PRECAUTIONS FOR GOOD STUDY

PLEASE
•Do not memorize
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-Look for connections and relations
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need to clarify
•Your student handbook for Pathology is ROBBINS
PATHOLOGY STUDENT EDITION 1
 GENERAL PATHOLOGY OF
INFECTIOUS DISEASE
Part 1
Prof. Dr. Ayper Eribol 2
 GENERAL PRINCIPLES OF
MICROBIAL PATHOGENESIS
• Infectious diseases remain an important health
problem despite effective vaccines and antibiotics.

• 2 of the top 10 leading causes of death are

attributable to infection (pneumonia and septicemia).
• ***Infectious diseases are particularly important
causes of death among the elderly, persons with
chronic diseases, and patients receiving
immunosuppressive drugs.

3
 Categories of Infectious Agents
Infectious agents belong to a wide range of classes and vary greatly in
size, ranging from prion protein aggregates of under 20 nm to 10-m
tapeworms

4
 PRIONS

• Prions are composed of abnormal forms of a host protein

termed prion protein (PrP).

• PRNP (prion protein) is the human gene encoding for the

major prion protein PrP (protease-resistant-protein, Pr for
prion, and P for protein), also known as CD230 (cluster of
differentiation 230).

5
 PrP

• PrP is found normally in neurons.

Expression of the protein is most predominant in the nervous system but occurs
in many other tissues throughout the body.

• Diseases occur when the PrP undergoes a

conformational change (misfolding) that confers
resistance to proteases.

6
WHY??

The exact mechanisms which lead to the conformational

change are still unknown.

It also remains to be brought to light how a polypeptide

chain can adopt at least two stable conformations.

Environmental change?
Chemical pollution?
Genetically modified food?
Lab investigations?

7
 Prions spread between animals through body fluids
like feces, saliva, blood, or urine, either through direct
contact or indirectly through environmental
contamination of soil, food or water.

To destroy a prion it must be denatured to the point that

it can no longer cause normal proteins to misfold.
Sustained heat for several hours at extremely high
temperatures (480 C and above) will reliably destroy a
prion.

8
 consequences
• Prions attack nerve cells producing neurodegenerative brain
disease.
• Accumulation of abnormal PrP leads to neuronal damage and
distinctive spongiform pathologic changes in the brain.
• They characterize several fatal and transmissible
neurodegenerative diseases (spongiform encephalopathies) in
humans and many other animals including;
–Kuru (associated with human cannibalism),
–Creutzfeldt-Jakob disease (CJD),
–Bovine spongiform encephalopathy (BSE) (better known as “mad cow
disease”), and
–Variant Creutzfeldt-Jakob disease (vCJD) (probably transmitted to humans
through consumption of meat from BSE-infected cattle).
9
Affected animal(s) Disease
Sheep, Goat Scrapie
Cattle Mad cow disease
Camel Camel spongiform encephalopathy (CSE)
Mink Transmissible mink encephalopathy (TME)
White-tailed deer, elk, mule deer, moose Chronic wasting disease
Cat Feline spongiform encephalopathy (FSE)
Nyala, Oryx, Greater Kudu Exotic ungulate encephalopathy (EUE)
Ostrich Spongiform encephalopathy
(unknown if transmissible)

Human Creutzfeldt–Jakob disease (CJD)

Iatrogenic Creutzfeldt–Jakob disease (iCJD)
Variant Creutzfeldt–Jakob disease (vCJD)
Familial Creutzfeldt–Jakob disease (fCJD)
Sporadic Creutzfeldt–Jakob disease (sCJD)
Gerstmann–Sträussler–Scheinker syndrome (GSS)
Fatal familial insomnia (FFI)
Kuru
Familial spongiform encephalopathy
Variably protease-sensitive prionopathy (VPSPr)
10
Normal

11
Prion protein (stained in
red) revealed in a
photomicrograph of
neural tissue from a
scrapie-infected
mouse.

12
This micrograph of brain tissue reveals the cytoarchitectural
histopathologic changes found in bovine spongiform
encephalopathy. The presence of vacuoles, i.e. microscopic “holes”
in the gray matter, gives the brain of BSE-affected cows a sponge-
13
like appearance when tissue sections are examined in the lab.
Classic CJD is a human prion disease.
It is a neurodegenerative disorder with characteristic clinical
and diagnostic features. This disease is rapidly progressive
and always fatal. Infection with this disease leads to death
usually within 1 year of onset of illness.

