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1. Chapter Five : Systemic
Mycoses

2. Acknowledgments
• Addis Ababa University
• Jimma University
• Hawassa University
• Haramaya University
• University of Gondar
• American Society for Clinical Pathology
• Center of Disease control and Prevention in Ethiopia.
2

3. Outline of presentation
• Objective
• Blastomycoses
• Coccidiodomycoses
• Paracoccidiodomycoses
• Histoplasmosis
• Summary

4. Objective
• Upon completion of this chapter , the students are able
to :
• List the clinical important pathogens of systemic
mycoses
• Describe the type of suitable specimen
• Mention the various Laboratory techniques that used to
diagnose systemic mycoses

5. Systemic mycoses
Introduction
• Systemic mycoses are fungal diseases that may affect
internal organs and may disseminate to multiple sites of
the body
• some times known as deep mycoses.
• They are difficult to eradicate and many patients with
deep mycoses die in spite of extensive medical care
especially when the central nervous system is involved.

6. Introduction cont’d
• Systemic mycoses occur in three disease forms:
• acute self limited pulmonary (asymptomatic to
severe)
• chronic (pulmonary or disseminated)
• disseminated.

7. Intro. cont’d…
• Since many fungi that cause superficial or cutaneous
mycoses are skin parasites by nature, the source of
infection is usually humans or animals.
• The disease is transmitted directly from an infected
person to a susceptible host.
• The agents of subcutaneous and systemic mycoses,
however, are normally saprophytic fungi growing in the
soil.
• These fungi do not need human or animal hosts for
survival because the primary source of infection is soil.

8. Introduction cont’d
• Humans acquire these mycoses only when the spores of the
soil organisms are either inhaled or introduced in to the body
through a break in the skin.
• The systemic fungi include
• Blastomyces dermatitis (blastomycosis),
• Coccidioides immitis (coccidioidomycosis),
• Histoplasma capsulatum (Histoplasmosis),
• Paracoccidioides brasiliensis (paracoccidioidomycosis).
• These various systemic mycoses and their respective
etiologic agents will be considered separately despite their
similarities.

9. 5.1. Blastomycosis
Definition
• Blastomycosis refers to the disease caused by the
endemic, saprophytic, dimorphic fungus Blastomyces
dermatitidis.
• It grows at room temperature as a white or tan mold but
grows within the host or at 37 0c as budding, round yeast
like cells.
• The fungus can be identified on the bases of its
appearance, its dimorphism the small spores borne as
hyphae of the mold form, or the results of nucleic
hybridization.

10. Epidemiology
Geographic Distribution:
• The disease is endemic in South Eastern and South
Central states of North America, along the Mississippi
and Ohio Rivers.
• Source of infection:
• Outbreaks have been associated with occupational or
recreational activities around streams or rivers with high
content of moist soil enriched with organic debris and/ or
rotting wood.

11. Pathogenesis and clinical picture
• Blast mycosis is acquired via inhalation of the conidia,
which transforms in to the yeast form once in the lungs.
• After 30 to 45 days an acute pulmonary disease
indistinguishable from a bacterial pneumonia may occur.
• However, at least 50% of primary infections are
asymptomatic.
• Inmost cases the disease manifest during a chronic and
indolent phase that may affect the lungs, the skin, the
bones, the genitourinary tract and other
reticuloendothelial system.

12. Pathogensis cont’d
Fig. Cutaneous blastomycosis. (Courtesy of Dr M. James,Royal Berkshire Hospital,
Reading, UK.)

13. Patho cont’d
• A few patients have acute, self-limited pneumonia; fever
productive cough, myalgia, and malaise are common
and are usually resolve within a month.
• The infection has three forms:
 Acute pulmonary blastomycosis, even though it may
resolve with out treatment; however treatment is
often used.
 Chronic pulmonary blastomycosis usually has
granulomatous lesions in the upper lobe often
misdiagnosed as carcinoma.

