appendicular skeleton.

Aspergillus osteomyelitis is a particularly important cause of morbidity and mortality in

immunocompromised hosts, especially in patients with chronic granulomatous disease,

solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT),

diabetes mellitus, and chronic corticosteroid use. Furthermore, Aspergillus osteomyelitis

also is reported in alcoholics and illicit intravenous drug

use.Notably, Aspergillus osteomyelitis is reported in patients without apparent

immunodeficiency.

Symptoms and signs of Aspergillus osteomyelitis tend to be non-specific.

Histopathology and microbiologic examination of bone tissue biopsy are the gold

standards for identification of Aspergillus spp.

Vertebral osteomyelitis with or without discitis is the most common osteoarticular

manifestation of Aspergillus. It may be caused by hematogenous dissemination from a

distant site, by traumatic or surgical inoculation, or more rarely, from adjacent tissues,

usually lungs. Host factors associated with Aspergillus vertebral osteomyelitis include

chronic granulomatous disease, primary monocyte killing deficiency, chronic obstructive

pulmonary disease treated with inhaled or systemic corticosteroids, intravenous

corticosteroid treatment, solid organ transplantation, HSCT, diabetes mellitus,

hematological malignancies, illicit intravenous drug use, pulmonary aspergillosis, and

prior back surgery.

Sternal aspergillosis is strongly associated with thoracic surgery. The route of infection

is likely through direct inoculation of the wound through surgery, post-operative wound

care or airborne transmission of conidia. Aspergillus sternal osteomyelitis may present in

the immediate post-operative period, or later in the ensuing weeks, months, or even years

after the operation

Osteomyelitis of the Ribs and Long Bones is common in patients with chronic

granulomatous disease, immunosuppressed recipients of solid organ transplants and

individuals who have experienced traumatic or surgical inoculation of affected bones.

89

Primary rib and long bone aspergillosis in immunocompetent individuals may rarely

occur.

Invasive Aspergillus mastoiditis may develop as an extension of infection from the

middle ear into the mastoid process, petrous and temporal bones. It is characterized by

otalgia, otorrhea, hearing loss and cranial nerve deficits, Aspergillus mastoiditis may be

indolent or rapidly progressive. Chronic fungal otitis media and regional surgical

procedures may progress to invade the petrous bone, transverse venous sinus, and

the cranial nerves, resulting in sinus thrombosis, cranial nerve palsies, and high mortality.

Aspergillus osteomyelitis of the cranium and mandible may occur as a sequela of other

infectious processes, e.g., invasive Aspergillus otitis or sinusitis. There are different

routes via which Aspergillus may invade the temporal bone and lateral skull base, such as

the external ear or the tympanic cavity during the process of acute or chronic otitis media.

Aspergillosis of the skull may also develop secondary to traumatic or surgical

inoculation.

Joint space infection caused by Aspergillus spp. (Septic arthritis) is uncommon.

Septic arthritis may be subsequent to hematogenous dissemination of pulmonary

aspergillosis, dissemination fromadjacent bone tissue infection, or associated with

traumatic or surgical manipulation. Aspergillus septic arthritis is commonly associated

with intra-articular corticosteroid injections and orthopedic hardware. Other host factors

that may exist simultaneously, with or independent of iatrogenic inoculation

of Aspergillus into the joint, include malignancy and its management, neutropenia, solid

organ or HSCT receipt, GVHD, alcoholic cirrhosis, and chronic granulomatous

disease. Aspergillus septic arthritis rarely occurs in an otherwise healthy host,

independent of any potential iatrogenic inoculation. Aspergillosis of the joint space also

may occur in the context of disseminated aspergillosis.

Axial high-resolution computed tomography scan of temporal bone demonstrating R (right) - soft tissue lesion (*) involving the tympanic membrane (TM) and external auditory canal (EAC) L (left) - soft tissue lesion (a) involving the TM and EAC with

erosion of posterior canal wall and facial canal near 2nd

genu (b). Bradoo RA, Shah KD, Gayathri H, Kapadia MA. Invasive

aspergillosis of the temporal bone. Indian J Otol 2012;18:30-3

91

Recently reported cases:

1. A rare case of spontaneous Aspergillus spondylodiscitis with epidural abscess in a 45-year-old

immunocompetent female.341

2. Does surgery influence the outcome of Aspergillusosteomyelitis? 342

3. Osteomyelitis caused by Aspergillusspecies. 343

4. Sternal osteomyelitis caused by Aspergillus fumigatus following cardiac surgery: Case and review. 344

5. Aspergillosis of bones and joints - a review from 2002 until today.345

6. Aspergillus osteomyelitis: epidemiology, clinical manifestations, management, and outcome. 346

7. Costochondritis caused by Aspergillus flavus following cardiac surgery.347

8. Osteomyelitis caused by Aspergillus species: a review of 310 reported cases.348

9. Case report of Aspergillus osteomyelitis of the ribs in an immunocompetent patient.349

10. A case-based discussion on a patient with non-otogenic fungal skull base osteomyelitis: pitfalls in

diagnosis.350

11. Vertebral osteomyelitis and epidural abscess due toAspergillus nidulans resulting in spinal cord

compression: case report and literature review. 351

12. Involvement of the opportunistic pathogen Aspergillustubingensis in osteomyelitis of the maxillary

bone: a case report. 352

13. Aspergillus osteomyelitis of the proximal humerus: a case report.353

14. Aspergillus vertebral osteomyelitis in immunocompetent patients. 354

15. Skull base osteomyelitis and potential cerebrovascular complications in children.355

16. Aspergillus osteomyelitis of the lumbar spine complicated with orbital apex syndrome: A potential role

of the Batson's plexus in disease propagation.356

17. Successful treatment of Aspergillus flavus spondylodiscitis with epidural abscess in a patient with

chronic granulomatous disease.357

18. Aspergillus fumigatus spondylodiskitis in renal transplant patient: voriconazole experience. 358

19. Osteomyelitis due to Aspergillus species in chronic granulomatous disease: an update of the literature. 359

20. Aspergillus vertebral osteomyelitis and ureteral obstruction after liver transplantation. 360

21. Aspergillus fumigatus osteomyelitis in a patient receiving alemtuzumab for B-cell chronic lymphocytic

leukaemia. 361

22. Aspergillus vertebral osteomyelitis in immunocompetent hosts: role of triazole antifungal therapy. 362

91

6. Aspergillosis in animals

6.1. Aspergillosis in dogs

In dogs, the three major forms of aspergillosis are nasal, bronchopulmonary, and disseminated

infections.

1. Nasal aspergillosis

o Nasal aspergillosis frequently accompanied by invasive sinusitis, occurs most

commonly in medium to large, dolichocephalic or mesaticephalic breeds.

o The primary etiologic agent is A. fumigatus, followed by A. flavus and A. niger.

o The clinical signs include sneezing, unilateral or bilateral nasal discharge,

rhinalgia, epistaxis, frontal sinus osteomyelitis, anorexia, and lethargy. In advanced

cases, ulceration of the nares, facial deformity due to paranasal extension, and

ocular involvement may be evident.

o Radiographs may show turbinate tissue destruction with large radiolucent spaces.

Fungal plaques in the nasal cavity may be observed by rhinoscopy.

2. Bronchopulmonary aspergillosis

o Bronchopulmonary aspergillosis is a rare disease in dogs.

o The causative agents and the breeds being affected are similar to those seen in the

nasal form of aspergillosis.

o The clinical signs are nonspecific, including depression, fever, and cough. Chest

radiographs can demonstrate diffuse nodular lesions in the lung.

3. Disseminated aspergillosis

o Disseminated aspergillosis is a relatively infrequent but potentially fatal disease in

dogs.

o The two most common etiologic agents are A. terreus and A. deflectus, followed

by A. fumigatus, A. niger, and A. flavipes in order of decreasing frequency.

o The majority of the reported cases of disseminated aspergillosis in dogs involve

young to middle-aged females.

o The German shepherd dog is the most commonly affected breed; however, other

breeds, including the Dalmatian, English setter, pug, Rhodesian ridgeback,

springer spaniel, and whippet, have occasionally been affected.

o Clinical signs of disseminated aspergillosis may develop suddenly or slowly over a

few months.

o Clinical presentations of disseminated aspergillosis may include diskospondylitis,

osteomyelitis, spinal hyperpathia, vestibular abnormalities, ataxia, paraparesis,

weight loss, anorexia, uveitis, lameness, renal failure, and respiratory distress.

o Common clinicopathologic features are leukocytosis, hyperglobulinemia,

azotemia, and hypercalcemia are.

o Granulomatous inflammation in multiple organs, including bone, kidney, and

spleen, is frequently observed.