14
Creutzfeldt-Jakob disease (CJD)
Spontaneous; sporadic
Inherited; familial

CJD can be transmitted from person to person

iatrogenically, by surgery, organ transplantation, or blood
transfusion.

15
 VIRUSES
• Viruses are obligate intracellular parasites
• Depend on the host cell’s metabolic machinery for their
replication.
• They consist of a nucleic acid genome surrounded by a protein
coat (a capsid).
• Viruses are classified by;
– Nucleic acid genome (DNA or RNA)
– The shape of the capsid (icosahedral or helical)
– The presence or absence of a lipid envelope
– Their mode of replication
– The preferred cell type for replication (called tropism)
16
– The type of pathology they cause.
Viruses account for a large number of acute infections.

Because of their small size (20–300 nm), single virus

particles can only be detected by electron
microscopy.

Some viruses may form aggregates in the nucleus

and/or cytoplasm of the infected cells. Such viral
inclusion bodies may be visible by light microscopy
and definitely by electron microscopy.

17
18
 Pathologic diagnosis of Viral Infections
•Viruses induce characteristic morphologic changes during
their replication in the cells they infect.
•Morphologic features indicative of a viral infection include
the formation of inclusion bodies in the host cell nucleus,
cytoplasm or both.
•For some viruses, formation of multinucleate giant cells,
presence of a perinuclear halo around the infected cell,
lymphocytic infiltration or even cellular necrosis can be
useful.

19
Inclusions
They typically represent sites of viral multiplication in a
bacterium or a eukaryotic cell and usually consist of viral
capsid proteins.

Inclusion bodies can also be hallmarks of genetic diseases, as

in the case of neuronal inclusion bodies in disorders like
frontotemporal dementia and Parkinson's disease.

They have been reported to be reversible.

There is a growing body of information indicating that formation of

inclusion bodies occurs as a result of intracellular accumulation
of partially folded expressed proteins which aggregate
through non-covalent hydrophobic or ionic interactions or a
combination of both.
20
Characteristic inclusion bodies:
–Seen with the light microscope and are useful for diagnosis. The
three most commonly recognized viral infections by
histopathological changes are those caused by herpes simplex virus
(HSV), cytomegalovirus (CMV) and Human papilloma virus (HPV).
•CMV infected cells;
–Enlarged cell
–A large eosinophilic nuclear inclusion
–Smaller basophilic cytoplasmic inclusions;
•Herpesviruses form a large nuclear inclusion surrounded by a clear
halo.
•HPV form koilocytes (a clear halo surrounding the nuclei)
•Smallpox and rabies viruses form characteristic cytoplasmic inclusions.
21
22
Examples of viral inclusions

A, Cytomegalovirus infection in the lung. Infected cells show

distinct nuclear (long arrow) and ill-defined cytoplasmic (short
arrows) inclusions.

B, Varicella-zoster virus infection in the skin. Herpes simplex

virus and varicella-zoster virus both cause characteristic
cytopathologic changes; fusion of epithelial cells, which produces
multinucleate cells with molding of nuclei to one another (long
arrow), and eosinophilic haloed nuclear inclusions (short arrow).

C, Hepatitis B viral infection in liver. In chronic infections,

infected hepatocytes show diffuse granular (“ground-glass”)
cytoplasm, reflecting accumulated hepatitis B surface antigen
(HBsAg).
23
Warthin-Finkeldey cells in measles

24
Negri bodies in rabies

25
Koilocyte

Perinuclear halo in HPV

26
 Viral mechanisms of disease
Viruses can cause illnesses in several ways:

- Many viruses cause transient illnesses (e.g., colds, influenza)

- Other viruses are not eliminated from the body and persist
within cells of the host for years either:
• Continue multiplying (e.g., chronic infection with hepatitis B
virus [HBV])
or
• Survive in some nonreplicating forms (termed latent infection)
with the potential to be reactivated later.
27
 Charachteristics of Diseases caused by
viruses
•Latent infection: Herpes zoster virus, the cause of chickenpox
–Enter dorsal root ganglia
–Establish latency
–Periodically activated to cause shingles, a painful skin condition.
•Neoplastic disease: Some viruses are involved in transformation of a
host cell into a benign or malignant tumor (e.g., human
papillomavirus [HPV]-induced benign warts and cervical carcinoma).
•Acute infections: (e.g., upper respiratory infection)

•Chronic infections: (e.g. Hepatitis B,C)

28
 Charachteristics of Diseases caused by
viruses

•Different species of viruses can produce the same clinical

picture (e.g., upper respiratory infection)

•Conversely, a single virus can cause different clinical

manifestation depending on host age or immune status
(e.g.,CMV).

