IMMUNOLOGY ANDSEROLOGY

INTRODUCTION TO IMMUNOLOGY
BASIC CONCEPTS OF IMMUNOLOGY

IMMUNE DISORDERS
DISEASESAND SEROLOGIC TESTS
“IF YOU WANT TO LIVEA
HAPPY LIFE, TIE ITTO A GOAL.

NOT TO PEOPLE OR THINGS.”

-Albert Einstein
DISEASESAND SEROLOGIC TESTS
BACTERIAL DISEASES
VIRAL DISEASES
PARASITIC DISEASES
FUNGAL DISEASES
Whathappenswhenaparasiteenters thebody?
1. No infection - host’s innate immunity prevents the parasite from
establishing an infection.
2. Parasite may invade the host, become established, and then be
killed and eliminated by host defense mechanisms.
3. Parasite may overwhelm and kill the host due to: rapid
multiplication, invasion of vital organ, inadequate immune
system.
4. Long-lasting infection - host cannot remove parasite
completely; may lead to death of host
5. Autoimmunity
Howdoesthebodyfightoffparasitic infections?

• Neutrophils act as phagocytes to destroy both intracellular

and extracellular parasites.
• Hydrogen peroxide kills extracellular organisms while
neutrophilic granules destroy ingested organisms.
• NK cells can be activated without prior exposure; produce
IFN-γ, which inhibits the replication of intracellular parasites.
• Complement - chemotaxis, opsonization for enhanced
phagocytosis, and lysis of extracellular parasites.
How does thebodyfightoffparasitic infections?
• Cytokines and chemokines - IFN-γ, migration inhibitory factor
(MIF), IL-3 and granulocyte monocyte colony-stimulating
factor (GM-CSF)
• Immunoglobulins – specific antibody can damage protozoa,
blocks parasite attachment to the host cell, prevent the spread
of the parasite, promote complement lysis, and enhance
phagocytosis and destruction through ADCC.
• Helminth infections in particular are characterized by
eosinophilia and high levels of IgE which binds to mast cells
and basophils.
 Howdoparasitesevadeimmune responses?