14. Patho cont’d
Disseminated blastomycosis is a progressive disease that
follows hematogenous spread from unresolving pulmonary
lesions by macrophages to remote sites, most commonly
skin and bones.

15. Laboratory Diagnosis
Direct Examination
• Clinical material, such as prostate fluid, or tissue, is
examined in 10% KOH.
• It reveals the characteristic yeast form of B. dermatitidis.
• The fungus usually occurs as thick- walled, globose
yeast that measures 8-15µm in diameter.
• Each blastoconidium is attached to the parent cell by
broad base.

16. Lab diagnosis cont’d
• Owing to their size, the yeasts of B. dermatitidis could be
confused with C immitis or C. neoformans under some
circumstances.
Culture and identification
• Culture is performed in the laboratory onto sabouraud
glucose agar, brain heart infusion agar, yeast extract-
phosphate agar, and a medium with cycloheximide, and
then incubated.
• The culture should be kept 4 weeks (7-28 days) before
discarding as negative:

17. Lab dx cont
Colonial appearance
• Mycelia phase: on sabouraud glucose agar, colony first
white, waxy, yeast like and later cottony with white aerial
mycelium; turns tan to brown with age.
• Yeast phase: on brain heart infusion agar, colony cream
to tan, waxy and wrinkled; inhibited chloramphenicol or
cyclohexmide.

18. Lab Dx cont’d
Microscopic appearance
• Mycelia phase: delicate, septate hyphae with round or
pyriform conidia borne singly on conidiophores or directly
on hyphae.
• Yeast phase: thick-walled large yeast cells with single
bud on a broad base

19. Lab Dx cont’d
Fig. Giemsa stain of Blastomyces
dermatitidis showing broad-based
budding yeast.
Blastomyces dermatitidis mould phase

20. Serologic Tests
Immunodifussion test: antigen - yeast culture filtrate
• Serologic tests for specific antibodies against yeast-
phase antigen A using immunodiffusion technique is of
some diagnostic value but more importantly serves as
indicator of the effect of therapy; antibodies disappear a
few months after successful treatment.
• However anti-A antibody appears after infection is not
known.
• The specificity of the ‘K’ antigen remains to be
determined.

21. Serology cont’d
• Complement Fixation test: antigen – yeast form
• Titers of 1:8 to 1: 16 ---------- suggestive of active
infection
• Titers of 1:32 or greater ------ indicative of the
infection
Note; cross-reaction occurs in patients having
coccidiodomycosis or histoplasmosis ; however titers
are usually lower

22. Serology cont’d
Skin Tests
• Skin tests with blastomycin are usually of no
diagnostic value because the test is often
falsely negative or falsely positive due to
cross reactivity with other pathogenic fungi,
such as Histoplasma capsulatum

23. 5.2. Coccidioidomycosis (Desert fever,
valley fever, San Joaquin fever)
Definition
• Coccidioidomycosisis, which is caused by C. immitis,
is also referred to as valleys fever or San Joaquin
fever because of the geographic distribution of C.
immitis and the associated area of occurrence of this
disease.
• It is seen often in the San Joaquin valley of California
that it has gained the common name of “valley fever or
San Joaquin Fever”.

24. Coccidioidomycosis cont’d
• The organism reproduces in a host tissue by forming
small endospores within mature spherules.
• After rupture of spherule, the released endospores
enlarge and repeat the cycle.
• The agent has two forms, growing as a white fluffy mold
on most culture media but as a non-budding spherical
form (a spherule) in host tissue or under special
condition.

25. Epidemiology
 Geographic Distribution: Coccidioidomycosis is
endemic in the semi-arid region of Central America,
Southwestern U.S.A., small portion of South America,
Mexico, which offer the unique climate essential for
the growth, and dissemination of C. immitis.
 Although the geographical distribution is well defined,
cases of coccidioidomycosis may be seen in any part
of the world because of the case of travel.