29
30
31
 BACTERIA

Bacteria are prokaryotes; have a cell membrane but lack

membrane-bound nuclei and other membrane-enclosed
organelles.

32
33
• There are two common forms of cell wall structure:

– a thick wall that retains crystal-violet stain (gram-positive

bacteria)

– a thin wall surrounded by an outer membrane (gram-negative

bacteria)

34
35
• Bacteria are classified by

– Gram staining (positive or negative),

– Shape (spherical ones are cocci; rod-shaped ones are bacilli)

– Need for oxygen (aerobic or anaerobic).

36
37
 Bacteria characteristics
• Motile bacteria have flagella, that rotate and move the bacteria.
• Some bacteria possess pili, that can attach bacteria to host cells or
extracellular matrix.
• Bacteria synthesize their own DNA, RNA, and proteins.
• They depend on the host for favorable growth conditions:
- many bacteria remain extracellular when they grow in the host,
- while others survive and replicate either outside or inside of host
cells (facultative intracellular bacteria)
- some grow only inside host cells (obligate intracellular bacteria).

38
 Normal microbiome

Normal healthy people can be colonized by as many as 1012

bacteria on the skin, 1010 bacteria in the mouth, and 1014
bacteria in the gastrointestinal tract.

Bacteria colonizing the skin include Staphylococcus

epidermidis and Propionibacterium acnes, the cause of
acne.

Aerobic and anaerobic bacteria in the mouth, particularly

Streptococcus mutans, **a major cause of tooth decay.
39
 Function of microbiome

**In the intestinal tract, the microbiota are responsible for

absorption of digested foods, maintaining the integrity of the
epithelium and the normal functioning of the intestinal immune
system.

And for competitively inhibiting invasion and colonization by

potentially pathogenic microbes.

Depletion of the microbiome or change in its composition has been

implicated in inflammatory bowel disease, the development of
allergies, and increased incidence of various systemic autoimmune
diseases.
40
41
The microbiome is the largest organ you may have never heard of, weighing
42
up
to three kilograms.
43
44
Histopathologic diagnosis of bacterial infections
•Bacteria are the most difficult microorganisms to detect in
routine H and E-stained histologic sections.
•Tissue diagnosis of a bacterial infection begins with the
recognition of a consistent pattern of inflammation.
• Individual bacteria are generally not detected in H and E-
stained tissue sections. Granules can be visualized by H and
E stain in cases of actinomycosis.
• Several modifications of Gram stains can be used for the
detection of bacteria in tissue sections. Gram-negative
bacteria are particularly difficult to visualize, mainly
because of a lack of contrast between the bacteria and the
counter stain.
45
• Silver precipitation stains, such as the Warthin-Starry, Steiner
or the Dieterle stains, may be used to detect these bacteria.

• Various other special stains have proven useful for detection of

selected bacteria in tissue sections.

- H. pylori may be demonstrated with modified Giemsa, Warthin-

Starry stain.

- Histopathological diagnosis of mycobacteria is quite common

and staining procedures most commonly used for visualization
of these acid fast bacilli in smears are EZN and Auramine O.

46
Tissue diagnosis of a mycobacterial infection begins by examination of H and E-stained tissue sections.
Although organisms cannot be seen, the pattern of granulomatous inflammation provides the first indication
47
that mycobacteria should be considered among the differential diagnoses.
Sarcoidosis
Nonnecrotizing
Granulomas

48
Syphylis
Endothelial cell proliferation + plasmacyte rich
chronic inflammation
İn the appropriate clinical setting.

51
 Sputum
Pneumonia
Str. Pn.

BAL
Gr - bacilli

The variety of bacterial morphology.