• Antigenic concealment
 Intracellular sequestration
• Antigenic variation
 Developing new antigens through: random mutation, genetic
recombination, gene switching/antigen switching
• Antigen shedding
• Antigenic mimicry
• Immunologic subversion
• Immunologic diversion
Toxoplasmosis
• Causative agent: Toxoplasma gondii, protozoa
Transmission: Ingestion of infective cysts
(oocysts), blood transfusions or organ
transplantation.
• Definitive host: Cats
• Asymptomatic or may present with a mild
lymphadenopathy; rarely reaches CNS or the eye.
• One of the more common opportunistic infections
seen in individuals with AIDS.
Toxoplasmosis
• Pathophysiology
• Encysted organisms can be found in any tissue and
may remain viable for years
• Greatest concern is that Toxoplasma species can cross
the placenta; however, women exposed to before
pregnancy do not transmit the infection to the fetus.
• T. gondii is capable of replicating inside human
macrophages.
• Antibody-coated Toxoplasma organisms trigger
phagocytosis, which ultimately destroys the organism.
Toxoplasmosis
• Laboratory Diagnosis
• Enzyme immunoassays (EIA) for IgM, IgG, or IgA
 Method of choice; more sensitive, less difficult to
perform, and easier to interpret.
• Indirect fluorescent antibody (IFA) assays for
IgG
 Widely used
• PCR
 For prenatal congenital toxoplasmosis; detects T.
gondii DNA on amniotic fluid
Protozoans
• Entamoeba histolytica
• E. histolytica/dispar
• Cryptosporidium parvum
• Giardia lamblia
• Trichomonas vaginalis
Protozoans
• Laboratory Diagnosis
• Direct fluorescent antibody (DFA)
 Use monoclonal antibody labeled with fluorescein isothiocyanate
against the parasite’s cell wall to visualize the initial antibody-
parasite complex
• ELISA
 Employ enzyme conjugated to either antigen or antibody, depending
on the assay being performed.
• PCR
 To identify species of Plasmodium, Toxoplasma, Giardia,
Trypanosoma, Leishmania, Cryptosporidium, Entamoeba,
Microsporidia, Babesia, and Cyclospora
Helminths
• Immune Response
 Phagocytosis by macrophages and neutrophils can destroy
larval stages of helminths.
 Eosinophils and mast cells play an important role in the
defense against tissue parasites.
 IgE-coated mast cells recruit eosinophils to the site of
infection. Eosinophils degranulate and kill the organisms
through oxygen-dependent and oxygen-independent
mechanisms.
Helminths
• Laboratory Diagnosis
• IFA tests
• Slide, tube, and precipitin tests
• Complement fixation
• Particle agglutination tests
• Enzyme-linked immunoassays
• Immunoblot methods – to diagnose cysticercosis,
echinococcosis, paragonimiasis, and schistosomiasis.
ParasiteDiagnosis
• Limitations
 Economical
 Specificity and sensitivity of the method
 Lack of proficiency testing
 Timing of testing for IgM
 Cross-reactivity of antibodies
DISEASESAND SEROLOGIC TESTS
BACTERIAL DISEASES
VIRAL DISEASES
PARASITIC DISEASES
FUNGALDISEASES
Fungi
• Eukaryotic cells with nuclei and rigid cell walls.
• Appearing either as filamentous molds or yeasts or
both
• Molds – aka mycelial fungi; hyphae and conidia.
 Hyphae - filamentous tubular branching structures that
intertwine to form a dense mat (mycelium)
 Conidia – aka spores; asexual reproductive structures
produced at the tip or along the sides of fertile hyphae.
• Yeasts - unicellular and reproduce asexually by budding.
Fungi
• Most fungi are monomorphic, but some exhibit thermal
dimorphism (reproduce both as molds, at 25°C to 30°C,
and as yeasts, at 35°C to 37°C).
• Mycelial mold form is the saprophytic state found in
nature
• Yeast form is the parasitic or pathogenic state found in
tissue in disease (except Coccidioides immitis, which
grows as a spherule at 35°C to 37°C)
• Thermal dimorphs are the etiologic agents of serious
systemic mycoses that can be life-threatening.
Fungi
• Nonpathogenic but may cause mild to very severe
pathology in immunocompromised individuals.
 Immunocompromised individuals (AIDS, increased use of
antibiotics, immunosuppressive agents for treatment of
cancer, transplantation patients)

• Pneumonia – caused by Pneumocystis carinii aka

Pneumocystis jiroveci; one of the most common
opportunistic diseases in AIDS patients
 Howdoesthebodyfightoff fungalinfections?
• Ability of the organism to survive at 37°C at a neutral pH and
the production of toxins are factors that affect the virulence
of the fungus.