26. Epidemiology cont’d
• Source of infection:
• The infective fungal spore is found in the soil.
• Patients with a history of exposure to soil acquire the
disease by inhalation of wind borne arthrospores from
dust generated when contaminated soil is agitated.

27. Pathogenesis and clinical picture
• The usual course of primary pneumonia is complete healing,
although an area of pnuemonitis (detected on radiographs)
may heal by formation of a coin like lesions called a
coccidioidoma.
• Less commonly, a single thin-walled cavity remains as a
chronic sequela in areas of consolidation.
• A chronic, progressive, granulomatous disease occurs in less
than 1% of those infected, especially immuno suppressed
individuals; this is granulomatous less resemble the tubercles
of tuberculosis and cavitations may occur.

28. Pathogenesis cont’d
• The infection may disseminate to different parts of the
body, including bones, viscera, and the CNS;
• however, the gastrointestinal tract is remarkably resistant
to infection
• The acute and most common forms of the disease
resemble influenza.
• Approximately 60% of the cases remain asymptomatic
and self-limiting.

29. Pathogenesis cont’d
• The initial symptoms of pulmonary infection consist of
cough, fever, and malaise.
• One to two weeks later, about 3 to 5% of patients
develop complications attributable to a hypersensitivity
reaction, the most frequent being skin lesions (erythema)
or joint symptoms (desert rheumatism).
• The symptoms of a chronic thin-walled cavity include
cough or hemoptysis in half of cases; the other half is
asymptomatic.
• Chronic pulmonary coccidiodomycosis cause cough,
sputum production, variable degree of fever, and weight
loss

30. Laboratory Diagnosis
Direct Examination
• Sputum, lung biopsy, pus from lesions and other clinical
specimens can be examined microscopically by means
of a KOH wet mount slide preparation, which reveals
characteristic spherules.
• It appears as large non-budding, thick-walled spherical
cells of various sizes (2 to 5µm in diameter) containing
small endospores or granular material.

31. Lab dx cont’d
Coccidioides immitis mould phase
Coccidioides immitis spherule
Fig. Coccidioidomycosis tissue
section. Spherules of various
sizes are stained black with
GMS. In the top left-hand part
of the slide

32. Culture and identification
Colonial appearance
• Mycelia phase: Culturing on Sabouraud’s glucose agar,
mature colonies may appear with 3 to 5 days of incubation.
• C. immitis often appear as delicate, some portion of the
colony will exhibit aerial hyphae, where as others will have the
hyphae adherent to the agar surface.
• It must only be carried out in a mycology laboratory with
facilities for the safe handling of cultures.
• The arthroconidia produced by C. immitis molds are highly
infectious.

33. Culture cont’d
• Yeast phase: on brain heart infusion test have similar
appearance to mycelia phase.
Microscopic appearance
• Mycelia phase: coarse, septate, branched hyphae that
produce thick-walled, barel-shaped, rectangular
arthroconidia that alternates with empty disjucture cells.
• Yeast phase: large, round, thick-walled spherules with
endospores as observed in tissue and direct
examination. Not true yeast.

34. Serologic tests
 Serologic testing on spaced serum specimens is highly
valuable for the diagnosis and prognosis of
coccidioidomycosis.
Tube precipitin
• Tube precipitin is the most sensitive test result, which
becomes positive 1 to 3 weeks after onset of infection,
with about 80% of cases positive by 2 weeks but the test
usually reverts to negative by 6 months after onset of
infection.

35. Latex particle agglutination
• antigen - coccidiodin
• Latex particle agglutination and immunodiffusion tests
are other serologic tests used as screening tools in
endemic areas and can detect 93% of cases.
• It is slightly more sensitive than tube precipitin, but with
6% to 10% false positive results.

36. Complement fixation tests
• Antigen - coccidiodin
• Complement fixation tests are also widely used,
especially for spinal fluid specimens for confirmation and
quantification of serum antibody detected by screening
tests.
• In the CSF specimen it detects more than 90% of active
coccidioidomycosis infection, whereas the tube precipitin
test is not reliable on spinal fluid.
• Titer of 1:2 to 1: 4 ------- active infection; low titers should
be followed by repeat testing at 2 to 3 weeks intervals.