A, Gram stain preparation of sputum from a patient with
pneumonia. Gram-positive, elongated cocci in pairs and
short chains (Streptococcus pneumoniae) and a
neutrophil are evident. Brain
B, Gram stain preparation of a bronchoalveolar lavage Meningoencephalitis
specimen showing gram-negative intracellular rods Borr. Burg.
typical of members of Enterobacteriaceae such as
Klebsiella pneumoniae or Escherichia coli.
C, Silver stain preparation of brain tissue from a patient
with Lyme disease meningoencephalitis. Two helical
spirochetes (Borrelia burgdorferi) are indicated by arrows.
A, B, and C are at different magnifications.
(B, Courtesy of Dr. Karen Krisher, Clinical Microbiology Institute, Wilsonville,
Oregon. A and C, Courtesy of Dr. Kenneth Van Horn, Focus Diagnostics, Cypress,
52
.)
California
53
Helicobacter Pylori Gastritis
Giemsa preparation 54
 FUNGI

Fungi are eukaryotes that possess thick, chitin-containing

cell walls and ergosterol-containing cell membranes.

Fungi can grow either as

- rounded yeast cells (unicellular fungi species:
Saccharomyces cerevisiae, Cryptococcus neoformans,
etc.)
or
- as slender, filamentous hyphae ( multicellular: Alternaria,
Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus,
Trichophyton, etc.)
55
Hyphae may be septate or aseptate.

Some fungi exhibit thermal dimorphism; that is,

- they grow as hyphal forms at room temperature
- but as yeast forms at body temperature.

Fungi may produce sexual spores or, more commonly,

asexual spores called conidia.
56
 Fungi may cause superficial or deep infections.

• Superficial infections involve the skin, hair, and nails.

Fungal species that cause superficial infections are called
dermatophytes.
Infection of the skin is called tinea; thus, tinea pedis is “athlete’s
foot” and tinea capitis is scalp ringworm.

Certain fungi invade the subcutaneous tissue, causing abscesses or

granulomas sometimes called mycetomas.

• Deep fungal infections can spread systemically and invade tissues,

destroying vital organs in immunocompromised hosts.

57
Histopathology remains one of the major tools of diagnosis
in mycology.

The major advantages of histopathology are speed, low cost

and the ability to provide a presumptive identification of the
infecting fungus as well as demonstrating the tissue reaction.
However, definitive species identification of the aetiologic
agent by histopathology may be difficult.

58
A number of histologic stains are available that are routinely
used to visualize fungi in tissue sections.

GMS* and PAS are very efficient to visualize the fungi.

H and E stain, on the other hand, is very useful to visualize the

host’s response but is not a special fungal stain. It does not
stain most of the fungi.

Thus, a combination of GMS and H-E is usually employed to

visualize both the tissue reaction and the infecting fungus.

59
 Site of sampling

Achieving a successful histopathologic diagnosis begins with

selection of the tissue sample to be examined.

While some fungi are more readily located at the periphery of the
infected lesion, others are more prominent at the centre.

60
 Fungal yeasts

Fungal hyphae

mucor

61
62
63
64
C.Alb.

65
Gomorhi Methenamin Silver St
Candida Albicans
66
Pseudomembranous tracheitis
caused by
Aspergillus fumigatus

in the setting of high grade T-cell

lymphoma

67
A) Right
upper lobe
apico-
posterior

B) Bronchus intermedius
Bronchoscopy
shows mucous-
like layer in the
C) Secondary carina
bronchotrachial right side.
tree.
68
Microscopic view of biopsy shows strains of Aspergillus
fumigatus with characteristic hyphae. 69
70
GMS stain : Aspergillus hyphae

71
PAS stain: Aspergillus hyphae 72
 PARASITIC INFECTIONS
The specimen most frequently submitted for
detection of parasites is stool and the usual request
is examination for ova and cyst.

Specimens other than stool submitted for detection

of parasites include
-peripheral blood film (thick and thin smear),
-bone marrow smear,
-duodenal aspirates,
-sputum samples,
-skin, muscle biopsy, etc. 73
The direct wet mount (saline) is a useful first step in
the examination of these specimens
and
if parasites are detected, the specimen is fixed and
stained to confirm the diagnosis.

74
Giemsa staining is recommended for detection of many parasites,
including:

- blood pathogens (Plasmodium species, Babesia species,

Trypanosoma species, L. donovani and microfilariae) in thick and thin
blood films,
- L. donovani in bone marrow and splenic aspirates
- Leishmania species in aspirates or imprints of cutaneous or
mucocutaneous lesions,
- trypanosomes in lymph node imprints,
- Toxoplasma gondii in imprints of brain, lung or other tissues
- G. lamblia, microsporidia, Entamoeba histolytica in imprints of
gastrointestinal biopsy.

