Lecture on Serological Diagnosis of Infectious Diseases And

LECTURE ON SEROLOGICAL LECTURE ON SEROLOGICAL DIAGNOSIS OF INFECTIOUS DIAGNOSIS OF INFECTIOUS

DISEASES AND TUMOR MARKERSDISEASES AND TUMOR MARKERS

ROBERTO D. PADUA JR., MD, DPSPROBERTO D. PADUA JR., MD, DPSPDEPARTMENT OF PATHOLOGY AND LABORATORY DIAGNOSISDEPARTMENT OF PATHOLOGY AND LABORATORY DIAGNOSIS

FATIMA COLLEGE OF MEDICINEFATIMA COLLEGE OF MEDICINE

SEROLOGICAL DIAGNOSIS OF INFECTIOUS SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASESDISEASES

• SEROLOGYSEROLOGY•The scientific study of blood sera and their The scientific study of blood sera and their

effectseffects

•Subdivision of immunology concerned with Subdivision of immunology concerned with in-vitro Ag-Ab reactionin-vitro Ag-Ab reaction

•Concerned with the laboratory study of the Concerned with the laboratory study of the activities of the components of serum that activities of the components of serum that contribute to immunitycontribute to immunity


• IMMUNOLOGYIMMUNOLOGY•The study of the molecules, cells, organs The study of the molecules, cells, organs

and systems responsible for the recognition and systems responsible for the recognition and disposal of foreign (non-self) materialand disposal of foreign (non-self) material

•The study of how the body components The study of how the body components respond and interactrespond and interact

•The desirable and undesirable consequences The desirable and undesirable consequences of immune interactionsof immune interactions

•The ways in which the immune system can The ways in which the immune system can be manipulated to protect or treat diseasebe manipulated to protect or treat disease


• IMMUNITYIMMUNITY•The ability of an organism to resist infection The ability of an organism to resist infection

by means of the presence of circulating by means of the presence of circulating antibodies and white blood cellsantibodies and white blood cells

•Distinctive characteristics of the immune Distinctive characteristics of the immune systemsystem

SpecificitySpecificity MemoryMemory MobilityMobility ReplicabilityReplicability cooperativitycooperativity


• METHODS OF DETECTION OF ANTIBODIESMETHODS OF DETECTION OF ANTIBODIES

1.1. Immuno-precipitation AssaysImmuno-precipitation Assays

= detect antibodies in solution= detect antibodies in solution

= qualitative indication of the presence = qualitative indication of the presence of of antibodiesantibodies

= end-point is visual flocculation of the = end-point is visual flocculation of the antigen and antibody in antigen and antibody in

suspensionsuspension



2. Complement Fixation2. Complement Fixation

= based on the activation or fixation of = based on the activation or fixation of complement following complement following

binding of binding of complement factors to complement factors to Ag-Ab immune Ag-Ab immune complexescomplexes



3. Neutralization3. Neutralization

= the ineffectivity of an organism or = the ineffectivity of an organism or the the activity of toxin is activity of toxin is neutralized by neutralized by specific antibodyspecific antibody

= rarely used for diagnostic purposes= rarely used for diagnostic purposes

= mainly used to detect antibody = mainly used to detect antibody formation formation after vaccinationafter vaccination


• METHODS OF DETECTION OF ANTIBODIESMETHODS OF DETECTION OF ANTIBODIES4. Particle Agglutination4. Particle Agglutination

= relatively simple and fast= relatively simple and fast= capable of detecting lower concentration of = capable of detecting lower concentration of antibodiesantibodies= designed to detect antibodies to viruses, = designed to detect antibodies to viruses, subsequent to interaction or vaccinationsubsequent to interaction or vaccination= utilize Ag coated latex particles, coal particles, = utilize Ag coated latex particles, coal particles,

bentonite particles or erythrocytesbentonite particles or erythrocytes= direct and indirect methods= direct and indirect methods



5. Immunofluorescence5. Immunofluorescence

= requires use of microscope equipped = requires use of microscope equipped to to provide ultraviolet provide ultraviolet illumination or an illumination or an instrument instrument capable of irradiating the capable of irradiating the assay assay with UV light and detecting the with UV light and detecting the resultant fluorescence with a resultant fluorescence with a fluorometerfluorometer


• METHODS OF DETECTION OF ANTIBODIESMETHODS OF DETECTION OF ANTIBODIES6. Enzyme Immunoassay6. Enzyme Immunoassay

= the most sensitive = the most sensitive

= usually indirect assay that depends on the use = usually indirect assay that depends on the use of of an antihuman IgG or IgM antibody an antihuman IgG or IgM antibody conjugateconjugate

= the antibody conjugate (if present) is made to = the antibody conjugate (if present) is made to attach to enzyme which catalyzes attach to enzyme which catalyzes

conversion conversion of the substrate to a colored product of the substrate to a colored product which which will then be read with the use of a will then be read with the use of a spectrophotometerspectrophotometer



7. Radioimmunoassay7. Radioimmunoassay

= high sensitivity= high sensitivity


• Microbial antigen detection provides Microbial antigen detection provides direct evidence of infection, and is direct evidence of infection, and is preferred for diagnosis of infection over preferred for diagnosis of infection over antibody detection (indirect evidence of antibody detection (indirect evidence of infection)infection)

• However, not all infectious agents have However, not all infectious agents have available antigen assays or culture available antigen assays or culture techniques making the detection of techniques making the detection of specific antibodies diagnostically usefulspecific antibodies diagnostically useful


• Infectious Disease Indicators, Non-Infectious Disease Indicators, Non-specificspecific

• Acute phase reactantsAcute phase reactants

• Limulus lysate assayLimulus lysate assay– Detects trace amounts of endotoxin from all Detects trace amounts of endotoxin from all

gram (-) bacteriagram (-) bacteria– Presence in CSF = gram (-) bacterial meningitisPresence in CSF = gram (-) bacterial meningitis– Rapid clearance from blood makes serum test Rapid clearance from blood makes serum test

unreliableunreliable


• Molecular BiologyMolecular Biology• Nucleic acid amplificationNucleic acid amplification

• DNA sequencing and typingDNA sequencing and typing

• Direct molecular probe (in situ Direct molecular probe (in situ hybridization)hybridization)

• Nucleic acid quantitationNucleic acid quantitation


• Molecular BiologyMolecular Biology– Uses:Uses:

• Cases requiring increased sensitivity and specificity Cases requiring increased sensitivity and specificity of identificationof identification

• Cases requiring faster report turnaround timeCases requiring faster report turnaround time• Confirmation of cultureConfirmation of culture• Identification of organisms that are non-viable or Identification of organisms that are non-viable or

cannot be culturedcannot be cultured• Identification of fastidious, slow growing organismsIdentification of fastidious, slow growing organisms• Identification of organisms that are dangerous to Identification of organisms that are dangerous to

cultureculture• Identification of organisms in small numbers or in Identification of organisms in small numbers or in

small volume specimenssmall volume specimens


• Molecular BiologyMolecular Biology– Uses:Uses:

• Density of amplifiable DNA correlates with microbial Density of amplifiable DNA correlates with microbial densitydensity

• Monitoring of disease progression or initiation or Monitoring of disease progression or initiation or modification of therapymodification of therapy

• Drug susceptibility testingDrug susceptibility testing• Differentiation of antigenically similar organismsDifferentiation of antigenically similar organisms• Molecular epidemiology and infection controlMolecular epidemiology and infection control• Disease diagnosis by characterization of genetic Disease diagnosis by characterization of genetic

materials without direct identification of infectious materials without direct identification of infectious agentagent

• Determination of virulence of antimicrobial Determination of virulence of antimicrobial resistance genesresistance genes


• SYPHILISSYPHILIS• The most commonly acquired spirochete disease in The most commonly acquired spirochete disease in

the U.S.the U.S.