37. CFT cont’d
• Titer of > 1:16 ---------- usually indicates active infection.
• Titer of 1:32 or higher--- usually reflects disseminated
disease; a continuing rise in titer above 1:32 signifies
progressing infection and poor prognosis.
Note: Cross-reaction occurs in patients with histoplasmosis
and negative reaction result occurs in patient with
pulmonary lesions.

38. Skin Tests
• Skin test is used to screen a population for individuals
who are infected with a pathogen but who have not
developed clinical symptoms.
• It is very useful, being equally as sensitive as serologic
tests.
• Skin test does not produce an antibody response that
would interfere with the other tests.
• Generally skin tests are available for the diagnosis of
coccidioidomycosis, using occidioidin; an antigen derived
from C. immitis.

39. 5.3.Paracoccidioidomycosis(paracoccidi-
oidl granuloma, Lutz-splendore-almeida’s
disease)
Definition
• Paracoccidioidomycosis formerly called South American
blastomycosis, which is a chronic granulomatous
systemic mycosis caused by a saprophytic P.
brasiliensis.
• A dimorphic fungus, P. brasiliensis grows as budding
yeast in tissue but may grow as either yeast or a mold on
culture medium.
Epidemiology
• Geographic Distribution

40. Paracoccidiods Cont’d
• Paracoccidioidomycosis is found in Brazil and also in the
other South American countries,
• Central America and Mexico, but its extreme indolence
may delay its recognition until many years after the
patient has left the endemic area.
• Source of infection: The exact mechanism by which
paracoccidioidomycosis is acquired is unclear; however,
it is speculated that is the origin is pulmonary which is
acquired by inhalation of organisms from the
environment.

41. Pathogenesis and clinical picture
• The infection begins as primary pulmonary infection. It is
often asymptomatic and then disseminates to produce
ulcerative lesions of the mucous membrane.
• Ulcerative lesions are commonly present in the nasal
and oral mucous, gingival, and less commonly in the
conjunctiva.
• Lesion occurs most commonly on the face is associated
with Oral membrane infection.
• The lesions are characteristically ulcerative, with
serpinous active border and a crusted surface.

42. Pathogenesis cont’d
• Pulmonary infection is seen most often, and progressive
chronic pulmonary infection is found in approximately
50% of the cases.
• Dissemination to other anatomic site, including the
lymphatic system, spleen, intestine, liver, brain
meninges, and adrenal glands occur in some patients
• Common signs include indurated ulcers of mouth,
oropharynx, larynx, and nose; enlarged and draining
lymph nodes;lesions of the skin and genitalia; and
productive cough, weight loss, and some times fever.

43. Laboratory Diagnosis
• Diagnosis rests on microscopic, examination of
specimens for multiple budding yeast forms, serologic
tests, and culture with or without antibacterial agents.
Direct Examination
• The yeast appears large, round to oval measuring 10-
40µm in diameter are usually recognized in direct wet
mount microsocopic examination of sputum, mucousal
biopsy, and other exudates.

44. Microscopy cont’d
• large, round to oval, thick-walled yeast cells with multiple
buds, which attach to mother cells by narrow
constrictions; resemble a “ ship’s wheel” will be seen
under microscope.

45. Culture and identification
 Colonial appearance
 Colonies of P. brasiliensis grows very slowly (21 to 28
days) on culture and has wrinkled and moist
appearance
Mycelia phase: on Sabouraud glucose agar, white,
leathery colony; turns tan-brown with age; short aerial
mycelium.
Yeast phase: on blood agar, cream to tan, moist, wrinkled
colony; turns waxy with age.