75
 Protozoa
Protozoa are single celled parasites. They can replicate

• intracellularly within a variety of cells (e.g., Plasmodium in

red cells, Leishmania and Trypanasoma in macrophages)

- Bloodborne protozoa (e.g., Plasmodium, Trypanosoma,

Leishmania) are transmitted by insect vectors.

or
- extracellularly in the urogenital system, intestine, or blood.
(Trichomonas vaginalis are sexually transmitted protozoa that
colonize the vagina and male urethra; Intestinal protozoans,
Entamoeba histolytica and Giardia lamblia, are ingested as cysts
in contaminated food or water and become motile trophozoites
that attach to intestinal epithelial cells).
76
77
Plasmodium falciparum, blood, giemsa 78
79
80
81
Entemoeba histolytica (amebiasis), colon, giemsa

82
83
Giardia lamblia, duodenum biopsy, giemsa

84
85
Trepanosoma, blood, giemsa 86
Trepanosoma, blood, giemsa

87
Leishmaniasis
Şark çıbanı
Leishmaniasis map
89
90
Leishmania
91
 92
Leishmania in blood, giemsa
Leishmnia, giemsa, skin touch imprint

93
Leishmaniosis, biopsy
Toxoplasma
Gondii

95
Brain tissue biopsy reveals bradyzoite of T. Gondii.
96
Toxoplasma, lymph node touch print, giemsa

97
 Helminths
Parasitic worms are highly
differentiated multicellular
organisms.

Their life cycles are complex.

• Helminths comprise three groups:
–Roundworms (nematodes),
–Flukes (trematodes),
–Tapeworms (cestodes)

98
ROUNDWORMS (NEMATODES) are circular in cross-
section and nonsegmented.

Intestinal nematodes include Ascaris lumbricoides,

Strongyloides stercoralis, and hookworms.

Nematodes that invade into tissue include the filariae

and Trichinella spiralis.

99
100
101
102
•FLUKES (TREMATODES) are leaf-shaped flatworms
with prominent suckers that are used to attach to the
host.

–They include liver and lung flukes and schistosomes.

103
 Schistosoma

Fasciola hepatica
Liver-lung flux
104
•TAPEWORMS (CESTODES) have a head (scolex) and a
ribbon of multiple flat segments (proglottids).

–They include the fish, beef, and pork tapeworms, found

in the human intestine.
–The larvae that develop after ingestion of eggs of
certain tapeworm can cause cystic disease within
tissues
–Echinoccus granulosus larvae cause hydatid cysts
–Pork tapeworm larvae produce cysts called cysticerci

105
Hydatid cyst

106
107
 Ectoparasites
• Ectoparasites are insects (lice, bedbugs, fleas) or
arachnids (mites, ticks, spiders) that attach to and
live on or in the skin.
• Diseases are characterized by itching and
excoriations, such as pediculosis caused by lice
attached to hairs, or scabies caused by mites
burrowing into the stratum corneum.
• At the site of the bite, mouth parts may be found
associated with a mixed infiltrate of lymphocytes,
macrophages, and eosinophils.
• Can serve as vectors for other pathogens, such as
Borrelia burgdorferi, the agent of Lyme disease,
which is transmitted by deer ticks.

108
DIAGNOSIS OF INFECTIOUS
DISEASES
109
The diagnosis of complex diseases, infectious or otherwise,
requires the collaborative efforts of clinicians, radiologists,
and pathologists.

The differential diagnosis generated at the bedside

through patient history and physical examination is narrowed
through consultation and thoughtfully ordered radiographic and
laboratory studies.

The anatomic pathologist, by providing the morphologic

interpretation of biopsies and cytologic preparations, is an
important member of the diagnostic team.

110
Collaboration between the anatomic pathologist and
the microbiologist is necessary to provide optimal
patient care, reduce waste, and prevent medical errors.

The anatomic diagnosis of disease may be used to

clarify complex microbiologic cultures, whereas cultures
may be used to reveal the identity of microorganisms
seen in tissue sections.

111
- first task of the anatomic pathologist is to examine the
specimen to determine whether normal or abnormal
histology (histopathology) is present.