• A complex sexually transmitted disease that has a A complex sexually transmitted disease that has a highly variable clinical coursehighly variable clinical course

• Over 50,000 cases reported in 1990 in the U.S.Over 50,000 cases reported in 1990 in the U.S.

• Causative agent is Treponema pallidumCausative agent is Treponema pallidum

• No natural reservoir in the environment, requires No natural reservoir in the environment, requires living hostliving host

• Spiral shaped and motile due to peri-plasmic flagellaSpiral shaped and motile due to peri-plasmic flagella

• Variable lengthVariable length


• SYPHILISSYPHILIS• Three other pathogens in the group Three other pathogens in the group

Treponema which are morphologically and Treponema which are morphologically and anti-genetically similar to T. pallidum, anti-genetically similar to T. pallidum, differences are in characteristics of lesions, differences are in characteristics of lesions, amount of systemic involvement and course amount of systemic involvement and course of the diseaseof the disease

T. pertenue (Yaws)T. pertenue (Yaws) T. endemicum (non-venereal syphilis)T. endemicum (non-venereal syphilis) T. carateum (pinta)T. carateum (pinta) T. cuniculi (rabbit syphilis)T. cuniculi (rabbit syphilis)


• SYPHILISSYPHILIS• Mode of TransmissionMode of Transmission

– Organism is very fragile, destroyed rapidly by Organism is very fragile, destroyed rapidly by heat, cold and dryingheat, cold and drying

– Sexual transmission most common, occurs when Sexual transmission most common, occurs when abraded skin or mucous membranes come in abraded skin or mucous membranes come in contact with open lesioncontact with open lesion

– Can be transmitted to fetusCan be transmitted to fetus– Rare transmission from needle stick and blood Rare transmission from needle stick and blood

transfusiontransfusion


• SYPHILISSYPHILIS - - Stages of the Disease - - Stages of the Disease1.1. Primary stagePrimary stage

= primary lesion is chancre= primary lesion is chancre= the lesion heals spontaneously after 1-5 = the lesion heals spontaneously after 1-5 weeksweeks= swab of chancre smeared on slide, = swab of chancre smeared on slide, examined under dark-field microscope, examined under dark-field microscope, spirochetes will be presentspirochetes will be present= 30% become serologically positive one = 30% become serologically positive one week after appearance of chancre, 90% week after appearance of chancre, 90% positive after three weekspositive after three weeks


• SYPHILIS SYPHILIS - - Stages of the Disease- - Stages of the Disease2. Secondary Stage2. Secondary Stage

= occurs 6-8 weeks after initial chancre, = occurs 6-8 weeks after initial chancre, becomes systemic, patient highly infectiousbecomes systemic, patient highly infectious= characterized by localized or diffuse = characterized by localized or diffuse mucocutaneous lesions, often with mucocutaneous lesions, often with generalized lymphadenopathygeneralized lymphadenopathy= primary chancre may still be present= primary chancre may still be present= secondary lesions subside in about 2-6 = secondary lesions subside in about 2-6 weeksweeks= serology tests nearly 100% positive= serology tests nearly 100% positive


• SYPHILIS SYPHILIS - - Stages of the Disease- - Stages of the Disease3. Latent Stage3. Latent Stage

= stage of infection in which organisms = stage of infection in which organisms persists in the body of the infected person persists in the body of the infected person without causing symptoms or signswithout causing symptoms or signs= this stage may last for years= this stage may last for years= one-third of untreated latent stage = one-third of untreated latent stage individuals develop signs of tertiary syphilisindividuals develop signs of tertiary syphilis= after 4 years it is rarely communicable = after 4 years it is rarely communicable sexually but can be passed from mother to sexually but can be passed from mother to fetusfetus


• SYPHILISSYPHILIS - - Stages of the Disease - - Stages of the Disease4. Tertiary Stage4. Tertiary Stage

= occurs anywhere from months to = occurs anywhere from months to years after secondary stage, typically years after secondary stage, typically between 10 to 30 yearsbetween 10 to 30 years

= gummatous syphilis= gummatous syphilis

= cardiovascular syphilis= cardiovascular syphilis

= neurosyphilis= neurosyphilis


• SYPHILISSYPHILIS• Congenital SyphilisCongenital Syphilis

Transmitted from mother to fetusTransmitted from mother to fetus Fetus affected during the second or third trimesterFetus affected during the second or third trimester 40% result in syphilitic stillbirth40% result in syphilitic stillbirth Live-born infants show no signs during first few Live-born infants show no signs during first few

weeksweeks= 60-90% develop clear or hemorrhagic rhinitis= 60-90% develop clear or hemorrhagic rhinitis= skin eruptions (rash) especially around mouth, = skin eruptions (rash) especially around mouth, palms of hands and soles of feetpalms of hands and soles of feet= general lymphadenopathy, = general lymphadenopathy, hepatosplenomegaly, jaundice, anemia, painful hepatosplenomegaly, jaundice, anemia, painful limbs & bone abnormalitylimbs & bone abnormality


• SYPHILISSYPHILIS - - DIAGNOSIS - - DIAGNOSIS• Evaluation based on 3 factorsEvaluation based on 3 factors

Clinical findingsClinical findings Demonstration of spirochetes in clinical Demonstration of spirochetes in clinical

specimenspecimen Present of antibodies in blood or CSFPresent of antibodies in blood or CSF

= more than one test = more than one test should be performedshould be performed

= no serological test = no serological test can distinguish between can distinguish between other other treponemal infectionstreponemal infections


• SYPHILISSYPHILIS - - DIAGNOSIS - - DIAGNOSIS• Laboratory TestingLaboratory Testing

A.A. Direct examination of clinical specimen by dark-Direct examination of clinical specimen by dark-field microscopy or fluorescent antibody testing of field microscopy or fluorescent antibody testing of samplesample

B.B. Non-specific or non-treponemal serological test to Non-specific or non-treponemal serological test to detect reagin, utilized as screening test only, not detect reagin, utilized as screening test only, not diagnosticdiagnostic= Reagin is an antibody formed against cardiolipin= Reagin is an antibody formed against cardiolipin= Found in sera of patients with syphilis as well as = Found in sera of patients with syphilis as well as

other diseases other diseases= Non-treponemal tests become positive 1-4 = Non-treponemal tests become positive 1-4 weeks weeks after appearance of primary after appearance of primary chancre, in secondary chancre, in secondary stage may have false stage may have false positive due to prozone, in positive due to prozone, in tertiary 25% are tertiary 25% are negative, after successful negative, after successful treatment will treatment will become non-reactive after 1 to 2 become non-reactive after 1 to 2 years years


• SYPHILISSYPHILIS - - DIAGNOSIS - - DIAGNOSIS• Laboratory TestingLaboratory Testing

C. Specific Treponemal antibody tests are used as a C. Specific Treponemal antibody tests are used as a confirmatory test for a positive reagin testconfirmatory test for a positive reagin test

SEROLOGICAL DIAGNOSIS OF INFECTIOUS SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = DISEASES = SYPHILISSYPHILIS

• NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN TESTTEST

1.1. Venereal Disease Research Laboratory=VDRL Venereal Disease Research Laboratory=VDRL = Flocculation test, antigen consists of very fine particles = Flocculation test, antigen consists of very fine particles that that precipitate out in the presence of reaginprecipitate out in the presence of reagin= Utilizes antigen consists of cardiolipin, cholesterol and = Utilizes antigen consists of cardiolipin, cholesterol and

lecithinlecithin= serum must be heated to 56 C for 30 minnutes to = serum must be heated to 56 C for 30 minnutes to remove remove anti-complimentary activity which may anti-complimentary activity which may cause false cause false positivepositive= reported as Non-reactive, weakly reactive and reactive= reported as Non-reactive, weakly reactive and reactive= used primarily to screen CSF= used primarily to screen CSF