92

o The disease can generally be diagnosed based on clinical, radiographic, and

pathological findings.

www.pethealthnetwork www.germanshepherds.com www.springerrescue.org www.njmoldinspection

VCA Animal Hospitals www.njmoldinspection.com

Nasal aspergillosis due to A. fumigatus Nasal infection by Aspergillus terreus.Dr. R. Mallik, Sydney, Australia

Disseminated aspergillosis due to A. terreus, Saggital section of kidney ,Saggital section of the vertebral column Dr. Michael Day,

University of Bristol

93

Higher magnification showing marked granulomatous inflammation with giant cell formation (arrowhead) surrounding septate fungal

hyphae and bulbous spore-like structures (arrow)m Grocott's methenamine silver-stained section demonstrating prominent fungal

hyphae and terminal conidiophores (arrows).. Dr. Michael Day, Bristol Univ

Cases recently reported:

1. Diagnostic value of MRI in dogs with inflammatory nasal disease.

363

2. What causes canine sino-nasal aspergillosis? A molecular approach to species identification. 364

3. Spontaneous pneumothorax associated with Aspergillus bronchopneumonia in a dog. 365

4. Cytokine and transcription factor expression by Aspergillus fumigatus-stimulated peripheral blood

mononuclear cells in dogs with sino-nasal aspergillosis. 366

5. A novel case of canine disseminated aspergillosis following mating. 367

6. Sinonasal aspergillosis in dogs: a review. 368

7. Toll- and NOD-like receptor mRNA expression in canine sino-nasal aspergillosis and idiopathic

lymphoplasmacytic rhinitis.369

8. Clotrimazole and enilconazole distribution within the frontal sinuses and nasal cavity of nine dogs with

sinonasalaspergillosis. 370

9. Analysis of gene expression in canine sino-nasal aspergillosisand idiopathic lymphoplasmacytic rhinitis:

a transcriptomic analysis. 371

10. Disseminated aspergillosis in a dog due to Aspergillus alabamensis. 372

11. Sensitivity and specificity of a blood and urine galactomannan antigen assay for diagnosis of

systemic aspergillosis in dogs. 373

12. A novel case of canine disseminated aspergillosis following mating. 374

13. Aspergillus fumigatus bronchopneumonia in a Hellenic shepherd dog. 375

14. Cavitating pulmonary lesions in German shepherd dogs. 376

15. Clinical resolution of nasal aspergillosis following therapy with a homeopathic remedy in a dog. 377

16. Aspergillus versicolor, a new causative agent of canine disseminated aspergillosis.

378

17. Frontal sinus depth at four landmarks in breeds of dog typically affected by sinonasal aspergillosis. 379

18. Aspergillus fumigatus Bronchopneumonia in a Hellenic Shepherd Dog.

380

19. Acute phase protein concentrations in dogs with nasal disease. 381

20. Long term survival in two German shepherd dogs with Aspergillus-associated cavitary pulmonary

lesions. 382

21. Otomycosis due to Aspergillus spp. in a dog: case report and literature review.

383

22. Efficacy of intrasinusal administration of bifonazole cream alone or in combination with enilconazole

irrigation in canine sino-nasal aspergillosis: 17 cases. 384

23. Chronic monolateral otomycosis in a dog caused by Aspergillus ochraceus. 385

24. Repeated rhinoscopic and serologic assessment of the effectiveness of intranasally administered

clotrimazole for the treatment of nasal aspergillosis in dogs. 386

94

25. Roland Kozdrowsk, Aspergillosis of a dog genital tract—Case report, 387

26. Aspergillosis of a dog genital tract-Case report. 388

6.2. Aspergillosis in cats

Feline aspergillosis includes

o Sinonasal aspergillosis (SNA) can be invasive or noninvasive and is most

commonly caused by Aspergillus fumigatus and Aspergillus niger. SNA has a

favorable prognosis with treatment

o Sino orbital aspergillosis (SOA) is an invasive mycosis that is being increasingly

recognized, and is most commonly caused by a recently discovered pathogen

Aspergillus felis. The prognosis for SOA remains poor.

Veterinary images, Nasal, sinus and orbital aspergillosis in a cat.

95

Severe bone erosion rostrally through the nasal bone.

Martin L. Whitehead, & Peter W. Kettlewell,. Chipping Norton Veterinary Hospital, Chipping Norton,Oxon

Cases recently reported:

1. Aspergillosis in cats: ABCD guidelines on prevention and management. 389

2. Aspergillus felis sp. nov., an emerging agent of invasiveaspergillosis in humans, cats, and dogs. 390

.

3. Feline aspergillosis. 391

4. Otomycosis due to Aspergillus spp. in a dog: case report and literature review. 392

5. Chronic monolateral otomycosis in a dog caused by Aspergillus ochraceus. 393

6. Invasive mould infections of the naso-orbital region of cats: a case involving Aspergillus fumigatus and

an aetiological review. 394

7. Intranasal infusion of clotrimazole for the treatment of nasalaspergillosis in two cats. 395

8. Bilateral orbital and nasal aspergillosis in a cat. 396.

9. Computed tomographic findings of fungal rhinitis and sinusitis in cats. 397

10. Aspergillus flavus keratomycosis in a cat treated with topical 1% voriconazole solution. 398

11. Isolation of Aspergillus udagawae from a fatal case of feline orbitalaspergillosis. 399

12. Feline sino-orbital aspergillosis:an emerging clinical syndrome. 400

13. Use of posaconazole in the management of invasive orbitalaspergillosis in a cat. 401

96

6.3. Aspergillosis in horses

Aspergillosis in horses is a fungal disease caused by Aspergillus spp Aspergillus spp are very

common in the environment, especially in moldy feed and bedding. They are opportunistic

pathogens and often cause disease in horses that are immunosuppressed from debilitating

conditions (e.g., enterocolitis, septicemia, neoplasia, Cushing's disease, equine protozoal

myeloencephalitis) or major surgery or that have been treated with immunosuppressive drugs.

They can infect internal organs, most commonly affects the guttural pouches but infection may also lead to abortion, keratomycosis, rhinitis and rarely pulmonary aspergillosis.

1. Equine Aspergillus Rhinitis, , Pulmonary Aspergillosis

Historical

Rivolta (1856) recorded the first case of aspergillosis in horses, but was frequently restricted to infection of the nasal sinuses and the guttural pouches. In a case described

Thary and Lucet (1895) described a rapidly developed rapidly disease that showed generalized interstitial

haemorrhage with caseous nodules in the kidneys in a four-year-old mare.

Foulerton (1899) described the lung of a horse with typical aspergillotic nodules

Noller & Krause in 1924, and Tscherniak (1928) gave details of several more cases Romanov (1928) reported meningeal involvement.

Infection is by inhalation of an overwhelming number of spores or by translocation of

organisms across an inflamed gastrointestinal tract. Aspergillus pneumonia is almost uniformly

fatal, often with no or mild respiratory signs. The two forms of Aspergillus pneumonia probably

reflect the two portals of entry, with fungal proliferation and invasion of the small airways

occurring secondary to inhalation, and angioinvasive aspergillosis with lesions centered around

large blood vessels likely due to hematogenous infection originating from the gastrointestinal

tract. In two retrospective studies of invasive pulmonary aspergillosis, 41 of 49 cases were

associated with enterocolitis.

Pulmonary aspergillosis is characterized grossly by multiple nodules throughout the lungs. On

histologic examination, there is often necrosis and purulent inflammation. Necrosis is due to

toxin and enzyme production as well as vascular obstruction. Fungal invasion of blood vessels

is common and results in vasculitis, thrombosis, infarction, and necrosis. Chronic lesions are

granulomatous, with macrophages, neutrophils, and multinucleated giant cells predominating.

97

2. Guttural Pouch Aspergillosis

Aspergillosis most often affects the guttural pouch. The infected guttural pouch is characterized

by a necrotizing inflammation and is thickened, haemorrhagic, and covered by a friable

pseudomembrane. There is no age, sex or breed predisposition for guttural pouch mycosis and

both left and right pouches are affected with equal frequency.

Guttural pouch mycosis is characterised by spontaneous epistaxis (often in a resting horse) as a

result of fungal erosion of the internal carotid artery. Other clinical signs include nasal

discharge and dysphagia. Mycotic plaques are usually located on the caudodorsal aspect of the

medial guttural pouch.In some instances, fungal plaques may be multiple or diffuse.

Guttural pouch mycosis in a horse (endoscopic image). Gutteral pouch aspergillus Dr. Sameeh M. Abutarbush.

Guttural pouch aspergillus (PAS) Dr. Michael Day, University of Bristol)

98

Recently reported cases:

1. A Case of Equine Aspergillosis: A Novel Sampling Procedure for Diagnosis. 402

2. Guttural pouch mycosis in six horses in New Zealand403

3. Aspergillus fumigatus guttural pouch mycosis in a horse which died from acute pulmonary oedema. 404

4. Guttural pouch mycosis in a donkey (Equus asinus): a case report.405

5. Guttural pouch mycosis in a 6-month-old filly.406

.

6. Diagnosis and management of guttural pouch mycosis.407

7. Atypical guttural pouch mycosis in three horses. 408

6.4. Aspergillosis in cattle

Aspergillus spp are a group of soil mould which are an aerosol cause of mycotic abortion,

respiratory diseases and aflatoxicosis in cattle worldwide. Species of Aspergillus which are

pathogenic to cattle include:

Aspergillus fumigatus

Aspergillus terreus

Aspergillus flavus

In affected cattle, infections with Aspergillus may be asymptomatic. In respiratory aspergillosis,

respiratory symptoms such as coughing, dyspneaand hemoptysis may be apparent. In some

cattle, this can be rapidly fatal as dissemination of spores occurs through the pulmonary

circulation.

Mycotic placentitis, also caused by Aspergillus spp, is usually a sporadic cause of abortion

affecting a small percentage of cattle in a herd. Cattle housed in the winter can experience an

abortion rate of up to 30% due to mycotic placentitis, if feed or bedding is heavily contaminated

with molds. Ingested mold is thought to localize in the cows' intestinal tract and then spread to

the placenta through the blood. High rates of mycotic placentitis have also been correlated with

heavy rainfall during the haymaking season, episodes of subclinical grain overload and with

prolonged intensive antibiotic treatment.

On postmortem, affect lungs contain multiple discrete granulomas, and the disease grossly

resembles tuberculosis.

Aspergillosis has a number of clinical manifestations in the cow including mastitis, placentitis,

diarrhoea, ocular infection and mycotic pneumonia. Abortion in the second or third trimester of

pregnancy has also been described.

In the case of pulmonic disease, clinical signs may include pyrexia, cough, dyspnoea and

tachypnoea but may be limited to vague signs such as weight loss or signs of mild

respiratory disease.

In aborting cattle, the foetus and placenta are retained and foetal lesions such as

bronchopneumonia and dermatitis may be seen.

99

Mastitic cows may display depression, weight loss and pyrexia with purulent mammary

secretions and a hot, swollen udder.

