Introduction to Serology - Immunology, HSV & H.pylori

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Introduction toserology - immunology


What do we mean by immunology ?What do we mean by immunology ? The production of immunoglobulins (antibodies) as a

reaction of the human body when infected with an agent.

Immunoglobulins: glycoprotein molecules that are produced by plasmacells in response to an immunogen and which function as antibodies.

Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host.

All lab tests where Ag is used to detect Ab or Ab to capture the Ag, present in human specimen (blood, faeces, urine, CSF, etc. …)


What do we mean by serology ?What do we mean by serology ?

All immunology tests performed on human serum

Immuno-serology:

all tests designed for the detection of human antibodies in human fluids

all tests using monoclonal or polyclonal antibodies for capturing antigens


Structure of an immunoglobulinStructure of an immunoglobulin

four chain structure as basic unit

two identical light chains and two identical heavy chains

Variable and Constant regions

Inter- & intra-disulfide bindings

Hinge region Oligosaccharides

(Carbohydrates)


Fab & FCFab & FC


Different immunoglobulins: IgGDifferent immunoglobulins: IgG

IgG is the major Ig in extra vascular spaces placental transfer - IgG is the only class of Ig that crosses the placenta.

Monomeer major Ig: 75% of total Ig’s 4 subclasses IgG1 to IgG4)


Different immunoglobulins: IgMDifferent immunoglobulins: IgM

IgM is the first Ig to be made by the fetus and the first Ig to be made by virgin B cells when stimulated by antigen. pentameric structure => IgM is a good complement fixing Antibody IgM antibodies are very good in clumping microorganisms for eventual elimination from the body.

pentamer (can also exist as monomeer)third most common Ig


Different immunoglobulins: IgADifferent immunoglobulins: IgA

IgA is major class Ig in secretions - tears, saliva, colostrum, mucus. Therefore important in local (mucosal) immunity.Normally IgA does not fix complement, unless aggregated.

monomeer but in secretions a dimersecond most common Ig


Other immunoglobulinsOther immunoglobulins

IgEIgEmonomer with extra domain in the constant regionleast common Iginvolved in allergic reactionsIgE also plays a role in parasitic helminth diseases does not fix complement

IgDIgDmonomerlow levels in serumuncertain roledoes not fix complement


ImmunoreactionImmunoreactionprimary humoral immune response to an antigen:

- following exposure, IgM is the first antibody to appear

- followed by a much higher titre of IgG

Reinfection:

- IgM remains the same or rises slightly

- IgG shoots up rapidly and earlier than in primary infection


Criteria for diagnosing primary InfectionCriteria for diagnosing primary Infection

Presence of IgM:- offers rapid diagnosis- might be negative at convalescent phase - possible interference with RF, polyclonal stimulation (other antigen)- also with re-infection and sometimes unexplained persistence of

IgM years after primary infection

Seroconversion: changing from a previously antibody (Ab) negative status (seronegative) to a positive status (seropositive). Negative IgM & IgG do not exclude exposure to antigen (incubation time).

Presence of IgG:- significant rise between acute and convalescent (recovery) phase - titre of IgG depending on individual - single high titre of IgG is a very unreliable means of serological

diagnosis


Criteria for diagnosing Criteria for diagnosing re-infection/re-activationre-infection/re-activation

In general:

- sharp rise of IgG

- low or absent IgM

Importance of diagnosing:

- diseases like e.g. Rubella where re-infection cause no fetal damage in pregnant women, whereas a primary infection in the first trimester does

- often very difficult to differentiate re-infection/re-activation from primary infection


Recovery primary infection or old infection ?Recovery primary infection or old infection ?

How to differentiate ? - Low avidity IgG recovery primary infection & recent infection - High avidity IgG old infection

Importance of differentiation: - primary infection of TORCH (Toxo, Rubella, CMV) during first trimester of pregnant women causes fetal damage- checking if vaccination has been succesfull

Screening population: - IgG present at acute & recovery phase and will remain detectable for mainy years, even for life time


Avidity & Affinity Avidity & Affinity The difference ?The difference ?

Avidity: - avidity is a measure of the overall strength of binding of an antigen (Ag) with many antigenic determinants and multivalent Ab’s … with other words: the eagerness of the Ab to match with an Ag- avidity is more than the sum of the individual affinities- the older the infection, the higher the avidity- the higher the avidity, the stronger the Ag-Ab binding

Affinity: - Ab affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody …. in other words: the degree of match between Ag & Ab


IgG Avidity

Avidity increases with the maturation of the immune response


Polyclonal and monoclonal Ab’sPolyclonal and monoclonal Ab’s

Monoclonal Ab’s: - derived from a single plasma cell and specific for one epitope- useful for in vitro & in vivo diagnostic products