• NON-TREPONEMAL SEROLOGICAL TESTS – NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN TESTREAGIN TEST

2. Rapid Plasma Reagin – RPR2. Rapid Plasma Reagin – RPR= general screening test= general screening test= can not be performed on CSF= can not be performed on CSF= the VDRL cardiolipin antigen is modified with choline = the VDRL cardiolipin antigen is modified with choline

chloride to make it more stable and is chloride to make it more stable and is attached to attached to charcoal particles to allow macroscopic charcoal particles to allow macroscopic reading, the reading, the antigen comes prepared and is very antigen comes prepared and is very stablestable= serum or plasma may be used for testing, serum is = serum or plasma may be used for testing, serum is not not heatedheated= results are read macroscopically= results are read macroscopically= appears to be more sensitive than the VDRL = appears to be more sensitive than the VDRL


• NON-TREPONEMAL SEROLOGICAL TESTS NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN TEST– REAGIN TEST

3. Other tests which use modified VDRL Ag3. Other tests which use modified VDRL AgA. USR – unheated serum reagin testA. USR – unheated serum reagin test

= modified VDRL Ag, uses choline = modified VDRL Ag, uses choline chloride/EDTA chloride/EDTA= microscopic flocculation test= microscopic flocculation test

B. RST – reagin screen testB. RST – reagin screen test= modified VDRL Ag with Sudan = modified VDRL Ag with Sudan

BlackBlack= Sudan Black makes flocculation = Sudan Black makes flocculation

reaction reaction macroscopically visible macroscopically visible


• SPECIFIC TREPONEMAL TESTSSPECIFIC TREPONEMAL TESTS

1.1. Treponema pallidum Immobilization Treponema pallidum Immobilization Test – TPITest – TPI

= live T. pallidum become immobilized = live T. pallidum become immobilized by by antibody in serum of antibody in serum of infected personsinfected persons

= cumbersome and expensive, no = cumbersome and expensive, no longer longer used in U.S.used in U.S.



2. Treponema pallidum Hemagglutination – 2. Treponema pallidum Hemagglutination – TPHATPHA

= adapted to microtechniques (MHA-TP)= adapted to microtechniques (MHA-TP)

= tanned sheep RBC’s are coated with T. = tanned sheep RBC’s are coated with T. pallidum antigen from Nichol’s pallidum antigen from Nichol’s

strainstrain

= positive result is agglutination of RBC’s= positive result is agglutination of RBC’s


• SPECIFIC TREPONEMAL TESTSSPECIFIC TREPONEMAL TESTS3. Fluorescent treponemal antibody absorption test (FTA-ABS)3. Fluorescent treponemal antibody absorption test (FTA-ABS)

= one of the most used confirmatory test= one of the most used confirmatory test= diluted, heat inactivated serum added to Reiter’s strain = diluted, heat inactivated serum added to Reiter’s strain of T. of T. pallidum to remove cross reactivity due to pallidum to remove cross reactivity due to other other TreponemesTreponemes= slides are coated with Nichol’s strain of T. pallidum and = slides are coated with Nichol’s strain of T. pallidum and add add absorbed patient serumabsorbed patient serum= slides are washed and incubated with Ab bound to a = slides are washed and incubated with Ab bound to a fluorescent tagfluorescent tag= after washing again the slides are examined for = after washing again the slides are examined for fluorescencefluorescence= requires experienced personnel to read= requires experienced personnel to read= highly sensitive and specific, but time consuming to = highly sensitive and specific, but time consuming to performperform



4. ELISA4. ELISA

= tubes coated with T. pallidum antigen= tubes coated with T. pallidum antigen

= antibody in serum attaches to antigen= antibody in serum attaches to antigen

= following washing, add an anti-= following washing, add an anti-antibody antibody tagged with enzyme alkaline tagged with enzyme alkaline phosphatasephosphatase

= detectable color changes occur= detectable color changes occur

Sensitivity and Specificity of Serologic Tests Sensitivity and Specificity of Serologic Tests for Untreated Syphilis at Different Stages for Untreated Syphilis at Different Stages

Serologic Test for Syphilis in Various Serologic Test for Syphilis in Various ConditionsConditions

Algorithm for Positive Serologic Test for Algorithm for Positive Serologic Test for SyphilisSyphilis


• PROBLEM AREASPROBLEM AREAS1.1. Biologic False Positives (BFP)Biologic False Positives (BFP)

A. Collagen diseases such as arthritis, A. Collagen diseases such as arthritis, LE, LE, etc., sometimes result in etc., sometimes result in increased increased amount of reaginamount of reagin

B. Certain infections : IM, malaria, B. Certain infections : IM, malaria, leprosyleprosy

C. Other treponemal infectionsC. Other treponemal infections


• PROBLEM AREASPROBLEM AREAS2. False negatives2. False negatives

A. Very early in disease or latent, A. Very early in disease or latent, inactive inactive stagestage

B. Immunosuppressed patientsB. Immunosuppressed patients

C. Consumption of alcohol prior to C. Consumption of alcohol prior to testing testing (temporary)(temporary)


• PROBLEM AREASPROBLEM AREAS3. Congenital syphilis3. Congenital syphilis

A. Non-treponemal tests on cord blood or A. Non-treponemal tests on cord blood or baby serum detect IgG baby serum detect IgG

antibody, antibody, maybe of maternal originmaybe of maternal originB. Detection of IgM lacks sensitivityB. Detection of IgM lacks sensitivityC. Western blot has demonstrated high C. Western blot has demonstrated high sensitivity and specificitysensitivity and specificityD. Recommended that all mothers be D. Recommended that all mothers be testedtested


• PROBLEM AREASPROBLEM AREAS4. Cerebrospinal Fluid tests4. Cerebrospinal Fluid tests

A. Used to determine if Treponemes A. Used to determine if Treponemes have have invaded the CNSinvaded the CNS

B. VDRL utilized to confirm B. VDRL utilized to confirm neurosyphilisneurosyphilis

C. Lacks sensitivityC. Lacks sensitivity


• CORRELATION OF TREATMENT WITH CORRELATION OF TREATMENT WITH TEST RESULTSTEST RESULTS

A.A. Treatment at the primary stage, serology Treatment at the primary stage, serology tests become non-reactive after 6 monthstests become non-reactive after 6 months

B.B. Treatment at secondary stage, tests Treatment at secondary stage, tests usually non-reactive after 12-18 monthsusually non-reactive after 12-18 months

C.C. If treatment is not initiated until 10 or If treatment is not initiated until 10 or more years, the reagin tests probably more years, the reagin tests probably positive for lifepositive for life


• LYME’S DISEASELYME’S DISEASE= Disease first recognized in 1977 in Lyme, = Disease first recognized in 1977 in Lyme, ConnecticutConnecticut= Causative organism is Borrelia burgdorferi= Causative organism is Borrelia burgdorferi= Can be cultured but it is very difficult= Can be cultured but it is very difficult= Organism has been isolated from blood, CSF, skin = Organism has been isolated from blood, CSF, skin lesions lesions and joint fluidand joint fluid= Can be transmitted perinatally, causing intrauterine = Can be transmitted perinatally, causing intrauterine deathdeath= Vector of transmission is the Ixodes tick= Vector of transmission is the Ixodes tick= Must remain attached a minimum of 24-48 hours for = Must remain attached a minimum of 24-48 hours for

transmission to occurtransmission to occur

SEROLOGICAL DIAGNOSIS OF INFECTIOUS SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = DISEASES = LYME’S DISEASELYME’S DISEASE