46. Culture cont’d…
Microscopic appearance
Mycelial phase: small, septate, branched hypha with
intercalary and terminal chlamydoconidia; few periform
micoconidia.
Yeast phase: large, round to oval, thick-walled yeast cells
with multiple buds, which attach to mother cells by narrow
constrictions; resemble a “ ship’s wheel”.
Serologic Tests
• The immuno-diffusion test for specific antibodies is
highly valuable for diagnosis, prognosis and disappears
with regression.
• It is also valuable for identifying the fungus.

47. 5.4. Histoplasmosis (Darling ‘s disease,
Reticuloendotheliosis, Reticulo-enthelial
cytomycosis)
• Histoplasmosis is the most common type of systemic
fungal infection caused by Histoplasma capsulatum
(classic or small form) and Histoplasma duboissi (African
or large form).
• Histoplasma capsulatum is a dimorphic fungus that
grows as a mold in nature or on sabourauds agar at
room temperature.
• Hyphae bear both large and small spores, which are
used for identification.

48. Histoplasmosis cont’d
• Samuel Darling, who discovered H. capsulatum in
around 1900, so-named the organism because he
mistook it for a capsulated animal parasite.
• Since it is now known to be a non- capsulated
saprophytic soil fungus, its present name is obviously
inappropriate. Histoplasmosis, caused by H. capsulatum,
shared many characteristics with coccidioidomycosis.
• Both diseases extend over a spectrum from acute self-
limited to chronic, granulomatous infection, and both are
endemic in man. However, histoplasmosis occurs
worldwide.

49. Epidemiology
• Geographic Distribution
• Histoplasma capsulatum
• infection occurs in many parts of the world,
• notably in the USA along the Mississippi river, and in
many areas along the East cost
• It is common in areas with optimal growth conditions like
accumulation of bird or bat droppings enrich the soil with
nutrient (eg. Nitrogen) that encourages the growth of H.
capsulatum.

50. Epidemiology cont’d
• Histoplasma duboissi
• It occurs in tropical Africa.
• It is often referred to as large cell histoplasmosis
because the intracellular yeast forms are three to four
times larger than H. capsulatum.
Source of infection
• Histoplasmosis is acquired via inhalation of the infective
units from the environment; particularly soil enriched by
dropping of certain birds and bats that are deposited in
the lungs.

51. Pathogenesis and clinical picture
• Infection begins with small primary focus of lung infection
much like the early lesions of tuberculosis.
• The causative agents are ingested by alveolar
macrophage in the lung.
• These macrophages carry the fungi to nearby lymph
nodes, usually the hilar nodes.
• The fungi multiply within the node tissue and ultimately
are released into a circulating blood, from which they go
on to seed other organs or destroyed by the body
defenses.

52. Pathogenesis cont’d
• The initial lung infection is usually in apparent.
• Clinical disease develops in some 5% of those infected.
• The majority of the infections are either asymptomatic or
mild, and the diagnosis is elusive cough fever, malaise,
and chest X-ray findings of hilar adenopathy with or with
out one or more areas of pneumonitis is typical features.
• Chronic pulmonary histoplasmosis is characterized by a
gradual onset (over weeks or months) of increasing
productive cough, weight loss and some times night
sweats.

53. Pathogenesis cont’d
• Acute disseminated histoplasmosis may be mistaken
milary tuberculosis.
• Common findings include fever, hepatosplenomegaly,
lymphadenopathy, Jaundice, anemia leukocytopenia and
thrombocytopenia
• In such individuals the infection may occur either as an
acute disease, as a chronic cavitary disease or as a
severe blood born disseminated disease

54. Pathogenesis cont’d
• Acute pulmonary histoplasmosis ranges from
asymptomatic to a severe self-limiting disease in which
symptoms resemble a mild cold
• Chronic pulmonary histoplasmosis is the outcome of the
continued progression of the initial infection or the result
of reactivation of an old quiescent lesion; cavitation is
frequent.
The disease resembles chronic pulmonary tuberculosis
clinically.