Mycotic abortion, Rycroft, BSc, PHD, C. Biol.F.I.Biol., FRCPath

B o vi n e P l a c e n t a l Co t y l e d o n - mu l t i f o c a l n e c r o s i s ( As p e r g i l l o s i s ) , Ro c h a r d J a l o w s k i

111

Recently reported cases:

1. Involvement of fungal species in bovine mastitis in and around .

409

2. Application of real-time PCR for detection of Aspergillus species in aborted ruminant foetuses. 410

3. Mycotic abortion in cattle .411

4. Abortus by Aspergillus fumigatus and A. nigerin cattle in southern Brazil. 412

5. Mastitis by Aspergillus fumigatusin sheep. 413

6. [Mycotic placentitis in cattle]. 414

7. Pathogenesis of experimental bovine mycotic placentitis produced by Aspergillus fumigatus. 415

8. Aspergillus nidulans and Aspergillus fumigatus as causal agents of bovine mastitis. 416

9. Cases of Aspergillus mastitis in cattle].417

10. Caprine mastitis due to aspergillosis and zygomycosis: a pathological and immunohistochemical

study418

6.5. Aspergillosis in Sheep and goats

There have been only five reports of aspergillosis in sheep. The lesions in lambs consist of

small (2-3 mm. Diam.) bluish-grey nodules surrounded by a narrow haemorrhagic zone, and

closely resemble those caused by the lungworm Muellerius capillaris (Andersen 1927). The

most recent report of the disease is in the Annual Report of the New Zealand Department of

Agriculture for 1955.

Van Hellens (1902-03) investigated in Finland one of the few epidemics of aspergillosis on

record among sheep. The animals were adult and showed chronic bronchitis and catarrhal

pneumonia closely resembling the symptoms of tuberculosis.

Death occurred up to one year after the appearance of symptoms. Conidiophores

of A.fumigatus were found in the sputum of affected sheep and numerous discrete and

sometimes well calcified nodules 1-7 mm. in diam. were present in the lungs. Nobel & Shamir

(1956) described a rather different condition in which granulomatous lesions containing the

hyphae of A.fumigatus were found in the lungs of a day-old lamb, and they considered the

infection had taken place in utero.

Recently, two cases of systemic aspergillosis were described by Pérez, 1999, in dairy sheep

from a flock in which fungal mastitis appeared subsequent to the antibiotic treatment of animals

before parturition. Lesions characterized by necrosis and a pyogranulomatous exudate were

observed in the mammary glands, supramammary and mediastinal lymph nodes, kidneys, lung,

liver, heart, forestomachs and brain. The intense vasculitis with thrombosis observed in various

organs, but especially in the mammary glands, suggested a haematogenous dissemination of the

infection from this organ. The aetiological diagnosis was accomplished by the

immunohistochemical staining of the fungal structures seen in the histological sections by the

specific Aspergillus monoclonal antibody Mab-WF-AF-1 together with the isolation of

Aspergillus fumigatus in pure culture from affected tissues.

111

In goats, nasal and cutaneous aspergillosis was reported by do Carmo et al.,2014, The clinical

signs were severe respiratory distress due to partial nasal obstruction, bilateral mucopurulent

nasal discharge, skin nodules on the ears and dorsal nasal region and focal depigmentation of

the ventral commissure of the right nostril. At necropsy examination, sagittal sectioning of the

head revealed a yellow irregular mass extending from the nasal vestibule to the frontal portion

of the nasal cavity. Microscopically, there was pyogranulomatous rhinitis and dermatitis, with

numerous intralesional periodic acid–Schiff-positive fungal hyphae morphologically suggestive

of Aspergillus spp. Aspergillus niger was isolated by microbiological examination.

Experimental aspergillosis in sheep and goats

Mycotic abortion was induced in pregnant ewes and goats by El-Naggar et al. (1997). Twelve

animals were inoculated i.v. with 10 ml of A. fumigatus suspension containing 2x107

viable

spores/ml and 5 pregnant animals were kept as control. All experimental animals aborted

between 19-30 days post inoculation. Uteri, maternal and fetal placenta and fetal tissues showed

extensive necrosis, infarction and invasion with A. fumigatus hyphae. Histological examination

revealed mycotic granulomatous inflammation in the lungs, heart, brain, spleen and kidneys.

Placenta of aborted ewe infected with A. fumigatus showing hemorrhages and necrosis, right: Hyphae of

A. fumigatus in the necrotic chorioallantoic villus

Placenta of aborted ewe, allantoic blood vessels showing hyphae of A. fumigatus penetrating the vascular wall (GMS), rigt: placenta of

aborted ewe showing congestion of maternal blood vessels and necrosis of fetal chorionic tissue (H&E)

112

Lung of aborted ewe showing large granulomatous mycotic abscess and multiple hyphae of A. fumigatus (PAS)

Left: Lung of aborted ewe showing typical dichotomous branching septated hyphae with reproductive vesicles

(GMSX400) Right: Lung of aborted ewe showing nodules of of A. fumigatus

Left: Spleen of aborted ewe showing the arrangement of hyphae of A. fumigatus within tissue (H&E), rigt:

mycotic granuloma with A. fumigatus

Placenta of aborted goat showing areas of coagulative necrosis (left) and A. fumigatus hyphae (right)

113

Placentome of aborted goat showing A. fumigatus hyphae in necrotic villus (GMSX400)

Experimental Aspergillus fumigatus mastitis in 4 goats and 4 ewes was induced by El-Naggar

et al. (1997). The left halves of the udders were inoculated intramammary with 2 ml of viable A.

fumigatus spore suspension (1.65x108

spores /ml). The right halves were inoculated with the

diluent. After 25 days the animal were sacrificed and examined. All animals showed

granulomatous inflammation of the udder. A. fumigatus was seen in the center of the lesion,

which was heavily infiltrated with neutrophils, macrophages and surrounded by fibrous

connective tissues. Diffuse necrosis and cavitation of the udder tissues were detected and

vesicles of Aspergillus fumigatus were also seen

Acute mycotic mastitis in ewe showing A. fumigatus hyphae stained by PAS (left) and GMS (right)

Supramammary lymph node of ewe showing area of hyperplasia with profileration of neutrophils and macrophages and spores of A.

fumigatus (PAS)

114

Cases recently reported:

1. Nasal and Cutaneous Aspergillosis in a Goat419

2. Generalized aspergillosis in dairy sheep. 420

3. Caprine mastitis due to aspergillosis and zygomycosis A pathological and immunohistochemical

study. 421

6.6. Aspergillosis in pigs

Records of aspergillosis in pigs are also rare. Berg (1898-99) first described the disease as a

persistent cough associated with a lobar pneumonia and nodule formation. The spleen and

kidney were enlarged and congested, and nodular lesions were found in the mesenteric lymph

nodes. Berg did not obtain any isolates from the lesions but assumed that an Aspergillus was the

cause of the disease. Generalized infection in a pig was also reported by Nuvoletti & Casella

(1903).

Aspergillus fumigatus infection of the lung in piglets, www.aspergillus.org.uk

6.7. Aspergillosis in camels

El-Khouly et al. (1992) reported on death in racing camels (Camelus dromedarius) associated

with a specific disease syndrome. Clinical signs included pyrexia, coughing, lachrymation,

oedema of the throat and submandibular region and enlargement of submandibular lymph

nodes. In terminal cases nervous signs were present and sometimes there was bloody diarrhoea

and vomiting. Of 480 camels at least 70 animals were affected with the disease and about 40

died. Morbidity and mortality was greater in camels recently imported. Consistent necropsy

findings were extensive petechial and ecchymotic haemorrhage beneath the epicardium,

endocardium and visceral pleura and in the mediastinal lymph nodes, and haemorrhagic oedema

115

of the pharyngeal and laryngeal areas. Haemorrhages occurred more variably in abdominal

organs and on the omasal and abomasal mucosa. Bronchopneumonia, omasitis and abomasitis

were observed on microscopic examination, together with liver and kidney lesions of presumed

toxic origin. Fungal hyphae and, occasionally, the characteristic conidial morphology of

Aspergillus fumigatus were seen in sections and direct smears from lesions in the respiratory

and alimentary tracts. A fumigatus was cultured from trachea, bronchi, bronchioles, lung tissue,

heart blood, omasum, abomasum, ileum and submandibular lymph nodes. Whether the role of

Aspergillus in the overall syndrome is primary or secondary has not been established; no other

potential aetiological agent has been identified.

Dehkordi et al. (2012) performed research for detection of Aspergillus species (A. fumigatus, A.

flavus, A. niger and A. terreus) in aborted bovine, ovine, caprine and camel foetuses by real-

time PCR in Iran. After modification of real-time PCR on abomasal contents, from the total

number of 970 samples, 141 (14.53%) gave positive results for Aspergillus species. Of them, 62

(17.71%), 33 (14.04%), 27 (12.05%) and 19 (11.8%) positive specimens were detected in

bovine, ovine, caprine and camel foetuses respectively. Statistical analysis showed significant

differences (P<0.05) between bovine and camel and bovine and caprine aborted foetuses.

Aspergillus abortion was the most prevalent in cattle whereas camels tended to be the most

resistant.

An invasive form of aspergillosis in an alpaca (Lama pacos) was described, with dissemination

causing small abscesses and multifocal areas of necrosis in the lung, heart, spleen and kidneys.

Histological sections showed hyphae morphologically compatible with an Aspergillus species.

Direct immunofluorescent testing confirmed the diagnosis of aspergillosis.( Severo et al., 1989).

6.8. Aspergillosis in American bisons

Rewell & Ainsworth (1947) showed how the respiratory passages of an American bison at the

London zoo were found on post-mortem examination to be lined with a greenish felt

of A.fumigatus with absence of severe lung lesions.

de los Monteros et al. (1999) reported on concomitant nasal zygomycosis and pulmonary

aspergillosis in a 3-mo-old female American bison calf (Bison bison) in Pennsylvania (USA).