• STAGES OF THE DISEASESTAGES OF THE DISEASE1.1. Localized rash – erythema chronicum migransLocalized rash – erythema chronicum migrans2.2. Dissemination to multiple organ systemDissemination to multiple organ system

= occurs by way of the bloodstream= occurs by way of the bloodstream= may occur weeks to months after infection= may occur weeks to months after infection= migratory pain may occur in the joints, tendons and = migratory pain may occur in the joints, tendons and bonesbones= neurologic = neurologic Bell’s palsy, peripheral neuropathy, Bell’s palsy, peripheral neuropathy, aseptic aseptic meningitismeningitis= cardiac include carditis and arrythmia= cardiac include carditis and arrythmia

3. Chronic disseminated3. Chronic disseminated= characterized by chronic arthritis= characterized by chronic arthritis= affects the large joints, especially the knee= affects the large joints, especially the knee


• Diagnostic criteriaDiagnostic criteria• Isolation of organism from clinical Isolation of organism from clinical

specimen specimen oror

• Diagnostic titers of IgG and IgM in serum or Diagnostic titers of IgG and IgM in serum or CSF CSF oror

• Significant change in serum titers of IgG or Significant change in serum titers of IgG or IgM in paired acute and convalescent seraIgM in paired acute and convalescent sera


• LABORATORY DIAGNOSISLABORATORY DIAGNOSIS• Diagnosed clinically, confirmed serologicallyDiagnosed clinically, confirmed serologically• Antibodies to antigens of B. burgdorferi can be detected by Antibodies to antigens of B. burgdorferi can be detected by

latex agglutination, IFA, ELISA, and Western Blotlatex agglutination, IFA, ELISA, and Western Blot• Serological tests are often falsely negative during early Serological tests are often falsely negative during early

weeks.weeks. Specific IgM Abs usually appear 2- 4 weeks after Specific IgM Abs usually appear 2- 4 weeks after

erythema migrans, peak after 3-6 weeks of illness, decline erythema migrans, peak after 3-6 weeks of illness, decline to normal after 4-6 months to normal after 4-6 months

IgG titers appears more slowly (4-8 weeks after the rash), IgG titers appears more slowly (4-8 weeks after the rash), peak after 4-6 months, may remain high for months or peak after 4-6 months, may remain high for months or yearsyears

• Western Blot is most sensitiveWestern Blot is most sensitive• IFA and ELISA are more commonly performed due to ease IFA and ELISA are more commonly performed due to ease

of procedure, but are subject to false positives due to either of procedure, but are subject to false positives due to either spirochete diseases and some autoimmune diseasesspirochete diseases and some autoimmune diseases

SEROLOGICAL DIAGNOSIS OF INFECTIOUS SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = DISEASES = STREPTOCOCCAL INFECTIONSTREPTOCOCCAL INFECTION

• STREPTOCOCCAL SEROLOGYSTREPTOCOCCAL SEROLOGY• Streptococci are gram (+), beta-hemolytic, Streptococci are gram (+), beta-hemolytic,

spherical, ovoid, or lancet-shaped organisms which spherical, ovoid, or lancet-shaped organisms which are catalase negative and seen in pairs or chainsare catalase negative and seen in pairs or chains

• Divided into groups or serotypes based on cell wall Divided into groups or serotypes based on cell wall components components Streptococcus pyogenes belongs to Streptococcus pyogenes belongs to Lancefield group A and it is believed the M protein Lancefield group A and it is believed the M protein is the chief virulent factor of this group is the chief virulent factor of this group

• Numerous exo-antigens are produced and excreted Numerous exo-antigens are produced and excreted as the cell metabolizes (Streptolysin O, DNase, as the cell metabolizes (Streptolysin O, DNase, Hyaluronidase, Nicotinamide, Adenine Hyaluronidase, Nicotinamide, Adenine dinucleotidase (NADase), Streptokinase)dinucleotidase (NADase), Streptokinase)

• Culture and rapid screening tests detect early Culture and rapid screening tests detect early infectioninfection

• Sequelae include Rheumatic Fever and Acute GN Sequelae include Rheumatic Fever and Acute GN


• GROUP A STREPTOCOCCAL GROUP A STREPTOCOCCAL INFECTIONINFECTION

• Two major sites of infection : upper Two major sites of infection : upper respiratory tract and skinrespiratory tract and skin

• Upper respiratory tract = sore throat, Upper respiratory tract = sore throat, tonsillar exudatetonsillar exudate

• Skin = pyoderma or impetigoSkin = pyoderma or impetigo• Suppurative complications = erysipelas, Suppurative complications = erysipelas,

scarlet fever, septic arthritis, meningitisscarlet fever, septic arthritis, meningitis• Non-suppurative complications = RF or Post-Non-suppurative complications = RF or Post-

streptococcal GNstreptococcal GN


• GROUP A STREPTOCOCCAL INFECTIONGROUP A STREPTOCOCCAL INFECTIONA.A. Rheumatic FeverRheumatic Fever

= only certain serotypes of S. pyogenes is involved= only certain serotypes of S. pyogenes is involved= develops as sequelae in 2-3% untreated upper = develops as sequelae in 2-3% untreated upper respiratory respiratory infectionsinfections= symptoms occur about 18 days after sore throat= symptoms occur about 18 days after sore throat= Group A streptococcus share antigenic determinants = Group A streptococcus share antigenic determinants with with host tissue, especially heart and even host tissue, especially heart and even jointsjoints= inflammation of mitral valve most serious= inflammation of mitral valve most serious= 30-60% of patients may suffer permanent disability= 30-60% of patients may suffer permanent disability


• GROUP A STREPTOCOCCAL INFECTIONGROUP A STREPTOCOCCAL INFECTIONB. Post-Streptococcal GlomerulonephritisB. Post-Streptococcal Glomerulonephritis

= follows Streptococcal infection of skin or pharynx= follows Streptococcal infection of skin or pharynx= occurs about 10 days following initial infection= occurs about 10 days following initial infection= characterized by damage to glomeruli of the kidneys= characterized by damage to glomeruli of the kidneys= renal function impaired due to reduction in = renal function impaired due to reduction in glomerular glomerular filtration rate, results in edema and HPNfiltration rate, results in edema and HPN= renal failure not typical= renal failure not typical= one theory is damage caused by antigen-antibody = one theory is damage caused by antigen-antibody

complexes depositing in kidneyscomplexes depositing in kidneys= complement is activated resulting in low levels= complement is activated resulting in low levels


• LABORATORY TESTINGLABORATORY TESTING• Most reliable test is culture and identification of the Most reliable test is culture and identification of the

organism from infected siteorganism from infected site• Rapid streptococcal screening tests from the throat Rapid streptococcal screening tests from the throat

exudates have high specificity but low sensitivity, exudates have high specificity but low sensitivity, 60-85%60-85%

• Detection of Streptococcal antibodies most useful in Detection of Streptococcal antibodies most useful in Streptococcal sequelaeStreptococcal sequelae

• The most useful antibodies are : ASO, anti-DNase B, The most useful antibodies are : ASO, anti-DNase B, anti-NADase, anti-Hyaluronidaseanti-NADase, anti-Hyaluronidase

• Serological evidence of disease is based on Serological evidence of disease is based on elevated or rising titer of Streptococcal antibodieselevated or rising titer of Streptococcal antibodies

• Four-fold (2 tube dilution) rise in titer is considered Four-fold (2 tube dilution) rise in titer is considered clinically significantclinically significant