Polyclonal Ab’s: - each B-cell clone produces Ab specific for one particular Ag epitope- but serum Ab’s are produced as a response to complex Ag, therefore contains a mixture of Ab’s raised against different Ag epitopes- each cell clone produce Ab’s specific for one of the various epitopes of the Ag this polyclonal response is advantageous for the immune system because it helps localization, phagocytosis, and complement mediated lysis of the Ag


Cross-reactivityCross-reactivitywith diagnostic productswith diagnostic products

« Non artificial » cross-reactivity:

- a polyclonal reaction after infection with other Ag

- Auto-immune diseases

- Other non viral, bacteriological or parasitological agentse.g. rheumatoid factor, anti-caseine, other proteins, …

« Artificial » cross-reactivity:

- caused by non specific monoclonals/polyclonals used in test kitcross-reactivity with related Ag’s, e.g. the herpesviridea

giving mainly false positive results

- appearance of the sample testede.g. haemolytic, icteric & lipemic samples

giving false positive or negative results


AntigensAntigensViruses – Bacteria - ParasitesViruses – Bacteria - Parasites

Bacteria:procariotic organisms

single-celled organisms able to reproduce by duplicating themselves

responsive to antibiotics typical bacterium: rigid cell wall, thin rubbery cell membrane surrounding the cytoplasma, various shapes (coccus, rod, vibrion etc)

contains all genetic information to copy itself – DNA in the chromosome

plasmids: extra circular bits of DNA

ribosomes: necessary for copying DNA

some have flagella


envelope: outermost spiky layer

protein coat

core of genetic material: DNA or RNA

The biggest viruses are only as large as the tiniest bacteria.

Virus:

lives within a cell (intracellular) to survive and needs host cell for multiplication

not responsive to antibiotics

protection through vaccination (not for all viruses)



Parasite: eukaryotic organisms

lives in or on the living tissue of a host organism

lives at the expense of the host

parasitism = biological interaction between host and parasite

Some parasites:

Cryptosporidium parvumoocytes

Giardia intestinalis


Toxoplasma gondii


Infectious diseases Infectious diseases Chorus Elisa lineChorus Elisa line

Kits currently available for the detection of Ab’s against:

TORCH panel:Toxoplasma gondiiRubella virusCytomegalovirus (CMV)

Epstein-Barr virus (EBV)

Measles

Mumps

Herpes Simplex virus type 1 & 2

Varicella zoster virus (VZV)

Helicobacter pylori

Treponema pallidum (Syphilis)


Viruses: Viruses: different families different families (1)(1)

HERPESVIRIDEA (DNA viruses)8 human herpes types

Alpha group HV-1 Herpes simplex 1 Fever blisters, encephalitis

HV-2 Herpes simplex 2 Genital, meningitis/enecephalitis (newborns)

HV-3 Varicella zoster Chicken pox, shingles

Beta group HV-4 Cytomegalovirus Infectious mononucleosis, neonatal fatality

Gamma group HV-5 Epstein-Barr virus Infectious mononucleosis, cancer (Lymphoma)

Other HV-6 HBLV (human B-lymphotropic virus)

B cells

HV-7 T cells (CD4+)

HV-8 Sarcoma, lymphoma


Viruses: Viruses: different families (2)different families (2)

PARAMYXOVIRIDEA (RNA viruses)

Subfamily Genus Human Species

Paramyxoviridea Paramyxovirus Human parainfluenza viruses types 1 & 3

Rubulavirus Human parainfluenza viruses types 2, 4a and 4b,

Mumps virus

Morbillivirus Measles virus

Pneumovirnea Pneumovirus Human respiratory syncytial virus



Viruses: Viruses: different families (3)different families (3)

TOGAVIRIDEA (RNA viruses)

*: rubella (German measles)

Genus Species

Alphavirus Sindbis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Ross River virus, O’nyong’nyong virus

Rubivirus Rubella virus*



BacteriaBacteriaHelicobacter pylori

slow-growing Gram – spiral shaped bacterium. It lives in the stomach and duodenum.

H. pylori is very common. About 2/3 of the world's adult population are infected with it. Almost everyone who has a duodenal ulcer has H. pylori, but not everyone that has H. pylori

develops an ulcer.

Treponema pallidum

It is a spirochete, a helical to sinusoidal bacterium

It is the causative agent of Syphilis

Despite the availability of effective therapy (penicillin), syphilis remains a commonly sexually transmitted disease worldwide


ParasitesParasitesToxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite

Toxoplasma gondii has very low host specificity, will probably infect almost any mammal.