- If abnormalities are seen, the pathologist then characterizes

the disease that is present:
Histologic and cytologic criteria are used to separate diseases into
a variety of categories, such as
• constitutional
• reactive or reparative,
• dysplastic and neoplastic diseases, and
• inflammatory conditions, which include infectious diseases.

- The separation of disease into such categories has both

therapeutic and prognostic implications.

112
When facing a solitary lung nodule

Carcinoma, for example, may be suspected in a

patient with a solitary lung nodule and a
history of long-term smoking.

However, if the excisional biopsy demonstrates

necrotizing granulomatous inflammation, the
diagnosis of a malignant tumor is excluded and the
search for an inflammatory or infectious etiology
would begin.

113
Tbc mimicking lung cancer 114
Infection mimicking cancer: Retrospective analysis of 147 cases,
emphasizing fungal etiology
European Respiratory Journal 2014 44:

Pulmonary infections mimicking cancer: a retrospective, three-

year review
Support Care Cancer 1997 Mar;5(2):90-3.

Fungal infections (histoplasmosis, cryptococcosis,

coccidiomycosis) accounted for 46%, mycobacteria for 27%,
bacteria for 22%, and parasitic lesions (dirofilariasis) for 5% of
these infections.
- the most common clinical manifestations were cough and
chest pain, and
- the most common radiographic finding was a solitary
pulmonary nodule.
115
Dermal fungal infection mimicking squamous cell carcinoma

116
(Granulomatous diseases
mimicking tbc)

117
Use of cytology and histopathology
in microbiologic diagnosis
118
MOST FREQUENT infective material sources in Pathology
routine practice

- Servical biopsy and smears for HPV, candida and TMV

assesment
- (smears, aspiration, biopsy) (skin and others) for Fungal
infections
- Gastric biopsy for Helicobacter pylori assesment
- Lymph node biopsy (tbc, tularemia, toxoplasma, cat
scratch, fungus and others)
- Liver biopsy (Chronic Hepatitis activity assesment)
- Colon biopsy for Entemoeba Histolitica
- Duodenal biopsy for Giardia Lamblia
- operation materials for Cyst hydatid disease
- Blood films and bone marrow aspirates from bloodborne
parasites (malaria, leishmania and others)
Diagnosis of infective disease in PATHOLOGY

WHAT TO LOOK AT ON FFPE blocks?

1)Tissue pattern of host reaction (tissue rxn type)

- Accompanying acute, chronic, granulomatous,
necrotic inflammation?
- Are there special morphologic features
suggesting a particular infection?

1)Special features about infective agent (viral

inclusions, bacterial clumps, fungus and parasite
highlighting with special stains )
Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens have been a
121
staple of research and therapeutic applications for decades.
Which one is what?

Cytology?
FFPE prep?

122
Cytology samples can be obtained using noninvasive and
minimally invasive techniques, and interpretation is affordable
and quick.

In fact, the increasing use of molecular‐based, diagnostic

techniques on fluid specimens, as well as FFPE material, is
expanding the role of cytology specimens for infectious disease
diagnostics.

123
The pathologist or clinician may aspirate palpable
lesions, whereas radiologic guidance is necessary for
deeper lesions.

An advantage of CT-guided aspiration of deep seated

lesions is that general anesthesia is not necessary.

Intraoperative cytologic diagnoses and rapidly stained

touch preparations have also been found to be useful.

124
CT Guided Aspiration of a pyogenic thalamic abscess

125
126
Liquid based cytology preparation

127
128
Touch imprint

129
130
131
 Cytologic samples of some common parasitic organisms

(A) Trichimonas vaginalis organisms

(arrows) seen on a cervical (B) Schistosoma haematobium ova with
Papanicolaou smear (Papanicolaou a central spine in a urine specimen
stain, magnification × 40). (Papanicolaou stain, magnification × 20).

C, D) Toxoplasma gondii cysts in the

cerebral spinal fluid (Papanicolaou stain,
132
magnification × 40).
Lastly….

•Many infective agents cause typical tissue

responses that may vary as the infection
progresses from acute to chronic or disseminated
phases.