• LABORATORY TESTINGLABORATORY TESTING1.1. Anti-Streptolysin O Titer (ASO Titer)Anti-Streptolysin O Titer (ASO Titer)

= two of the toxins produced are Streptolysin S, which is = two of the toxins produced are Streptolysin S, which is oxygen oxygen stable, non-antigenic and Streptolysin O stable, non-antigenic and Streptolysin O (SLO), which is (SLO), which is oxygen labile and antigenicoxygen labile and antigenic= SLO is a hemolysin which is toxic to many tissues, = SLO is a hemolysin which is toxic to many tissues, including heart including heart and kidneysand kidneys= evokes an antibody response (anti-SLO) which neutrolizes = evokes an antibody response (anti-SLO) which neutrolizes the the hemolytic action of SLOhemolytic action of SLO= the test is specific for ASO, it does not test for antibodies = the test is specific for ASO, it does not test for antibodies to any to any other Streptococcal exotoxinsother Streptococcal exotoxins= normal values will vary, <125 Todd units for adults, 5-125 = normal values will vary, <125 Todd units for adults, 5-125 Todd Todd units for children, recent Strep infections 250 units for children, recent Strep infections 250 Todd units for Todd units for adults, 333 Todd units for childrenadults, 333 Todd units for children= a single titer is of little significance unless extremely = a single titer is of little significance unless extremely elevated, titers performed over a period of time will give the elevated, titers performed over a period of time will give the most informationmost information


• LABORATORY TESTINGLABORATORY TESTING2. Anti-DNase B Testing2. Anti-DNase B Testing

= may appear earlier than ASO= may appear earlier than ASO= increased sensitivity for detection of = increased sensitivity for detection of glomerulonephritis preceded by streptococcal glomerulonephritis preceded by streptococcal

skin infectionskin infection= macro- and micro-titer, ELISA, and = macro- and micro-titer, ELISA, and neutralization neutralization techniques are availabletechniques are available= Neutralization technique has advantage of = Neutralization technique has advantage of

stability of reagentsstability of reagents


• LABORATORY TESTINGLABORATORY TESTING3. Anti-Hyaluronidase Testing3. Anti-Hyaluronidase Testing

= test patient serum for antibodies which = test patient serum for antibodies which inhibit inhibit action of Hyaluronidaseaction of Hyaluronidase= after performance of the test, a clot will = after performance of the test, a clot will form into form into the tubes where enzyme activity the tubes where enzyme activity of of Hyaluronidase has been Hyaluronidase has been neutralized by neutralized by patient antibodypatient antibody= Hyaluronidase produced by patients with = Hyaluronidase produced by patients with throat throat or skin infections, ASO produced or skin infections, ASO produced in response in response to throat infections onlyto throat infections only


• LABORATORY TESTINGLABORATORY TESTING4. Streptozyme Testing4. Streptozyme Testing

= hemagglutination procedure to detect = hemagglutination procedure to detect antibodies antibodies to numerous Streptococcal to numerous Streptococcal antigensantigens= sheep RBC’s are coated with Streptolysin, = sheep RBC’s are coated with Streptolysin,

Streptokinase, Hyaluronidase, Streptokinase, Hyaluronidase, DNase, and DNase, and NADaseNADase= patient serum diluted 1 : 100, mixed with = patient serum diluted 1 : 100, mixed with sheep sheep RBC’s and observed for RBC’s and observed for agglutinationagglutination= rapid and simple to perform, more false = rapid and simple to perform, more false positive positive and negative results occurand negative results occur


• SEROLOGY OF VIRAL INFECTIONSSEROLOGY OF VIRAL INFECTIONSA.A. HepatitisHepatitis

= general term meaning inflammation of the liver, = general term meaning inflammation of the liver, usually usually accompanied with fever, nausea, accompanied with fever, nausea, vomiting and vomiting and jaundicejaundice= can be caused by radiation, chemicals, disease = can be caused by radiation, chemicals, disease processes processes such as autoimmune disease, viruses such as autoimmune disease, viruses and cancerand cancer= 5 distinct viruses – A, B, C, D and E= 5 distinct viruses – A, B, C, D and E= all of these are RNA viruses except hepatitis B which = all of these are RNA viruses except hepatitis B which is a is a DNA virusDNA virus= initial infection may be clinically silent= initial infection may be clinically silent= chronic carrier state may develop and may result to = chronic carrier state may develop and may result to liver liver failure due to cirrhosis, hepatocellular failure due to cirrhosis, hepatocellular carcinoma, or carcinoma, or fulminant hepatitisfulminant hepatitis

SEROLOGICAL DIAGNOSIS OF INFECTIOUS SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = DISEASES = VIRAL HEPATITISVIRAL HEPATITIS

• Hepatitis A virus (HAV)Hepatitis A virus (HAV)• Transmitted by fecal oral routeTransmitted by fecal oral route• Occurs worldwideOccurs worldwide• Most hepatitis epidemics are due to HAVMost hepatitis epidemics are due to HAV• Progress of infection:Progress of infection:

Incubation of 2-7 weeks, may be asymptomatic or Incubation of 2-7 weeks, may be asymptomatic or may include jaundicemay include jaundice

Clinical illness develop abruptly and include fever, Clinical illness develop abruptly and include fever, anorexia, vomiting, fatigue and malaiseanorexia, vomiting, fatigue and malaise

Increase in serum transaminasesIncrease in serum transaminases RUQ pain, dark urine and pale stoolRUQ pain, dark urine and pale stool Recovery 2-4 weeks, no carrier stateRecovery 2-4 weeks, no carrier state Mortality 0-1%Mortality 0-1%

SEROLOGICAL DIAGNOSIS OF INFECTIOUS SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = DISEASES = VIRAL HEPATITISVIRAL HEPATITIS

• Hepatitis A virus (HAV)Hepatitis A virus (HAV)• Antibody and antigen markersAntibody and antigen markers

First and most clinically useful is IgM First and most clinically useful is IgM antibody to HAVantibody to HAV

IgM indicates acute infection, appears 4-5 IgM indicates acute infection, appears 4-5 weeks after exposureweeks after exposure

IgM disappears in 3-6 months, replaced by IgM disappears in 3-6 months, replaced by IgG anti-HAVIgG anti-HAV

IgG peaks during convalescence and may IgG peaks during convalescence and may remain detectable for liferemain detectable for life

Time course of Hepatitis A virus (HAV) Time course of Hepatitis A virus (HAV) infectioninfection

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = INFECTIOUS DISEASES = VIRAL VIRAL

HEPATITISHEPATITIS

• Hepatitis B virus (HBV)Hepatitis B virus (HBV)• Old term “serum hepatitis”, incubation period of 4-26 Old term “serum hepatitis”, incubation period of 4-26

weeksweeks

• Route of infection is usually parenteral, direct inoculationRoute of infection is usually parenteral, direct inoculation

• Incidence of infection is 140,000-320,000 cases per year Incidence of infection is 140,000-320,000 cases per year resulting in 5-6,000 deaths per yearresulting in 5-6,000 deaths per year

• Duration of acute infection ranges from 4-8 weeks with Duration of acute infection ranges from 4-8 weeks with symptoms similar to HAVsymptoms similar to HAV

• 10% progress to chronic10% progress to chronic

• One-third of chronic at risk of developing chronic active One-third of chronic at risk of developing chronic active hepatitis, cirrhosis and/or hepatocellular carcinomahepatitis, cirrhosis and/or hepatocellular carcinoma

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = VIRAL INFECTIOUS DISEASES = VIRAL

HEPATITISHEPATITIS

• Hepatitis B virus (HBV)Hepatitis B virus (HBV) = Lab Diagnosis= Lab Diagnosis• Involve the detection of three marker systemInvolve the detection of three marker system