55. Pathogenesis cont’d
• Disseminated Histoplasmosis is commonly a generalized
disease of the reticuloendothelial system that presents
lesions in almost every organ of the body.
• It is rare, occurring mostly in individuals with patients of
depressed cellular immune response (e.g. Patients with
AIDS, T-cell defects, or lymphoma)
• In children younger than age one year who appear to
have reticuloendothelial defect (fulminant disease of
child hood)

56. Laboratory Diagnosis
Direct Examination
• The direct microscopic examination of respiratory tract
specimens is usually unsatisfactory for the detection of
H.capsulatum and H. duboissi.
• The organism however may be detected when
examining wrigght’s or Giemsa- stained specimen of
bone marrow or peripheral blood.
• The yeast can be seen in the cytoplasm of endothelial
and mononuclear cells.

57. Lab dx cont’d
• H. capsulatum is found intracellular with in mononuclear
cells as small, rounded oval yeast cells 1 to 4 µm in
diameter.
• They require differentiation from the amastigotes of
leishmania donovani, which can also be found in
mononuclear cells.
• H. duboissi appear as large, round to oval cells
measuring 7-15µm in diameter.
• The cells may appear nucleated.

58. Lab dx cont’d
Giemsa stain of Histoplasma Capsulatum
Histoplasma capsulatum mould phase
showing tuberculate macroconidia.
R.J.Hay&M.k,Moore, mycology

59. Lab Dx cont’d
Culture
• Histoplasma can be easily cultured from clinical
specimens. It is usually considered to be a slow growing
mold and commonly requires 2 to 4 or more weeks for
colonies to appear at 250c to 300c
Colonial appearance
• Mycelia phase: on sabouraud’s glucose agar the
colonial morphology produces mycelial growth with
characteristic warty conidia when cultured at room
temperature.

60. Culture cont’d
• Yeast phase: on brain heart infusion agar small
microconidia and characteristic large, round, spiny
macroconidia are produced.
• At 370C on certain media it is possible to induce the
yeast phase of this dimorphic fungus.

61. Microscopic appearance
Mycelia phase:
 Septate hyphae with round to periform microconidia on
short branchs or directly on hyphal stalk; later large,
round thick-walled, knobby tuberculate macroconidia
forms.
Mycelia phase:
• small budding round to oval yeast cells.

62. Serologic Tests
Complement fixation test (CFT):
antigen – histoplasmin or its yeast form.
• Complement fixation is the most commonly used
diagnostic and prognostic test in histoplasmosis.
• Specific CF antibodies arise in 90% of patients 2 to 4
weeks following initial infection.
• A titer of 1:8 to 1:16 highly suspicios of infection.
• A titer of 1:32 or greater indicative of active infection.
by about 9 months.

63. Serology cont’d
• In patients with an established diagnosis, titers of 1:16
are considered suspicious, and 1:32 or above and
persist for a few weeks indicate progressive disease.
• Latex agglutination;
• antige - Histiplasmin.
• The test is unreliable;may cause false positive and
false negative teste may be observed.any positive test
should be confirmed by the CFT test.

64. Serology cont’d
• Skin test
• Histoplasma skin tests are usually contraindicated in
histoplasmosis, first because of extensive cross-
reactivity with other fungal pathogens and, more
importantly, because skin tests alter the level of humoral
antibodies important in serologic diagnosis.

65. Fig. 5-1 Morphology of frequently isolated
systemic (dimorphic) fungi
Mycelial phase Yeast phase
Blastomyces dermatitidis
Coccidioides immitis

66. Histoplasma capsulatum
Paracoccidioides brasilensis
Mycelial phase
Yeast phase

67. Summary
• List the pathogens that cause systemic mycoses
• What are the sources of infection?
• What are the two forms of the pathogenic fungi and
where they found?
• Mention the three forms of systemic mycoses and
what make them different?

68. References
• Murry Medical microbiology 5th edn.
• Sherris Medical microbiology an introuction to infection.
• Jawetz, Melnick, & Adelberg's Medical Microbiology, 24th
Edition.