When considered with relevant clinical

information, the histological features seen in tissue
biopsies may provide sufficient information to
correctly identify a particular type of organism.
133
 SPECIAL TECHNIQUES FOR
IDENTIFYING INFECTIOUS AGENTS
• Many infectious agents can be seen in hematoxylin and eosin
(H&E)–stained sections
–Inclusion bodies formed by CMV and herpes simplex virus
(HSV);
– Bacterial clumps, which usually stain blue;
– Candida and Mucor among the fungi;
– Most protozoans; all helminths

Although many microorganisms can be visualized in H-E stained

sections, others can be visualized only with certain
histochemical stains. 134
Some infectious agents, are best visualized by special stains
—Gram, acid-fast, silver, mucicarmine, and Giemsa stains—
or after labeling with specific antibodies.

135
H-E clumps of bacteria

136
PAS
Fungal hyphae

137
Giemsa
Intracellular parasites (protozoa)

138
EZN mycobacteria

139
Some clues for correct diagnosis of infections

• Organisms are usually best visualized at the advancing edge of a

lesion rather than at its center, particularly if there is necrosis.

•Pathology lab practice mostly consist of chronic phase infection

samples.

• Acute infections can be diagnosed serologically by detecting

pathogen-specific antibodies in the serum.

•- The presence of specific immunoglobulin M (IgM) antibody shortly

after the onset of symptoms is often diagnostic.

- Assays for serum antibodies (Ig G) are very useful for the diagnosis
of hepatitis caused by viruses.
140
•Conventional isolation of the aetiological agent by
culture followed by its identification is the best
way to identify any pathogen to establish
infectious disease aetiology in any disease.
•Because of a number of reasons, e.g., improper
specimen collection, transportation and processing,
there is a poor isolation rate of microorganisms
from cultures in tissue biopsies.
•Specimen collection for culture and tissue
diagnosis should be separate.
141
Conventional culture confirmation of tissue biopsies
often fail to identify any pathogen as, first of all,

- invariably most of the tissue samples that are

collected in formalin, which prevents pathogen
growth in culture media.

- presence of inhibitors like dead tissue debris,

fibers, etc. also delays isolation.

- microbiologists often lack expertise in identifying

infectious pathogens directly from tissue biopsies by
microscopic visualization.
142
NEWER TECHNIQUES IN
DIAGNOSING INFECTIOUS AGENTS
143
Immunohistochemistry, In Situ Hybridization and In
Situ Polymerase Chain Reaction (PCR)

Molecular methods of detection may be particularly

useful

- when microorganisms are undetectable by means of

histochemical staining methods,
- are present in low numbers,
- stain poorly, are uncultivable or
- exhibit an atypical morphology.

144
Immunohistochemistry

This technique uses monoclonal or polyclonal antibodies directed

against specific microbial antigens.

It detects specific proteins in the tissue.

Once bound, the antibodies are detected by use of either

fluorescent or chromogenic signal amplification.

The specificity of this method is dependent on the specificity of

the antigen binding (Fab) portion of the immunoglobulin molecule
used.

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HPV IHC

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Fluorescent in situ hybridization (FISH)

is a technique that can be used to detect and localize

the presence or absence of specific DNA sequences
on chromosomes.

It uses fluorescent probes that bind to only those parts

of the chromosome with which they show a high degree
of sequence similarity.

Fluorescence microscopy is used to find out where the

fluorescent probe is bound to the chromosome.

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FISH is often used for finding specific features in DNA.
These features can be used in genetic counselling, medicine
and species identification.

Although it is most commonly used to detect viral targets,

FISH has also been used to detect common bacteria and fungi
as well as intracellular bacteria such as Chlamydia pneumonia
and Rickettsia species.

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Plasmodium genus-specific
FISH test identifying all
human malaria parasites.

Photographs showing
Plasmodium genus-specific
FISH test results with blood
smears from patients with
confirmed P. falciparum, P.
vivax, P. malariae, P. ovale
and P. knowlesi infections.

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Nucleic acid amplification techniques, such as Polymerase
chain reaction (PCR) and Transcription-mediated
amplification are used for diagnosis of gonorrhea, chlamydial
infection, tuberculosis, and herpes encephalitis and
coronavirus.
• Quantitative nucleic acid amplification tests are used to
guide the medical management of infections with HIV, HBV,
HCV.

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In situ PCR is a method that may be used to amplify the
nucleic acid of a specific target by use of PCR in an
intact tissue section to detect and localize the amplified
product.

This method has been used to detect viruses in

tissue sections. It is, however, technically difficult to
perform and prone to contamination because
amplification occurs on a slide rather than in a closed
tube.

151