•Hepatitis B surface antigen (HBsAg) is the first to Hepatitis B surface antigen (HBsAg) is the first to appear, appears 2-4 weeks during late incubation, appear, appears 2-4 weeks during late incubation, marker of choice for recent infectionmarker of choice for recent infection

•Anti-Hepatitis B surface antigen (anti-HBs) is the Anti-Hepatitis B surface antigen (anti-HBs) is the last antibody to appear, may persist for lifelast antibody to appear, may persist for life

•Between disappearance of HBsAg and appearance Between disappearance of HBsAg and appearance of anti-HBs is known as the core windowof anti-HBs is known as the core window


HEPATITISHEPATITIS

• Hepatitis B virus (HBV)Hepatitis B virus (HBV) = Lab = Lab DiagnosisDiagnosis

• IgM antibody to Hepatitis B core antigen IgM antibody to Hepatitis B core antigen (anti-HBc) may be the only detectable (anti-HBc) may be the only detectable marker during the core window, marker during the core window, differentiates recent infection from chronic differentiates recent infection from chronic carrier statecarrier state

•Third marker is Hepatitis Be antigen Third marker is Hepatitis Be antigen (HBeAg), appearance of HBeAg and anti-(HBeAg), appearance of HBeAg and anti-HBe, closely coincide with HBsAgHBe, closely coincide with HBsAg

Hepatitis B viral genomeHepatitis B viral genome

Spread of Hepatitis B virus (HBV) in the Spread of Hepatitis B virus (HBV) in the bodybody

Symptoms of typical acute viral hepatitis B Symptoms of typical acute viral hepatitis B infection correlated with the four clinical periods infection correlated with the four clinical periods of this diseaseof this disease

Clinical outcomes of Acute Hepatitis B Clinical outcomes of Acute Hepatitis B infectioninfection

The serologic events associated with The serologic events associated with the typical course of acute HBV the typical course of acute HBV infectioninfection

Interpretation of Serologic Markers of Interpretation of Serologic Markers of Hepatitis B Virus InfectionHepatitis B Virus Infection


HEPATITISHEPATITIS

• Hepatitis D virus (HDV)Hepatitis D virus (HDV)•Requires infection with Hepatitis BRequires infection with Hepatitis B

•Route of transmission the same as HBVRoute of transmission the same as HBV

•Can occur as coinfection or superinfectionCan occur as coinfection or superinfection

Consequences of delta virus infectionConsequences of delta virus infection


HEPATITISHEPATITIS

• Hepatitis D virus (HDV)Hepatitis D virus (HDV) = Serological = Serological markersmarkers

•HDAg found early, disappears rapidly, not very HDAg found early, disappears rapidly, not very usefuluseful

• IgM anti-D and total anti-HD (IgM and IgG) IgM anti-D and total anti-HD (IgM and IgG) detected during acute phasedetected during acute phase

•Presence of IgM anti-D and HBsAg together Presence of IgM anti-D and HBsAg together with IgM anti-HBc indicates co-infectionwith IgM anti-HBc indicates co-infection

•Absence of IgM anti-HBc indicates Absence of IgM anti-HBc indicates superinfectionsuperinfection

•Presence of anti-HD indicates chronic infectionPresence of anti-HD indicates chronic infection


HEPATITISHEPATITIS

• Hepatitis C virus (HCV)Hepatitis C virus (HCV)•Clinically and epidemiologically similar to Clinically and epidemiologically similar to

HBVHBV

•60-70% of HCV patients will develop chronic 60-70% of HCV patients will develop chronic hepatitis, 10-20% cirrhosis and 15% hepatitis, 10-20% cirrhosis and 15% hepatocellular carcinomahepatocellular carcinoma

•HCV and HBV may be present as co-HCV and HBV may be present as co-infectionsinfections


HEPATITISHEPATITIS

• Hepatitis C virus (HCV)Hepatitis C virus (HCV) = Serological = Serological MarkersMarkers

•Serological profile not fully developedSerological profile not fully developed

•Present of HCV antibodies only indicates Present of HCV antibodies only indicates present or past infectionpresent or past infection

•Can have false negative in some patientsCan have false negative in some patients

Outcomes of Hepatitis C infectionOutcomes of Hepatitis C infection


HEPATITISHEPATITIS

• Hepatitis E virus (HEV)Hepatitis E virus (HEV)• Similar to HAV in transmission and clinical courseSimilar to HAV in transmission and clinical course

• Found primarily in developing countries, Africa and Found primarily in developing countries, Africa and AsiaAsia

• Results in acute hepatitis, no risk of chronic hepatitisResults in acute hepatitis, no risk of chronic hepatitis

• Pregnant women with HEV may develop fulminant Pregnant women with HEV may develop fulminant liver failure and deathliver failure and death

• No distinctive markers, diagnosis based on symptoms No distinctive markers, diagnosis based on symptoms for exposed individuals in endemic countriesfor exposed individuals in endemic countries


HEPATITISHEPATITIS

• Hepatitis G virusHepatitis G virus• Independently discovered 1995-1996 by 2 Independently discovered 1995-1996 by 2

separate research groupsseparate research groups•RNA virusRNA virus•Transmissible by blood-borne routeTransmissible by blood-borne route•Found in patients with acute or chronic liver Found in patients with acute or chronic liver

dse.dse.•Exact clinical significance needs to be further Exact clinical significance needs to be further

defineddefined•ELISA and Western Blot methods have been ELISA and Western Blot methods have been

developeddeveloped

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = INFECTIOUS DISEASES = HERPES HERPES

VIRUSVIRUS

B. HERPES VIRUS GROUPB. HERPES VIRUS GROUP= includes EBV, CMV, Herpes simplex = includes EBV, CMV, Herpes simplex

virus type I and II, Varicella-virus type I and II, Varicella-zoster zoster virusvirus= DNA viruses that remain within = DNA viruses that remain within nucleus nucleus while completing life cyclewhile completing life cycle= most infections are subclinical and = most infections are subclinical and

result in latent stageresult in latent stage


VIRUS GROUPVIRUS GROUP

• Epstein-Barr Virus (EBV)Epstein-Barr Virus (EBV)•Spread through oral transmission of Spread through oral transmission of

infective saliva and is the cause of infectious infective saliva and is the cause of infectious mononucleosismononucleosis

•Other diseases – Burkitt’s lymphoma, Other diseases – Burkitt’s lymphoma, nasopharyngeal carcinoma, B-cell lymphomanasopharyngeal carcinoma, B-cell lymphoma

•Virus may become reactivated and is the Virus may become reactivated and is the suggested cause of chronic fatigue suggested cause of chronic fatigue syndromesyndrome



• Epstein-Barr Virus (EBV)Epstein-Barr Virus (EBV)– Characteristics of infectionCharacteristics of infection

•4-7 week incubation, acute self limiting 4-7 week incubation, acute self limiting

•Enlarged LN in the neck, sore throat, fever, Enlarged LN in the neck, sore throat, fever, rashrash

•Malaise, lethargy, extreme tirednessMalaise, lethargy, extreme tiredness

•Liver and spleen involvement and Liver and spleen involvement and enlargementenlargement

•Hematology : high WBC, over 20% atypical Hematology : high WBC, over 20% atypical reactive lymphocytesreactive lymphocytes



• Epstein-Barr Virus (EBV)Epstein-Barr Virus (EBV)– Serological testingSerological testing = may involve screening = may involve screening

tests to detect heterophile antibodiestests to detect heterophile antibodies• Heterophile antigens are a group of similar antigens Heterophile antigens are a group of similar antigens

found in unrelated animalsfound in unrelated animals

• Heterophile antibodies produced against heterophile Heterophile antibodies produced against heterophile antigens of one species will cross react with othersantigens of one species will cross react with others