Etiologic diagnosis was made by immunohistochemistry using a panel of monoclonal

antibodies and heterologously absorbed polyclonal antibodies. In the lungs fungal infection was

accompanied by hemorrhage, fibrin exudation, and infiltration with neutrophils. Fungi were

observed to penetrate apparently normal epithelial lining of the nasal turbinates, and there was

hemorrhage, edema, and invasion of blood vessels in the submucosa. In vessels fungi were

typically associated with thrombosis. The calf may have been infected due to a high level of

exposure to mouldy feed and litter in the environment in combination with a collapse of it's

natural defence mechanisms.

116

6.9. Aspergillosis in monkeys

Scott (1930) gave details of nine cases of mycosis (aspergillosis?) and a further nine associated

with tuberculosis in captive monkeys encountered at the London zoo. The lungs were the chief

organs involved but miliary nodules were occasionally found throughout the viscera.

Jurczynski (2012) reported on invasive aspergillosis in a putty-nosed monkey (Cercopithecus

nictitans) with adrenocortical Cushing's syndrome. An 18-year-old captive female putty-nosed-

monkey (Cercopithecus nictitans) with a history of long-term infertility and

hyperglucocorticism was euthanized because of perforating thoracic trauma induced by group

members and subsequent development of neurological signs.

Complete necropsy and histopathological examination of formalin-fixed tissue samples was

carried out. The monkey showed invasive pulmonary and cerebral infection with Aspergillus

fumigatus together with adrenocortical neoplasia and signs of Cushing’s syndrome, such as

alopecia with atrophic skin changes, evidence for diabetes mellitus and marked

immunosuppression.

6.10. Aspergillosis in deers

Outbreaks of pulmonary aspergillosis have been reported in hares in Sweden (Thjostta, 1933;

Hölphers & Lilleengen, 1947) and also in roe-deer in that country and Switzerland, in which the

conchal and ethmoid bones as well as the lungs and other organs became affected by tumour-

like growths containing hyphae (Krembs, 1937; Burgisser, 1955). When specific determination

had been carried out, A.fumigatus was the most frequent isolate but Burgeon (1929)

reported A.niger from nodular lesions in the lungs of a bull, a heifer and a stag in Indo-China.

117

During 1988, pulmonary mycosis was diagnosed in four of 116 farmed deer examined on

suspicion of tuberculosis. The histopathology showed allergic bronchopulmonary mycosis in a

red deer (Cervus elaphus) and the agent was identified as a zygomycete, probably Absidia

corymbifera, by immunofluorescence staining. Three fallow deer (Dama dama) had invasive

necrotizing mycotic pneumonia and progressive exudative mycotic alveolitis caused by

Aspergillus fumigatus. In the red deer, weakness due to paratuberculosis had probably

promoted the mycotic infection. The three fallow deer were bred on another farm, where

predisposing factors included mouldy straw and incorrect management.( Jensen et al., 1989)

Aspergillus pneumonia in lung of deer © Bristol Biomedical Image Archive. pathmicro.med.sc.edu

6.11. Aspergillosis in rabbits

Aspergillosis in domestic rabbits has been reported by Schöppler (1919) and Höppli (1923).

Patton (1975) described cutaneous and pulmonary aspergillosis in rabbits.

118

6.12. Aspergillosis in guinea-pigs

Ainsworth & Austwick (1955a) have recorded the disease in guinea-pigs. Recently, guinea-pigs

are used for assessment of Aspergillus fumigatus burden in pulmonary tissue of antimycotics.

Histological examination of guinea pig lungs exposed to and infected with aerosolized A. fumigatus aac.asm.org

4.13. Aspergillosis in lesser blind mole rats

Tamam and Refai (2013) reported on seven wild Egyptian lesser blind mole rat (Spalax

leucodon Egyptiacus) that died naturally in the wild. All the animals had large pulmonary

lesions that on microscopic, microbiological, and ultrastructural analysis were shown to contain

mixed infections with Alternaria alternata and Aspergillus candidus. Some of the lesions were

circumscribed with fibroblastic proliferation and inflammatory response. The lungs had

haemorrhage and chronic inflammatory response to the organisms, which is likely to have been

the cause of death.

(A) Thoracic cavity of Spalax leucodon showing large, tumour-like lesions replacing the apical left lung lobe and large, firm brown

lesions in the caudal and right lobes (yellow arrows). (B) Thoracic cavity containing brown, consolidating focal lesions in the centre of

the left lung lobe, emphysematous change (red arrows), and red hepatisation of the lower right lobe (violet arrow).

119

6.14. Aspergillosis in dolphins.

Reidarson et al. (1998) reported on a 4-yr-old male bottlenose dolphin (Tursiops truncatus) that

developed an Aspergillus fumigatus pneumonia. Fungal elements were identified by cytology

and microbiology from endoscopic bronchoalveolar lavage and brushings of a raised yellow

endobronchial lesion. The results of qualitative immunodiffusion serology, a technique that

identifies specific circulating antibodies to Aspergillus fumigatus, were suggestive of an active

infection. The dolphin was treated with itraconazole for over 2 yr, which resulted in remission

of clinical signs. Pneumonia caused by Aspergillus sp. accounts for the large majority of

pulmonary mycoses in marine mammals

The clinical history, laboratory data, and gross and histological findings are described and

discussed by Brian et al. (2005) for three cases of fatal pulmonary aspergillosis in three species

of dolphins.

Reorted cases

1. Invasive aspergillosis in a Putty-nosed monkey (Cercopithecus nictitans) with adrenocortical

Cushing’s syndrome. 422

2. Aspergillosis in camels affected with a specific respiratory and enteric syndrome. 423

3. Pulmonary mycosis in farmed deer: allergic zygomycosis and invasive aspergillosis424

4. Application of real-time PCR for detection of Aspergillus species in aborted ruminant foetuses. 425

5. Efficacy of voriconazole against invasive pulmonary aspergillosis in a guinea-pig model. 426

6. Experimental aspergillosis in guinea pigs: influence of itraconazole on fungaemia and invasive fungal

growth. 427

7. Occurrence of Aspergillus fumigatus Fresen. in the Lung of an American Bison. 428

8. Nasal zygomycosis and pulmonary aspergillosis in an American bison.429

9. Bronchoscopic and serologic diagnosis of Aspergillus fumigatus pulmonary infection in a bottlenose

dolphin (Tursiops truncatus). 430

10. Invasive aspergillosis in an alpaca (Lama pacos). 431

11. Pulmonary aspergillosis in three species of dolphin. 432

12. Cutaneous and pulmonary aspergillosis in rabbits.433

13. Dual mycotic pulmonary granulomas caused by Alternaria alternata and Aspergillus candidus in the

wild egyptian mole rat (spalax leucodon egyptiacus). 434

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7. Aspergillosis in birds

Aspergillosis was first described in a wild duck in 1833 and in turkeys as early as 1898.

All species of birds probably are susceptible.

Aspergillus fumigatus is a common cause of the disease. However, other species like A.

flavus, A. niger, A. nidulans, andA. terreus may also be isolated from avian cases of

aspergillosis (sometimes in mixed infections) but much less frequently than A. fumigatus

The bird comes in contact with the organisms through contaminated feed, litter or

premises. The disease is not contagious and does not spread from one bird to another.

Most healthy birds can withstand repeated exposure to these organisms. Inhalation of

large amounts of the infectious form of the mould or reduced resistance of the bird

apparently results in infection.

7. 1. Aspergillosis in poultry

Infection by Aspergillus sp. has been reported in almost all domesticated avian species and

production types: layer cockerels, pullets in cages, broiler breeders, and growers of chicken or

turkey poults, duck breeders and goslings. Aspergillosis is a disease, usually of the respiratory

system, of chickens, turkeys, and less frequently ducklings, pigeons and geese, In chickens and

turkeys, the disease may be endemic on some farms.

Aspergillus fumigatus can penetrate egg shells under ideal growth conditions and thus

infect the embryos. Such eggs will often appear green when candled (the embryo will be

dead). Infected embryos may hatch with well- developed lesions.

If infected eggs break in the hatchery, large numbers of spores are released which

contaminate the hatchery environment and air systems can lead to severe outbreaks in

very young birds (less than 3 weeks of age). Eggs punctured for in-ovo injection are

particularly susceptible to contamination. Even low-level contamination of hatchers or air

systems can result in mortalities of 50% or greater when in-ovo injection is used.

High mortality rates are seen in newly hatched chicks and poults that inhale large

numbers of spores during hatching in contaminated incubators. The diseased is called

brooder pneumonia. In older birds, infection is caused primarily by inhalation of spore

laden dust from contaminated litter or feed or dusty range areas.

Dyspnea, hyperpnea, somnolence and other signs of nervous system involvement,

inappetence, emaciation, and increased thirst may be seen. In chicks or poults up to 6

weeks, the lungs are most frequently involved.

Airsacculitis in young mature turkeys is a leading cause of postmortem condemnation.

Pulmonary lesions are characterized by cream-coloured plaques a few mm to several cm

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in diameter; occasionally, mycelial masses may be seen within the air passages on gross

examination. The plaques also may be found in the syrinx, air sacs, liver, intestines, and

occasionally the brain. The encephalitic form is most common in turkeys. An ocular

form, in which large plaques has been seen in chickens and turkeys.

Aspergillus infected eggs, www.thepoultrysite.com

Laboured breathing and gaping in birds suffering from aspergillosis, www.thepoultrysite.com and wildlifedisease.unbc..