• Natural or innate resistance (physical barriers: skin and

mucous membranes) to fungal infection is very high

• Cellular immunity - most important defense against fungal

infection.
MycoticInfections
• Most pathogenic fungi are found in water, soil, or decaying
organic matter; some exist in humans as normal flora.
• Transmission: traumatic entry into body tissues or accidental
inhalation into the lungs.
• Fungal diseases are generally not spread from person to
person.
• A few endogenous organisms (e.g. Candida albicans) can
cause infection when the host defense is deficient for only a
brief period.
MycoticInfections
• Mycotic infection based on site of infection:
• Superficial – restricted to outer layer of skin
• Cutaneous – involve keratinized body areas
• Dermatomycoses – hair and nails
• Subcutaneous – deep, ulcerated skin lesions
• Systemic – deep viscera and spread throughout the body
• Originate in lungs
• Opportunistic – in immunosuppressed individuals
• Commensalistic
MycoticInfections
• Clinical Manifestations:
• Hypersensitivity
• Allergic reaction to spores
• Mycotoxicosis
• Poisoning by food products contaminated with fungal toxins
• Aflatoxins, deoxynivalenol, fumonisins, zearalenone, T-2 toxin,
ochratoxin, ergot alkaloids
• Mycetismus
• Ingestion of toxin-producing mushrooms (containing neurotoxins)
• Amanita phalloides, A. verna, A. virosa
• Fungal growth in host tissues
MycoticInfections
• Aspergillosis
• An opportunistic infection
predominantly caused by
Aspergillus fumigatus, A.
flavus, A. niger, and A.
terreus found worldwide in
soil and air, in decaying
vegetation, and on stored
grains
MycoticInfections
• Aspergillosis
• Disseminated aspergillosis - usually occurs in severely
immunocompromised hosts; assoc. with CGD
• Pulmonary colonization - primary condition induced by
inhalation of large numbers of conidia.
• Allergic bronchopulmonary aspergillosis - allergic
reactions (allergy, asthma, pulmonary infiltrates) to the
toxins and the endotoxins of Aspergillus species in
hypersensitive individuals exposed repeatedly to conidia.
MycoticInfections
• Aspergillosis: LaboratoryDiagnosis
• Ouchterlony immunodiffusion (ID) or
counterimmunoelectrophoresis (CIE) for antibodies
 Aspergillus reference antiserum must demonstrate three or
more bands with Aspergillus reference antigen for validity.
• • Nonspecific banding can be caused by C-reactive protein.
EIA
 More sensitive than immunodiffusion tests and is therefore more
valuable for diagnosis of aspergillosis in immunocompromised
patients.
MycoticInfections
• Candidiasis
• Infections with Candida albicans and
several other Candida species are
collectively called candidiasis.

• Candida albicans - most common cause

of all serious fungal diseases; normal
microbiota alimentary tract and the
mucocutaneous regions of the body
MycoticInfections

• Candidiasis: LaboratoryDiagnosis
• ID and CIE methods for antibodies
 Give comparable results; detect antibodies in systemic
candidiasis
 Positive: Formation of one or more bands between the
reagent antigen and patient’s serum
 Patient’s symptoms, direct smears, and cultures should
be considered to validate positive ID and CIE tests
MycoticInfections
• Candidiasis: LaboratoryDiagnosis
• Latex agglutination
 Quantitative and can be of diagnostic and prognostic value.
 Latex particles sensitized with a homogenate antigen of C.
albicans are reacted with patient sera and control sera.
 When this screening test is positive, a serial dilution is
performed and reported as the highest dilution giving a 2+
reaction.
 1:4 titer - early infection, colonization, or a nonspecific reaction.
 1:8 or greater - invasive infection.
 A fourfold decrease in titer may indicate successful therapy
MycoticInfections
• Candidiasis: LaboratoryDiagnosis
• PNA-FISH
 Peptide nuclear acid fluorescent in situ hybridization
 Utilizes small PNA polymer probes labeled with a
fluorescent dye to identify PNA sequences on
chromosomes.
 Currently used to differentiate Candida albicans
from other Candida species.
MycoticInfections
• Coccidioidomycosis
• aka Valley fever
• Causative agent: Coccidioides immitis - mold found
primarily in alkaline desert soil in hot, semiarid regions.
• Vectors: Animals (desert rodents)
• Transmission: inhalation of dust containing the fungus
• Acute primary pulmonary disease a chronic progressive
pulmonary disease, or secondary infections
• Dissemination to the skin (erythema nodosum), bones,
subcutaneous tissues, lymph nodes, and meninges is rare.
MycoticInfections
• Coccidioidomycosis: LaboratoryDiagnosis
• Complement Fixation
 Most widely used quantitative serodiagnostic method for
identifying C. immitisinfections.
 1:2 to 1:4 titers – presumptive evidence of early infection;
should be repeated in 3 to 4 weeks.
 1:16 titer - active infection, along with positive ID test.
 Specimens: Serum, cerebrospinal fluid (CSF), pleura,
peritoneum, and joint fluids
 False-positive: Patients with acute histoplasmosis
MycoticInfections
• Coccidioidomycosis: LaboratoryDiagnosis
• Tube Precipitation (TP)Test
 Positive TP test - early indication of a primary infection.
 Highly specific with very few cross-reactions.