• Forssman antigen is an example of a heterophile Forssman antigen is an example of a heterophile antigen and is found on the RBC’s of many speciesantigen and is found on the RBC’s of many species

• Forssman antibodies formed against Forssman Forssman antibodies formed against Forssman antigens will agglutinate sheep RBC’santigens will agglutinate sheep RBC’s



• Epstein-Barr Virus (EBV)Epstein-Barr Virus (EBV)– Infectious Mononucleosis slide testsInfectious Mononucleosis slide tests

•Horse RBC’s possess antigens which react Horse RBC’s possess antigens which react with the antibody associated with IMwith the antibody associated with IM

•Patient serum mixed with horse RBC’s, Patient serum mixed with horse RBC’s, agglutination is positiveagglutination is positive

•Not diagnostic, must look at total clinical Not diagnostic, must look at total clinical picturepicture



• Epstein-Barr Virus (EBV)Epstein-Barr Virus (EBV)– EBV specific antibodies may be measuredEBV specific antibodies may be measured

• Must know pattern of appearance of EBV antigensMust know pattern of appearance of EBV antigens

• Most valuable is IgM antibody to viral capsid antigen Most valuable is IgM antibody to viral capsid antigen (VCA), indicates a current infection (best marker), (VCA), indicates a current infection (best marker), lasts about 12 weekslasts about 12 weeks

• Can also detect anti-early antigen (EA), recent Can also detect anti-early antigen (EA), recent infection and anti-EB nuclear antigen (EBNA), older infection and anti-EB nuclear antigen (EBNA), older infectioninfection

• ELISA and immunofluorescence techniques most ELISA and immunofluorescence techniques most commonly usedcommonly used



• CytomegalovirusCytomegalovirus•Transmission occurs from person to personTransmission occurs from person to person

•Symptoms resemble IM but has negative Symptoms resemble IM but has negative test for EBVtest for EBV

• In babies may cause life-threatening illness In babies may cause life-threatening illness resulting in CNS involvement, hearing loss, resulting in CNS involvement, hearing loss, and mental retardationand mental retardation

•Seen in patients with deficient immune Seen in patients with deficient immune system, AIDS, transplantationsystem, AIDS, transplantation



• CytomegalovirusCytomegalovirus– Immunologic responseImmunologic response

• For best diagnostic results, lab tests for CMV antibody For best diagnostic results, lab tests for CMV antibody should be performed by using paired serum samplesshould be performed by using paired serum samples

• One blood sample should be taken upon suspicion of One blood sample should be taken upon suspicion of CMV, and another one taken within 2 weeks. A virus CMV, and another one taken within 2 weeks. A virus culture can be performed at any time the pt. is culture can be performed at any time the pt. is symptomaticsymptomatic

• IgM antibodies produced against early and IgM antibodies produced against early and intermediate-early (IE) CMV antigens, last for 3 to 4 intermediate-early (IE) CMV antigens, last for 3 to 4 monthsmonths

• IgG appear shortly after and peak at 2 to 3 monthsIgG appear shortly after and peak at 2 to 3 months



• CytomegalovirusCytomegalovirus– Laboratory DiagnosisLaboratory Diagnosis

•Range from culture and cytologic techniques to Range from culture and cytologic techniques to DNA probes, PCR and serologic techniquesDNA probes, PCR and serologic techniques

•Detection of antibodies indicator of recent Detection of antibodies indicator of recent infectioninfection

•Viral culture lack sensitivity and are time Viral culture lack sensitivity and are time consuming and expensiveconsuming and expensive

•Microscopic examination of biopsy specimens, Microscopic examination of biopsy specimens, urine sediment or peripheral blood may reveal urine sediment or peripheral blood may reveal the typical cytomegalic cell with “owl’s eye” the typical cytomegalic cell with “owl’s eye” inclusion inclusion


VIRUS GROUPVIRUS GROUP• CytomegalovirusCytomegalovirus

– Laboratory DiagnosisLaboratory Diagnosis• Detection of CMV Ag in cells more appropriately detected Detection of CMV Ag in cells more appropriately detected

by immunofluorescent techniques using monoclonal by immunofluorescent techniques using monoclonal antibodiesantibodies

• ELISA is the most commonly available serologic test for ELISA is the most commonly available serologic test for measuring antibody to CMVmeasuring antibody to CMV

• The result can be used to determine if acute infection, The result can be used to determine if acute infection, prior infection, or passively acquired maternal antibody in prior infection, or passively acquired maternal antibody in an infant is presentan infant is present

• Other tests include various fluorescence assays, indirect Other tests include various fluorescence assays, indirect hemagglutination, and latex agglutinationhemagglutination, and latex agglutination

• Screening tests using coated latex particles compare Screening tests using coated latex particles compare favorably to more complex tests for antibody detectionfavorably to more complex tests for antibody detection

• False positives can occur = RA and Ebstein-Barr False positives can occur = RA and Ebstein-Barr antibodiesantibodies



• Herpes Simplex Virus (HSV)Herpes Simplex Virus (HSV)– Laboratory testingLaboratory testing

• Recovery of the virus in cell culture is considered the Recovery of the virus in cell culture is considered the “gold standard” for detection of this virus from “gold standard” for detection of this virus from sources other than CSF, culture helpful in sources other than CSF, culture helpful in differentiating types of HSVdifferentiating types of HSV

• Direct examination using immunofluorescence or Direct examination using immunofluorescence or immunoperoxidase staining of cells from lesionimmunoperoxidase staining of cells from lesion

• DNA probes, ELISA, latex agglutination, RIA and DNA probes, ELISA, latex agglutination, RIA and indirect immunofluorescenceindirect immunofluorescence

• Serology is not very useful because there is a high Serology is not very useful because there is a high prevalence of antibody in the normal populationprevalence of antibody in the normal population



• Varicella-Zoster VirusVaricella-Zoster Virus– Laboratory testingLaboratory testing important to distinguish important to distinguish

VZV from other infections, selection of VZV from other infections, selection of antiviral drugs, or determining immune antiviral drugs, or determining immune status of individualsstatus of individuals•PCR is now the routine testing method for VZVPCR is now the routine testing method for VZV•Direct fluorescent antibody staining and viral Direct fluorescent antibody staining and viral

culture techniques may be used for the culture techniques may be used for the detection of VZV in most specimen typesdetection of VZV in most specimen types

• IgG and IgM antibody tests by ELISA may be IgG and IgM antibody tests by ELISA may be usedused

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = INFECTIOUS DISEASES = GERMAN GERMAN

MEASLESMEASLES

• Rubella VirusRubella Virus– Laboratory testingLaboratory testing

• Performed primarily for diagnosis of acquired Performed primarily for diagnosis of acquired infections and to determine immune status of infections and to determine immune status of pregnant patientspregnant patients

• Some tests detect IgG antibodies, other IgMSome tests detect IgG antibodies, other IgM• Methods include : hemagglutination inhibition, Methods include : hemagglutination inhibition,

passive hemagglutination, neutralization, hemolysis passive hemagglutination, neutralization, hemolysis in gel, complement fixation, fluorescent in gel, complement fixation, fluorescent immunoassay, RIA, ELISA and latex agglutinationimmunoassay, RIA, ELISA and latex agglutination

• Method depends on volume of testing, turn around Method depends on volume of testing, turn around time, complexity, expense and whether a qualitative time, complexity, expense and whether a qualitative or quantitative test is neededor quantitative test is needed

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES =INFECTIOUS DISEASES = MEASLESMEASLES