Aspergillosis in a duck Aspergillosis in a pigeon, Bull vet inst pulawy 51, 2007

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Left: Aspergillosis in the air sacs of a chick, www.sandhillvet.co.uk

Right: Multiple nudules in the liver of 3 weeks old poult, due to aspergillosis., www.poultrymed.com

Aspergillus granulomas of the serous coats:www.thepoultrysite.com

Lung nodules =aspergillosis, OldVe T.com Multiple nodular lesions in the lung of a duck. Parasitology-Mycology, ENVA

7.2. Aspergillosis in wild birds:

A. fumigatus has been isolated from lesions in wild birds since the early 1800s. Major die-offs

of free-ranging wild birds have been reported from waterfowl, gulls, and corvids following

dumping of mouldy waste grains in areas where birds feed.. Aspergillosis has also been

reported in penguins, raptors, migratory , waterfowl, psittacines and zoologic specimens, such

as flamingos.

7.2.1. Aspergillosis in penguins

On Sunday, November 10th,2013, staff at the Calgary Zoo made the difficult decision to

euthanize a 14-year-old male Gentoo penguin Houdini. He had been sick for almost a month.

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The results of a necropsy confirmed severe aspergillosis – a fungal infection that affects the

respiratory system and is one of the most common causes of death in captive penguins and has

been recorded in wild penguins. Penguins are among several birds that are exquisitely sensitive

to acquiring the infection, with increased disease noted when the spore levels become

concentrated and/or the birds immune systems are depressed as when they moult.

Disseminated Aspergillosis in a Little Penguin, arwh , , forum.backyardpoultry.com

The necropsy of penguin showed that airsacs were diffusely thickened, opaque, and studded

with multiple off-white to pale green dull. Similar plaques were scattered across coelomic

viscera serosal surfaces and adherent to the lungs which were diffusely dark red purple and wet.

The histopathology revealed that granulomatous, heterophilic, and necrotizing inflammation

with myriad intralesional fungal hyphae morphologically consistent with Aspergillus spp. were

present in the lungs, airsacs, kidneys, oviduct, trachea, mesentery, and serosal surfaces of

coelomic viscera. There was significant atrophy of adipose tissue, skeletal muscle, liver, and

pancreas

In a retrospective studies in Gifo University, Yanai reported that, 10 of 42(28.3%) cases of

death in penguins in Japan were due to aspergillosis.

Aspergillosis (air sacs) in an adult male Macaroni penguin, Yanai, Gifu University

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Multiple necrotic areas around oesophagus and lungs,Yanai,Gifu multi-focal necrosis in the lungs,Yanai, Gifu

Histological sections: thickened air sac with conidial heads of A. fumigatusin the lumen of the air sac,Yanai, Gifu

7.2.2. Aspergillosis in parrots

Aspergillosis is less common in companion parrots; however, disease is more

prevalent in African grey parrots (Psittacus erithacus), Amazon parrots

(Amazona spp.), Pionus parrots (Pionus spp.) and macaws. The likelihood of a

fungal infection is increased if the bird is housed in an environment in which there

is poor sanitation, high relative humidity and high temperatures, which can increase

the load of fungal spores. A bird with a weakened immune system due to steroid

administration or concurrent illness (particularly when treatment involves long-term

antibiotics) is also at greater risk for aspergillosis.

Like humans, parrots and other large birds cough when they experience a respiratory irritation.

Coughing can be normal when it only occurs every now and then, but when parrot's cough

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becomes chronic, it could be a sign of aspergillosis. Spores released by the aspergillus can get

trapped in the bird's respiratory system, causing the cough.

In addition to coughing, when aspergillus spores get into parrot's throat and lungs its voice may

change and it may refuse to talk entirely.

Spores released by aspergillus affect different parts of the respiratory system in different ways.

Such symptoms include labored breathing or sudden fits of suffocation. Your bird's neck may

turn blue as it tries to talk or cough. This is indicative of choking, but the bird could also be

suffocating on an aspergillus spore. In some instances, parrots are known to suddenly drop dead

after inhaling an aspergillus spore.

African Grey parrot Amazon parrots Pionus parrots Blue Yellow Macaw

7.2.3. Aspergillosis in quails

Aspergillosis in quail is charachterized by the formation of yellowish white nodular growth in

lungs and intercostal areas with thickened air sacs. Histopathologically, lungs show severe

congestion with focal haemorrhages, multiple granulomatous inflammation with caesative

necrotic areas in centre. Various fungal elements like conidia, long septate hyphae with

mononuclear and heterophilic infiltration are seen in these areas. Microbiological study reveals

velvety bluish green colony of Aspergillus fumigatus.

Nodules in the air sacs and on the peritoneal serosa in a case of aspergillosis in a common quail. www.fmv.utl.

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Aspergillosis in quail crop heart , www.rarc.wisc.edu

7.2.4. Aspergillosis in ostrich

Aspergillosis in ostrich was reported by Perelman and Kuttin (1992), Katz et al. (1966), Pérez,

et al. (2003)Yokota et al. (2004), SANCAK A.A. and PARACIKOLU (2005), Khosravi

(2008), Shathele et al. (2009) and Tijani et al. (2012). ARAGHI et al. (20014) describes an

aspergillosis outbreaks in ostrich flocks of eastern Iran during 2010–2012. They reported that

signs of respiratory involvement, anorexia, depression, progressive emaciation and decreased

production were the most commonly seen in affected farms. Morbidity rate was 43% and

54.53% in breeding birds and chickens, respectively. Aspergillus fumigatus and Aspergillus

niger were identified.

www.doctorfungus.org, ostrich c with respiratory aspergillosis.

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7.2.5. Aspergillosis in red-tailed hawk (Buteo jamaicensis).

Aspergillosis in red-tailed hawk Multiple granulomas in the lungs A. fumigatus colony with spores in air sacs,MVS

7.2.6. Aspergillosis in Red-billed Toucan (Yanai, Gifu Univ)

Aspergillosis in Red-billed Toucan, Yanai, Gifu Univ.: multifocal lesions in the lung and liver

Hyphae radiating from a central necrotic nodule Conidia heads of Aspergillus in a pulmonary cavity

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7.2.7. Aspergillosis in Snow Owl (Yanai, Gifu)

Severely thickened air sacs of Snow Owl caused by A. fumigatus, Yanai, Gifu

7.2.8. Aspergillosis in Goshawk

Aspergillosis in Goshawk, Yanai, Gifu

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7.2.9. Aspergillosis in Cormorants

Aspergillosis in Cormorants, multifocal necrosis with Aspergillus hyphae in the lung

7.2.10. Aspergillosis in swans

Souza et al. (2005) reported on mortality in wild swans inNorthwest Washington State due to

aspergillosis.

Abou-Rawash et al. (2008) reported on disseminated aspergillosis in a Whooper Swan (Cygnus

Cygnus) in Japan. A Whooper Swan which usually migrates from Siberia to the north of Japan

in the winter season was found in Kume Island at the south of Japan in a state of pronounced

illness. Blood and serum analysis revealed hypoalbuminaemia elevated serum level of uric acid

(UA) and creatinin phosphokinase (CPK). The bird succumbed after unsuccessful attempts for

treatment. At necropsy, the bird had multiple focal and coalescent caseated granulomatous

nodules of whitish greenish color on the thoracic and abdominal air sacs, the lungs, and the

serosal surfaces of the spleen, liver and kidneys. At the inner side of the air sacs hyphal growth

with dark greenish color were scattered on most of the surface. Histopathologically, the present

case had a sever degree of chronic disseminated aspergillosis. The granulomas had a central

necrotic areas consisted of necrotic cell debris and hyphae with the microscopical features of

Aspergillus. A. fimigatus was identified in tissues by PAS, GMS, and immunohistochemically.

PCR was very successful to identify the causative fungus like other infectious agent.

On Jan. 27, 2014, 149 dead swans have been found in Whatcom County. The death was

attributed also here to aspergillosis. a disease which is being blamed for the deaths of trumpeter

swans spending the winter on Whatcom County's Wiser Lake.

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Whooper swan, multifocal white nodules in the air sacs with frequent fungi, Yanai, Gifu

Multiple mycotic lesions in the lungs and larynx of a whooper swan, Yanai, Gifu

7.2.11. Aspergillosis in kiwi

The Wellington Zoo reported on 30 September 2013 that, eight young rowi—the rarest

species of kiwi—have died from respiratory tract infections. The kiwi were being treated for

nematodes (a type of worm) in Wellington Zoo when they started to show signs of respiratory

problems.The respiratory tract infection was caused by the fungus Aspergillus spp. which is

commonly found in the environment. It is thought that the birds’ weakened immunity from the

nematodes made them susceptible to this.

Travis et al. (2014) reported on Isolation and Identification of Aspergillus spp. from Brown

Kiwi (Apteryx mantelli) Nocturnal Houses in New Zealand.

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A Rowi kiwi chick. Photo / File Eight Kiwi chicks have died from respiratory tract infections at Wellington Zoo.