2 life cycles: the intestinal and extraintestinal phases

intestinal phase occurs in cats only produces « oocysts »

extratestinal phase in all infected animals (including cats) produces tachyzoites and eventually bradyzoites & zoitocysts


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Infectious Diseases


Childhood diseasesChildhood diseases

The most common childhood diseases are: Viral infections

Measles Mumps Rubella* Chicken pox Respiratory syncytial virus (RSV) Rotavirus The fifth disease (Parvovirus)

Bacterial infections Wooping Cough (pertussis) Meningitis Scarlet fever

* Risk for pregnant women


Other infectious diseasesOther infectious diseases

Toxoplasmose (Toxoplasma gondii)* Cytomegalovirus (CMV)* Infectious Mononucleosis with Epstein-Barr virus (EBV) Herpes Simplex virus, type 1 (oral-facial lesions) Herpes Simplex virus, type 2 (genital lesions) Influenza A & B Hepatitis A & B Helicobacter Pylori Syphilis ………

* Risk for pregnant women


VaccinationsVaccinations

1. Aim is to prevent epidemics which caused many deaths in the past.

2. Vaccines help the body produce antibodies protection.3. Most common vaccines:

MMR: measles, mumps & rubella IPV: inactivated poliovirus DTaP: diphetheria, tetanus & pertussis Varicella vaccine HBV & HAV: hepatitis A & B Influenza PCV: pneumococcal conjugate vaccine ……….

4. Pipeline: hepatitis C, RSV, HIV, CMV, EBV, …..5. Far future: HSV 1&2, adenovirus, toxoplasmose


HSV-1HSV-1

• oral-facial lesions

• most common HSV

• usually acquired in childhood

• fever blisters inside mouth

• transmitted by infected saliva

• 90% of adults have Ab’s

HSV-1HSV-1

• oral-facial lesions

• most common HSV

• usually acquired in childhood

• fever blisters inside mouth

• transmitted by infected saliva

• 90% of adults have Ab’s

Herpes Simplex 1 & 2 Herpes Simplex 1 & 2 (1)(1)

both belonging to the Herpesviridea slight antigenic difference between HSV-1 and HSV-2

HSV-2HSV-2

• genital lesions (ulcers or sores)

• possible complications: meningoencephalitis or infection of the eye (acquired at burth, passing through infected mother’s birth canal)

• can also be asymptomatic

• sexually transmitted

• 25% of 25-45 years old of US population

HSV-2HSV-2

• genital lesions (ulcers or sores)

• possible complications: meningoencephalitis or infection of the eye (acquired at burth, passing through infected mother’s birth canal)

• can also be asymptomatic

• sexually transmitted

• 25% of 25-45 years old of US population

Cross-infection of type 1& 2 viruses may occur from oral-genital contact.



Human response to infection: primary infection often subclinal and rarely diagnosed primary infection ? first IgM followed by IgG virus stays in your system once you’ve been infected reactivation after latency period, may or may not rise lesions

Why testing ? Infection contracted during birth is of particular interest

important cause of morbidity and mortality

!! Important to determine the immunitary state of women during pregnancy in order to detect serum conversion !!



Recommended testing:

Seronegative

NEGATIVE

Acute/Recentin fection

IgMPO SITIVE

Old infection(Ig G p ositive)

IgMNEG ATIVE

Herpes Sim plex 1+2 IgM

POSITIVEIgG or IgM ?

HSV 1 Screenand

HSV 2 Screen(IgM + IgG)

Chorus line:- HSV-1 Screen (IgM & IgG)- HSV-2 Screen (IgM & IgG)- Herpes Simplex 1+2 IgM


Helicobacter pylori Helicobacter pylori (1)(1)

a bacteria responsible for most ulcers and many cases of chronic gastritis

weakens protective coating of stomach & duodendum digestive juices will iritate sensitive lining of these organs

H. pylori infection is linked to stomach cancer having H. pylori in gastrointestinal tract is not always

leading to ulcer or gastritis other factors increase risk



Human response to infection: most patients exhibit a strong immune response locally mostly IgA antibodies circulating antibodies are primarily IgG IgGs remain constantly high until infection is eliminated

(antibiotics)

Treatment of H. pylori: concentration of Ab’s does not correlate with disease severity but

succesful treatment can be monitored by decrease of Ab’s Efficacy of treatment decrease in IgG level over time

No treatment:

Spontaneous clearing of H. pylori by IgA & IgG is rare

elevated IgA & IgG levels, indicates current infection



Recommended testing: IgA and IgG

Chorus line:- H. pylori IgA- H. pylori IgG

Seronegative

H. Pylori IgGNEG AT IVE

H. Pylori IgANEG AT IVE

Case 1

Start prim ar infection

H. Pylori IgGNEG AT IVE

H. Pylori IgAPO SIT IVE

Case 2

Follow -up sam pleIncrease IgG = acute inf.

Decrease IgG = im pact drug or recoveryStable = past infection

Acute infectionor

past infection ?Drug m onitoring

H. Pylori IgGPO SIT IVE

H. Pylori IgAPO SIT IVE

Case 3

Treated infection ?F o llo w -up sam p le

H. Pylori IgGPO SIT IVE

H. Pylori IgANEG AT IVE

Case 4

Introduction to Serology - Immunology, HSV & H.pylori

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