• Immunodiffusion
 Agar gel double ID tests - as sensitive as complement fixation Most
commonly used screening test for the diagnosis of
coccidioidomycosis.
 Two or more bands usually indicate active disease or dissemination
 Serum dilutions - for quantification of coccidioidin antibodies.
MycoticInfections
• Coccidioidomycosis: LaboratoryDiagnosis
• Latex Agglutination
• Latex particles sensitized with coccidioidin are reacted with
inactivated patient serum to detect antibodies to the organism.
• CF test or an ID test should be performed for confirmation when LA
screening tests are positive.

• Enzyme Immunoassay
 EIA tests for IgG and IgM antibodies are available for use with serum
or CSF.
 Positive EIA tests should be confirmed with CF or TP tests, because
the EIA test is not absolutely specific.
MycoticInfections
• Cryptococcosis
• Causative agent: Cryptococcus
neoformans
• Vector: Pigeons; C. neoformans is found
where pigeons roost and defecate
• Transmission: Inhalation of yeast
• Virulence factors: Encapsulation and
melanin synthesis
• May be asymptomatic or may develop
as a symptomatic pulmonary infection.
MycoticInfections
• Cryptococcosis
• Disseminated cryptococcosis - yeast spread is hematogenous; any
organ or tissue of the body may be infected, but localization outside
the lungs or brain is relatively uncommon.
• Growth inhibiting substances present in body fluids resolves
infection in normal individuals
• Serious clinical disease is found in patients with debilitating
illnesses and immunosuppression (AIDS patients)
• Primary and secondary cutaneous cryptococcosis - encountered in
immunosuppressed patients.
• Meningitis - due to the absence of inhibitory factors in spinal fluid
and the minimal phagocytic response in CNS.
MycoticInfections
• Cryptococcosis: LaboratoryDiagnosis
• Latex Particle Agglutination (LPA) Antigen Test
 Recommended due to high sensitivity and specificity for
detection of C. neoformans capsular polysaccharide antigen
 Inactivated serum or spinal fluid and positive and negative
human reference sera are each mixed with latex particles
 Presence of any agglutination is considered a positive test if
controls are acceptable.
 Serial dilution – quantitative; titer is reported as the highest
dilution showing 2+ agglutination.
• 1:2 titer - infection; 1:4 or greater - active infection.
MycoticInfections
• Cryptococcosis: LaboratoryDiagnosis
• Enzyme Linked Immunoassay
 To detect antigens of C. neoformans in both serum and CSF.
 More sensitive than the latex agglutination procedure but takes
more time to perform.
MycoticInfections
• Cryptococcosis: LaboratoryDiagnosis
• Tube Agglutination Test (Antibody Detection)
 Can be used as qualitative screening test and semiquantitative
test to detect antibodies to C. neoformans.
 Screening Test:
 Serum or CSF from infected patients is heat inactivated to destroy
complement then incubated and refrigerated overnight with
aCryptococcus antigen suspension of weakly encapsulated yeast
cells along with controls.
 Semiquantitative test - serial dilution
• 1:2 titer or greater -current or recent infection
 MycoticInfections
• Cryptococcosis: LaboratoryDiagnosis
• Indirect Fluorescent Antibody Test
 Most valuable when antigen tests are negative; can be combined
with antigen tests to determine the patient’s prognosis.
 Positive test - recent or current infection with C. neoformans
• or a cross-reaction with another fungus.
 IFA tests have a 77% specificity and 50% sensitivity, but both false-
negative and false-positive results may occur.
 “INTELLECTUAL GROWTH
SHOULD COMMENCE AT BIRTH
AND CEASE ONLY AT DEATH.”

- A l b e r t Einstein