Recently reported cases:

1. Secondary Aspergillus fumigatus infection associated with coloidal goiter in a black-masked lovebird

(Agapornis personata). 435

2. Drug resistance of Aspergillus fumigatus strains isolated from flocks of domestic geese in Poland. 436

3. Isolation and identification of Aspergillus spp. from brown kiwi (Apteryx mantelli) nocturnal houses in

New Zealand. 437

4. Monitoring of fungal loads in seabird rehabilitation centers with comparisons to natural seabird

environments in northern California.438

5. Mutations in the Cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus isolated from

avian farms in France and China.439

6. Environmental contamination by Aspergillus spp. in laying hen farms and associated health risks for

farm workers. 440

7. Isolation and identification of Aspergillus spp. from brown kiwi (Apteryx mantelli) nocturnal houses in

New Zealand. 441

8. [Radiographic findings in raptors affected with a mycosis of the respiratory tract].442

9. A model for treating avian aspergillosis: serum and lung tissue kinetics for Japanese quail (Coturnix

japonica) following single and multiple aerosol exposures of a nanoparticulate itraconazole suspension. 443

10. Inflammatory marker profiles in an avian experimental model of aspergillosis.444

11. Opportunistic infection of Aspergillus and bacteria in captive Cape vultures (Gyps coprotheres). 445

12. Emerging and reemerging diseases of avian wildlife.446

13. Assessment of Aspergillus fumigatus pathogenicity in aerosol-challenged chickens (Gallus gallus)

belonging to two lineages. 447

14. Aspergillosis in Japanese quail (Coturnix coturnix japonica). 448

15. . The occurrence of aspergillosis in flock of turkey poults.449

16. Serum protein electrophoresis by using high-resolution agarose gel in clinically healthy and Aspergillus

species-infected falcons. 450

17. Innate Immunity and the Role of Epithelial Barrier During Aspergillus fumigatus Infection.451

18. A longitudinal study on the incidence of mortality of infectious diseases of commercial layer birds in

Bangladesh.452

.

19. Aspergillus fumigatus and other thermophilic fungi in nests of wetland birds. 453

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20. Microsatellite typing of Aspergillus flavus from clinical and environmental avian isolates.454

.

21. Genetic structure of Aspergillus flavus populations in human and avian isolates.455

22. Aspergillus fumigatus from normal and condemned carcasses with airsacculitis in commercial poultry.456

23. Retrospective study of aspergillosis and other fungi in poultry (1980-2008).457

24. Serologic testing for aspergillosis in commercial broiler chickens and turkeys.458

25. Aspergillosis in gamebirds and ducks. 459

26. Macroscopic and Microscopic Diagnosis of Aspergillosis in Poultry. 460

27. Aspergillosis in gamebirds and ducks. 461

28. Spinal aspergillosis in pheasants. 462

29. Aspergillus fumigatus in Poultry. 463

30. Aspergillus fumigatus in Poultry. 464

31. Diagnosis and Treatment of Aspergillosis in An Ostrich Flock.465

32. Microsatellite typing of avian clinical and environmental isolates of Aspergillus fumigatus. 466

33. Articular Aspergillosis of Hip Joints in Turkeys. 467

34. Molecular epidemiology and virulence assessment of Aspergillus fumigatus isolates from white stork

chicks and their environment. 468

35. Aspergillus infections in birds: a review. 469

36. Embryonated eggs as an alternative infection model to investigate Aspergillus fumigatusvirulence. 470

37. Pyogranulomatous Aspergillosis in An Endemic Formosan Blue Magpie. 471

38. Aspergillosis in turkey poults.472

39. Clinical, mycological and pathological findings in turkeys experimentally infected by Aspergillus

fumigatus. 473

40. Efficacy of voriconazole in Japanese quail (Coturnix japonica) experimentally infected withAspergillus

fumigatus. 474

41. Development of a method for the identification using the polymerase chainre action of Aspergillus

fumigatus isolated from ostriches. 475

42. Aspergillosis in ostriches. 476

43. Aspergillosis and gastric impaction in an ostrich. 477

44. Aspergillus fumigatus infection in an ostrich (Struthio camelus). 478

45. Outbreak of severe disseminated aspergillosis in a flock of ostrich (Struthio camelus).479

46. Fatal Aspergillosis in an Ostrich (Struthio camelus) Predisposed by Pulmonary Haemangioma in the

Kingdom of Saudi Arabia.480

47. Aspergillosis outbreaks in ostrich flocks of eastern iran during 2010–2012 . 481

48. Outbreak of aspergillosis in a flock of adult ostriches (Struthio camelus)482

49. Pulmonary Aspergillosis in an Adult Male Ostrich (Struthio camelus): A Case Report.483

50. Disseminated aspergillosis in a Whooper Swan (Cygnus Cygnus). 484

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8. Laboratory diagnosis of aspergillosis

8.1. Direct microscopic examination

8.1.1. Wet preparations

One of the simplest approaches to diagnose invasive aspergillosis is to examine appropriate specimens

microscopically. Samples are prepared as wet preparation in potassium hydroxide solution and

examined unstained for fungal hyphae.

8.1.2. Stained preparations

Preparations can be stained by various stains eg, Calcofluor white, Uvitex 2B, and

Blankophor—which are water-soluble colourless dyes

Calcofluor white stain of an aspirate of the right side of the neck revealing a mold with septate hyphae, morphologically

consistent with Aspergillus., www.theaidsreader.com Skin scraping showing filamentous hyphae with blankophor www.life-worldwide.org

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8.1.3. Histopathological examination

Histopathological examination of biopsy or autopsy material provides a diagnosis of proven

invasive fungal infection (IFI). Tissue sections are stained by standard haematoxylin and eosin

staining should reveal the presence of Aspergillus hyphae but stains such as period acid Schiff

and Grocott’s silver should be carried out whenever a fungal infection is suspected.

Aspergillus hyphae seen in tissue sections tend to be narrow (1–3 um in diameter) and septate

a Septate, branching hypha (arrow) of Aspergillus flavus in corneal scrape material from a patient with keratitis (Gram stain; 1,000×). Leema G,

Kaliamurthy J, Geraldine P, Thomas PA - Mol. Vis. (2010), b.Direct microscopic examination of corneal material by the method of Gram staining revealed the presence of fungal hyphae.b.Aspergillus keratitis:

Histopathology of the biopsied mass showed a giant cell granuloma with surrounding numerous branching, septate hyphae. (H and E, x 40)

Akhtar K, Mehdi G, Shahid M, Alam A. Tropical India. Ann Trop Med Public Health 2012;5:357-9

a.Angioinvasion – Aspergillus hyphae invading a blood vessel, GMS stain, www.stritch.luc.edu

b. Bronchoscopic biopsy demonstrated septate hyphae with branching at 45o (methenamine silver stain). www.aspergillus.org.uk

125

a. Aspergillus fumigatus,hyphae in pulmonary lesion of a horse. PAS stain, www.microbeworld.org

b. Aspergillus fumigatus in a patient with chronic granulomatous disease showing a 45 degree

branching hypha within a giant cell. www.life-worldwide.org

8.1.4. Immunohistochemistry (IHC) or immunocytochemistry

Immunohistochemistry (IHC) or immunocytochemistry is a method for localizing specific

antigens in tissues or cells based on antigen-antibodies recognition. Specificity should derive

from binding of an antibody with its counterpart antigen at a light microscopic level. The use of

an enzymatic label antibody such as horseradish peroxidase allows visualization of the labeled

antibody using conventional light microscopy in the presence of a suitable chromogenic

substrate system. IHC is now applied to routinely formalin-fixed paraffin-embedded tissues

sections. Depending on the colorimetric developer, fungi stain dark brown or red, usually with a

counterstain of hematoxylin. Immunohistochemical methods have the potential advantage of

providing a rapid and specific identification of several fungi, allowing pathologists to identify

unusual filamentous and yeast-like infections and accurately distinguish them from confounding

artefacts.

Fixed tissue sections can be stained by immunohistochemical labelling using Aspergillus

monoclonal antibody.

126

Aspergillus Polyclonal Antibody. Immunohistochemistry with anti-NY-BR-1 Monoclonal Anti, www.pierce-antibodies.com

Anti-Aspergillus antibody , www.abcam.com, Immunohistochemistry (Frozen sections) - Anti-Aspergillus antibody (ab20419)Image from Grahl N

et al., PLoS Pathog. 2011 Jul;7(7):e1002145

. Anti-Aspergillus antibody. ab20419, staining Aspergillus (green) in lung tissue from a murine model of corticosteroid-induced invasive pulmonary

aspergillosis, by Immunohistochemistry www.abcam.com.

127

8.1.5. Molecular diagnosis in formalin fixed paraffin-embedded tissue

Two methods for extracting fungal DNA from paraffin wax embedded tissue sections, based on

the TaKaRa DEXPAT™

kit and QIAamp® DNA mini kit, were optimised and compared by

Paterson et al. (2003). DNA was amplified by PCR using pan-fungal probes, and detected by

Southern blot hybridisation using a high stringency method with a probe specific

for Aspergillus fumigatus and A flavus.

DNA Ligation Kit Selection Guide www.clontech.com

Takara EIA Kit Principles. www.clontech.com3

Biwas et al. (2008) demonstrated Aspergillus fumigatus fungus by PCR-based RFLP technique

from paraffin section of an eyeball of an eight- month-old child removed for endogenous

endophthalmitis.

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Diagnosis of Aspergillus fumigatus endophthalmitis from formalin fixed tissue. Biwas et al. (2008) www.scienceopen.com...

Sometimes a fungus is seen in a specimen or tissue and not cultured. It is important to identify

such fungi to genus and preferably species level. Fresh non-embedded tissues have shown that

sensitivity for PCR detection of fungi exceeds 95%, while the sensitivity of paraffin-embedded

samples is currently ~60%. If fungal infection is strongly suspected prior to biopsy or resection,

retention of some of the sample fresh (ie not placed in formalin) may facilitate aetiological

diagnosis. Only for Aspergillus spp. is a commercially available technique to determine the

genus of fungi found in tissue sections published, but this method does not separate species

of Aspergillus. The majority of the published assays target specific rRNA genes (18S or D1-2

of 28S) or the intervening internal transcribed spacer (ITS1 and ITS2).

8.2. Isolation and identification

Most Aspergillus sp. grow relatively rapidly (typically within 48 hr) and on most microbiology

media including both mycological media such as Sabouraud’s agar and blood agars used for

general bacteriological culture.Identification of cultures of most species Aspergillus is generally

straightforward by colony and microscopic morphology.

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Aspergillus restrictus Aspergillus candidus

Aspergillus. niger Aspergillus carbonarius

Aspergillu ustus Aspergillus sydowii

A. versicolor A. glaucus

Aspergillus equitis Aspergillus hollandicus

131

Aspergillus terreus Aspergillus flavipes

Aspergillus ochraceus Aspergillus flavus

Aspergillus parasiticus Aspergillus oryzae

Aspergillus wentii Aspergillus fumigatus

131

Aspergillus felis Aspergillus clavatus

Aspergillus nidulans Aspeergillus avenaceous

Aspergillus carneus, Aspergillus giganteus

Aspergillus indologenus Aspergillus deflectus

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8.3.Molecuar identification of aspergilli

Sequencing of genes, such as actin, calmodulin, ITS, rodlet A (rodA) and/or β-tubulin (βtub),

has been used to distinguish A. fumigatus from related species. Multilocus sequence typing can

alternatively be used for the identification of those related species, which is a strategy that also

involves sequencing of several gene fragments. A few other techniques, such as random

amplified polymorphic DNA, restriction fragment length polymorphisms and a new proposed

microsphere-based Luminex assay, may enable molecular identification of A. fumigatus without

sequencing. However, these methodologies are quite time consuming and labour demanding

and are thus impractical in most clinical labs. In addition, they can be very expensive when

employed to study collections of large numbers of isolates (Serrano et al., 2011).

Electrophoretic profile of several species of sectionFumigati. F1 - Aspergillus fumigatiaffinis, F2 - Aspergillus lentulus, F3 -Aspergillus

novofumigatus, F4 - Aspergillus unilateralis, F5 - Neosartorya hiratsukae, F6 - Neosartorya pseudofischeri, F7 - Neosartorya udagawae; AF1, AF2

and AF3 -Aspergillus fumigatus strains.

Alignment of β-tubulin sequences from species of section Fumigati. Serrano et al. BMC Microbiology 2011 11:82 doi:10.1186/1471-2180-11-82

133

Alignment of rodlet A sequences from species of section Fumigati.

Serrano et al. BMC Microbiology 2011 11:82

As reported by Balajee et al. (2009) the molecular identification scheme was as follows:

Genomic DNA was extracted from aspergilli grown for 48 h on Sabouraud dextrose agar plates

by using a DNeasy tissue kit (Qiagen, Valencia, CA). Universal fungal primers directed to the

ITS1-5.8S-ITS2 and the β-tubulin regions were employed to amplify DNA from

all Aspergillus isolates, as described previously. The resultant PCR amplicons were purified by

using an ExoSAP-IT enzyme system (USB Corporation, Cleveland, OH), according to the

manufacturer's instructions. Sequencing of both strands (with the same primers used for PCR

amplification) was performed with a BigDye Terminator (version 1.1) cycle sequencing kit

(Applied Biosystems). All cycle sequencing reactions were performed on a GeneAmp PCR

system 9700 thermocycler (Applied Biosystems) by using an initial denaturation at 96°C for 5

s, followed by 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. The products were

purified with an Agencount CleanSEQ system (Beckman Coulter, Beverly, MA), dried,

resuspended in 0.1 mM EDTA, and run on a 3730 DNA analyzer (Applied Biosystems) using

of the protocols supplied by the manufacturer. The resultant nucleotide sequences were edited

by using the Sequencher program (Gene Codes Corporation, Ann Arbor, MI) and aligned by

using the program CLUSTAL W. Gene sequences derived from the ITS1-5.8S-ITS2 and the β-

tubulin regions of all the Aspergillus isolates were compared with sequences in the GenBank

database to identify isolates to the species complex level and to the species level within the

complex.

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Rapid detection of Aspergillus spp. DNA from respiratory and serum samples using Real-Time PCR

Effective extraction of fungal DNA from respiratory samples

Aspergillus niger PCR Detection Kit [32900] : Norgen Biotek Aspergillus niger PCR Primer Set and Controls

Aspergillus fumigatus PCR Detection Kit New rtPCR kit to detect Aspergillus infection,

135

8.4. Serological techniques

8.4.1. Antigen detection in the diagnosis of invasive aspergillosis

In his excellent review on the diagnosis of invasive aspergillosis, Verweij (2005) mentioned

that Aspergillus species are known to release exo-antigens during growth in vitro and in vivo.

Low levels of antigens may be present in body fluids of patients with

invasive Aspergillus infection such as serum, urine and CSF. A number of methods have been

developed and evaluated which employ antibodies directed against antigens produced by

Aspergillus. These methods include:

1. latex agglutination: Detection limit 15 ng/ml

2. radioimmunoassay: Detection limit 10 ng/ml

3. ELISA inhibition : Detection limit 5 ng/ml

4. sandwich ELISA : Detection limit 1 ng/ml

8.4.1.1. Detection of Aspergillus galactomannan

Two antigen detection kits are commercially available and have been evaluated in several

institutes.

The latex agglutination (LA) test (Pastorex Aspergillus, Sanofi Diagnostics Pasteur, Marnes-La-

Coquette, France) employs the rat monoclonal antibody EB-A2 to

detect Aspergillus galactomannan.

Recently, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed

(Platelia® Aspergillus, Sanofi Diagnostics Pasteur) which employs the same monoclonal

antibody as the latex agglutination test. The detection limit of the sandwich ELISA was lowered

10-fold by employing the monoclonal antibody as a captor and detector, thus allowing 0.5 to 1.0

ng/ml of galactomannan to be detected. Several investigations have shown that the sensitivity of

the sandwich ELISA was higher that that of the LA-test.

Detection of Aspergillus galactomannan antigen Aspergillus Galactomannan detection kits

136

The assay was originally designed for serum, but has also been used on bronchoalveolar lavage

fluid (BAL), sputum, CSF, pleural fluid, pericardial fluid and tissue. The generally accepted

cutoff in serum for positives is 0.5.

The higher the OD the greater the likelihood of invasive aspergillosis. No cutoff has been

defined for any other specimen type, but in BAL fluids different authors have recommended

cutoffs from 1.0 to 2.0. This variation partly reflects different patient populations and different

dilution factors during bronchoalveolar lavage.

Overall specificity of Aspergillus antigen detection using the galactomannan EIA is

approximately 80%. Several antibiotics and other patient factors may yield false positives.

Several beta-lactam antibiotics are manufactured using fungal fermentation as s first step. This

often results in carryover of galactomannan into the antibiotic preparation and ‘false positive’

galactomannan results that can persist for some days. The most common antibiotics to be

implicated are piperacillin/tazobactam (Tazocin) False positive reactivity of the ELISA was

found in up to 8% of serum samples and occurred especially with samples obtained within 30

days of BMT or cytotoxic chemotherapy, and in premature infants. False positive reactivity in a

single serum sample can be overcome by performing ELISA with additional serum samples

and most investigators now define positive as two separate positive samples. If positive, other

tests and procedures are required to localise and confirm the presence of infection.

8.4.1.2. Detection of 1,3 β-D-Glucan (BDG)

As mentioned by Barton (2013), the cell walls of Aspergillus contain relatively large amounts

of glucan of which 1,3 -D-glucan forms a large part. In vitro analysis of growing A.

fumigatus showed that, like GM, BDG is released during logarithmic growth, though slightly

later.

BDG can be detected through a pathway in the Limulus amoebocyte lysate (LAL) coagulation

cascade that has traditionally be used for the detection of bacterial endotoxin. Whilst endotoxin

interacts with LAL via factors B and C, factor G in the LAL interacts with BDG activating a

proclotting enzyme which can then cleave a chromogenic substrate to generate a product

detectable by spectrophotometry down to 10 pg/mL

There are four different commercial assays for the detection of BDG in clinical specimens. The

Associates of Cape Cod Fungitell kit uses amoebocyte lysates from Limulus polyphemus while

the Seikagaku Fungitec-G test uses reagents from Tachypleus tridentarius as does the Wako β-

Glucan test. Recommended cutoffs for reporting positive results vary between assays.

Manufacturers of the Fungitell assay requires >80 pg/mL to be detected for a positive with 60–

79 pg/mL Cutoffs for the T. tridentatus based assays are lower at 20 pg/mL and even lower

137

cutoffs of 11 pg/mL or two sera at 7 pg/mL have been proposed. A fourth assay, GKT-25-M has

been reported in the Chinese literature.

Fungitell® Assay, www.bioresearchonline.com Fungitech G-test, Seikagaku Co, www.pfdb.net

8.4.2. Aspergillus antibody detection

There are multiple marketed IgG antibody tests to detect A. fumigatus antibodies. A smaller

number of less well used and often usually incompletely validated tests are available for other

species of Aspergillus including A. flavus, A. terreus, A. niger, A. versicolor and A. clavatus. All

testing is on serum.There are multiple marketed IgG antibody tests to detect A.

fumigatus antibodies, including Immy,Serion/Virion, Bioenche, BioRad, Thermofisher, Elitech,

and Microgen.

8.4.2.1. Aspergillus Immunodiffusion test

The ID test is very helpful in the diagnosis of ABPA and aspergilloma, the two forms of

aspergillosis observed in immunocompetent individuals where infection can be correlated

with a rise in specific antibodies. Precipitins can be found in >90% of patients with

aspergilloma and in 70% of patients with ABPA.

In contrast to immunocompetent hosts, growth of Aspergillus in the tissues of an

immunosuppressed host does not correlate with an increase in anti-Aspergillus antibody

titers. The parallel use of ID and CF tests and clinical data is an effective means for

specific diagnosis of Aspergillosis Precipitins can be found in >90% of patients with

aspergillomas and in 70% of patients with ABPA.

The greatest number of Aspergillosis cases may be detected by the use of A. fumigatus,

A. flavus, A. niger, and A. terreus antigens in separate ID tests performed at the same

time. Precipitins are less frequent in patients with invasive Aspergillosis.

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In fact, the presence of anti-Aspergillus antibody in immunocompromised individuals is

more likely to represent antibody formed before the onset of immunosuppressive therapy

rather than as a result of invasive infection.

An increase in antibody titer at the end of immunosuppression is indicative of recovery

from IA, whereas absence of an antibody titer or declining antibody levels suggest a poor

prognosis. Thus, antibody detection can be used prognostically but not diagnostically for

IA.

Two or more distinct precipitin lines should be formed when A. fumigatus reference

antiserum is allowed to react with A. fumigatus antigen. One or more distinct precipitin

lines should be formed when A. flavus, A. niger, or A. terreus reference antiserum is

allowed to react with the homologous antigen.

Because the Aspergillus antigens of diagnostic significance have not been defined, any

precipitin band (whether identity, partial identity, or non-identity) is significant and the

number of bands should be reported.

The demonstration of one or more precipitins indicates infection, including aspergilloma.

Precipitating antibodies are often detectable in serum from patients with ABPA.

Although one or two precipitins can occur with any clinical form of Aspergillosis, the

presence of three or more bands is invariably associated with either an aspergilloma or

IA. The test may be negative for some patients receiving long-term antifungal or

corticosteroid therapy. When used with reference antisera the ID test is 100% specific.

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8.4.2.2. Aspergillus Antibody, Complement Fixation test

Single titers >=1:32 are indicative of recent infection. Titers of 1:8 or 1:16 may be indicative

of either past or recent infection, since CF antibody levels persist for only a few months. A

four-fold or greater increase in titer between acute and convalescent specimens confirms the

diagnosis. Sensitivity of the CF test for aspergillosis is lower than that of the immunodiffusion

test. Crossreactions may occur in patients with histoplasmosis and coccidioidomycosis. This test

is approved for New York patient testing.

Comments of Richard C. Barton (2013) on methods of laboratory diagnosis of aspergillosis

Of the wide variety of methods reviewed of diagnosing invasive aspergillosis, direct

microscopy and histopathology are undoubtedly the most subjective, skilled, and labour

intensive.

Though histopathological detection of Aspergillus of tissue will remain an essential

capacity and help define the highest level of certainty in diagnostic criteria, less invasive

and more rapid methods have already overtaken this approach in most settings. Some will

also consider that traditional culture-based methods may also be superceded by DNA and

antigen detection methods.

However, in the recent US PATH registry update culture of Aspergillus for the diagnosis

of IA was still the most frequent laboratory approach. Furthermore, cultures

of Aspergillus allow further analysis such as susceptibility to testing, recent

developments in direct molecular detection of resistance notwithstanding, and the

possibility of molecular typing in epidemiological investigations. Furthermore, new

species of Aspergillus causing invasive disease continue to be uncovered by culture

whose applicability to serological and molecular detection cannot be assumed. Culture of

respiratory specimens in clinical laboratories is unlikely to be abandoned in the near

future and culture of Aspergillus will remain an important if generally insensitive

approach to the diagnosis of IA.

The detection of galactomannan in clinical specimens is firmly established as the test of

choice for any laboratory providing diagnostic services for patients at risk of IA. Serum

detection remains a key approach and amenable to serial measurements whilst BAL

material appears to have a higher sensitivity but is often harder to obtain. False positives

are less of an issue than is often claimed with antibiotic sources of GM now rarely seen

and neonatal samples likely to account for a tiny fraction of samples analysed. Sensitivity

is clearly affected by antifungal treatment, though the biological basis for this is unclear,

this remains the main limitation to the use of the assay particularly in patients undergoing

antifungal prophylaxis. A plethora of meta-analyses have confirmed the clinical value of

GM testing and at least some analyses of GM serum kinetics point to the potential for this

141

assay to guide therapy beyond diagnosis. The BDG assay has yet to be widely adopted as

a diagnostic tool, perhaps suffering from the need to also require more specific diagnostic

methods to target therapy in positive patients. Meta-analysis suggests that problems with

specificity were probably overestimated in initial studies and sensitivity is comparable

with GM. The BDG assay will probably find its place in some centres in carefully

designed care pathways where the negative predictive value can be used to reduce

empiric therapy. The PCR assay to detect Aspergillus DNA held a large amount of

promise but has been limited up until recently by a lack of standardisation. The scientists

of EAPCRI have nobly met the challenge of technical diversity in PCR assays and

provided meticulously researched standard methods for extraction of DNA from whole

blood and serum.

9. Treatment of aspergillosis

As mentioned by Mayo Clinic Staff, the following guidelines are recommended:

1. Observation. Aspergillomas often don't need treatment, and medications aren't usually

effective in treating these fungal masses. Instead, aspergillomas that don't cause

symptoms may simply be closely monitored by chest X-ray.

2. Oral corticosteroids. The goal in treating allergic bronchopulmonary aspergillosis is to

prevent existing asthma or cystic fibrosis from becoming worse. The best way to do this

is with oral corticosteroids. Antifungal medications by themselves aren't helpful for

allergic bronchopulmonary aspergillosis, but they may be used in combination with

corticosteroids to reduce the dose of steroids and improve lung function.

3. Antifungal medications. These drugs are the standard treatment for invasive pulmonary

aspergillosis. Historically, the drug of choice has been amphotericin B, but the newer

medication voriconazole (Vfend) is now preferred because it appears more effective and

may have fewer side effects. All antifungals can cause serious problems, however,

including kidney and liver damage, and they frequently interact with other medications

given to people who have weakened immune systems.

4. Surgery. Because antifungal medications don't penetrate aspergillomas very well,

surgery to remove the fungal mass is the first-choice treatment when bleeding from the

mass in the lungs occurs. But the surgery is risky, and your doctor may instead suggest

embolization. In this procedure, your doctor, usually a radiologist, threads a small

catheter into the artery that supplies blood to the cavity containing the fungus ball, and

injects a special material that clogs the artery. Though this procedure can stop massive

bleeding, it doesn't prevent it from recurring, so it's generally considered a temporary

treatment.

141

Therapeutically Important Antifungal Agents (Khan, 2013)

1. Polyene Antibiotic (Amphotericin B):

Amphotericin B (AmB) is a polyene (containing multiple double bonds) macrolide

antibiotic originally isolated from a Streptomyces species. It binds to ergosterol, a

component of the fungal cell membrane. Binding to ergosterol destroys fungal

membrane integrity, resulting in leakage of cellular content and then cell death.

Being highly insoluble in water and in its original formulation, AmB was

complexed with deoxycholate.

AmB is poorly absorbed orally and requires parenteral administration. Following

IV administration, the drug is released slowly and is highly bound to protein

(>90%). AmB accumulates significantly in the liver and spleen, and it is present in

synovial, pleural, and peritoneal fluid. Central nervous system (CNS) penetration is

minimal. AmB has a half-life of 15 days and is excreted very slowly from the

kidney.

AmB adverse effects (AEs):

1. Infusion reactions include fever, shaking, chills, hypotension, and

tachypnea.

2. Renal toxicity is characterized by renal ischemia, hypokalemia, and

tubular acidosis.

3. Reduction of the production of erythropoietin (a glycoprotein that

stimulates bone marrow to produce RBCs) in the kidney.

AmB should not be administered concurrently with other nephrotoxic agents

(aminoglycosides, nonsteroidal anti-inflammatory drugs).

Serious AEs have prompted the development of lipid formulations of AmB. Lipid

formulations that minimize the renal toxicity of AmB are AmB colloidal dispersion

(ABCD), liposomal AmB (L-AmB), and AmB lipid complex (ABLC). These

formulations have largely replaced AmB.

2. Azoles (Voriconazole, Posaconazole, Itraconazole):

Chemically, azoles are triazole derivatives. These agents act by inhibiting fungal sterol-14-

alpha-demethylase, a cytochrome-dependent enzyme associated with ergosterol synthesis.

Inhibition of this enzyme enables the accumulation of methylsterol, thus impairing other

cellular functions. The main azoles are:

1. Voriconazole is formulated for oral and parenteral administration. Minimally bound to

plasma proteins, it is metabolized in the liver by CYP enzymes (CYP3A4, CYP2C9,

CYP2C19) and also inhibits these enzymes. Voriconazole is excreted renally. Transient

142

visual disturbances (blurred vision, changes in color vision or brightness) are associated

with this drug.

2. Posaconazole is administered orally as a suspension. Its absorption is increased with

food. Posaconazole is metabolized by glucuronidation and is excreted in the feces. The

drug inhibits CYP3A4 and is a substrate of P-glycoprotein (PgP). AEs include headache

and GI distress.

3. Itraconazole i. Itraconazole is available in oral and parenteral formulations. It is absorbed

well orally in the presence of food and low pH and is excreted fecally.

ii. Itraconazole is significantly bound to plasma proteins and is metabolized by

CYP3A4. Hydroxyitraconazole is an active metabolite of the parent drug.

iii. Itraconazole inhibits CYP enzymes and PgP, and it results in liver toxicity.

iv. Itraconazole has a negative inotropic effect on the heart and should not be

used in patients with ventricular dysfunction or congestive heart failure.

4. Echinocandins (Caspofungin, Micafungin):

These agents inhibit 1,3-beta-glucan synthase, an enzyme responsible for

fungal cell wall synthesis.

i. Echinocandins have selective toxicity against fungi because mammalian

cells do not possess a cell wall.

ii. Echinocandins are semisynthetic lipopeptides and are administered IV only.

These agents are significantly bound to plasma proteins (>95%), and CNS

penetration is inadequate.

iii. Echinocandins are excreted predominantly via the feces. Echinocandins do

not induce or inhibit CYP enzymes, nor do they interact with PgP. As a

result, the potential for drug interactions with other therapeutic agents is

negligible.

iv. Echinocandins are usually well tolerated, and the most common AEs are

elevated liver enzymes and creatinine (Cr), histamine-mediated effects (rash,

pruritus, facial swelling), GI distress, headache, and pyrexia.

143

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