• RubeolaRubeola• Serology testing provides best means of confirming a Serology testing provides best means of confirming a

measles diagnosismeasles diagnosis

• Methods to detect Rubeola antibodies include : Methods to detect Rubeola antibodies include : hemagglutination inhibition, endpoint neutralization, hemagglutination inhibition, endpoint neutralization, complement fixation, IFA and ELISAcomplement fixation, IFA and ELISA

• In addition to signs and symptoms, diagnosis In addition to signs and symptoms, diagnosis confirmed by presence of Rubeola specific IgM confirmed by presence of Rubeola specific IgM antibodies or four-fold rise in IgG antibody titer in antibodies or four-fold rise in IgG antibody titer in paired samples taken after rash to 10 to 30 days laterpaired samples taken after rash to 10 to 30 days later

• IgM test highly depended on time of sample IgM test highly depended on time of sample collection with 3-11 days after rash being optimalcollection with 3-11 days after rash being optimal

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES =INFECTIOUS DISEASES = MUMPSMUMPS

• MumpsMumps• Methods to detect mump antibodies include : Methods to detect mump antibodies include :

complement fixation, hemagglutination inhibition, complement fixation, hemagglutination inhibition, hemolysis-in-gel, neutralization assays, IFA and ELISAhemolysis-in-gel, neutralization assays, IFA and ELISA

• Current or recent infections indicated by presence of Current or recent infections indicated by presence of specific IgM antibody in single sample which can be specific IgM antibody in single sample which can be detected within 5 days of illnessdetected within 5 days of illness

• Fourfold rise in specific IgG antibody in 2 samples Fourfold rise in specific IgG antibody in 2 samples collected during acute and convalescent phasescollected during acute and convalescent phases

• Fluorescent antibody staining for mumps antigens Fluorescent antibody staining for mumps antigens developed but not widely useddeveloped but not widely used

• Cross-reactivity between antibodies to mumps and Cross-reactivity between antibodies to mumps and parainfluenza viruses has been reported in test for IgGparainfluenza viruses has been reported in test for IgG

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES =INFECTIOUS DISEASES = HIVHIV

• Human Immunodeficiency Virus (HIV)Human Immunodeficiency Virus (HIV)• Etiologic agent of AIDSEtiologic agent of AIDS• Discovered independently by Luc Montagnier of France Discovered independently by Luc Montagnier of France

and Robert Gallo of the US in 1983-1984and Robert Gallo of the US in 1983-1984• Former names of the virus include :Former names of the virus include :

Human T cell Lymphotrophic virus (HTLV-III)Human T cell Lymphotrophic virus (HTLV-III) Lymphadenopathy associated virus (LAV)Lymphadenopathy associated virus (LAV) AIDS associated retrovirus (ARV)AIDS associated retrovirus (ARV)

• HIV-2 discovered in 1986, antigenically distinct virus HIV-2 discovered in 1986, antigenically distinct virus endemic in West Africaendemic in West Africa

• One million people infected in US, 30 Million worldwide One million people infected in US, 30 Million worldwide are infectedare infected

• Leading cause of death of men aged 25-44 and 4Leading cause of death of men aged 25-44 and 4thth leading cause of death of women in this age group in leading cause of death of women in this age group in the USthe US


• Structural genesStructural genes•Gag is p55 from which three core proteins Gag is p55 from which three core proteins

(p15, p17 and p24) are formed(p15, p17 and p24) are formed

•Env gene codes for envelope proteins Env gene codes for envelope proteins gp160, gp120 and gp41gp160, gp120 and gp41

•Pol codes for p66 and p51 subunits of Pol codes for p66 and p51 subunits of reverse transcriptase and p31 an reverse transcriptase and p31 an endonucleaseendonuclease


• Immunologic ManifestationsImmunologic Manifestations• Early stage slight depression of CD4 count, few Early stage slight depression of CD4 count, few

symptoms, temporarysymptoms, temporary• Window of up to 6 weeks before antibody is detected, Window of up to 6 weeks before antibody is detected,

by 6 months 95% positiveby 6 months 95% positive• During window p24 antigen present, acute viremia During window p24 antigen present, acute viremia

and antigenemiaand antigenemia• Antibodies produced to all major antigensAntibodies produced to all major antigens

First antibodies detected produced against gag First antibodies detected produced against gag proteins p24 and p55proteins p24 and p55

Followed by antibody to p51, p120 and gp41Followed by antibody to p51, p120 and gp41 As disease progresses, antibody levels decreasesAs disease progresses, antibody levels decreases


• Immunologic ManifestationsImmunologic Manifestations• Immune abnormalities associated with Immune abnormalities associated with

increased viral replicationincreased viral replication Decrease in CD4 cellsDecrease in CD4 cells B cells have decreased response to antigenB cells have decreased response to antigen CD8 cells initially increase and may remain CD8 cells initially increase and may remain

elevatedelevated As HIV infection progresses, CD4 T cells drop As HIV infection progresses, CD4 T cells drop

resulting in immunosuppression and resulting in immunosuppression and susceptibility of patient to opportunistic susceptibility of patient to opportunistic infectionsinfections

Death comes due to immuno-incompetenceDeath comes due to immuno-incompetence


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection1. Methods utilized to detect1. Methods utilized to detect

•AntibodyAntibody

•AntigenAntigen

•Viral nucleic acidViral nucleic acid

•Virus in cultureVirus in culture


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection2. ELISA Testing2. ELISA Testing

= first serological test developed to = first serological test developed to detect HIV infectiondetect HIV infection

= antibodies detected include those = antibodies detected include those directed against p24, gp120, gp160 and gp41, directed against p24, gp120, gp160 and gp41, detected first in infection and appear in most detected first in infection and appear in most individualsindividuals

= used for screening only, false positives = used for screening only, false positives do occurdo occur


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection4. Western Blot Testing4. Western Blot Testing

= most popular confirmatory test= most popular confirmatory test

= antibodies to p24 and p55 appear earliest = antibodies to p24 and p55 appear earliest but decrease or become but decrease or become undetectableundetectable

= antibodies to gp31, gp41, gp120, and = antibodies to gp31, gp41, gp120, and gp160 appear later but are present gp160 appear later but are present throughout all stages of the disease throughout all stages of the disease


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection4. Western Blot Testing 4. Western Blot Testing = interpretation of result= interpretation of result

no bands, negativeno bands, negative

in order to be interpreted as positive a in order to be interpreted as positive a minimun minimun of 3 bands directed against the of 3 bands directed against the following antigens following antigens must be present : must be present : p24, p31, gp41 or gp120/160p24, p31, gp41 or gp120/160

CDC criteria require 2 bands of the CDC criteria require 2 bands of the following : following : p24, gp41 or gp120/160 p24, gp41 or gp120/160


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection4. Western Blot Testing 4. Western Blot Testing = interpretation of result= interpretation of result

indeterminate results are those samples that indeterminate results are those samples that produce bands produce bands but not enough to be positive, but not enough to be positive, may be due to the following:may be due to the following:

1. prior blood transfusions, even with non-HIV-1 1. prior blood transfusions, even with non-HIV-1 infected bloodinfected blood 2. prior or current infection with syphilis2. prior or current infection with syphilis 3. prior or current infection with malaria3. prior or current infection with malaria 4. autoimmune diseases4. autoimmune diseases

5. infection with other human retroviruses5. infection with other human retroviruses6. second or subsequent pregnancies in women6. second or subsequent pregnancies in women*** run an alternate HIV confirmatory assay*** run an alternate HIV confirmatory assay


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection5. Indirect immunofluorescence assay 5. Indirect immunofluorescence assay

= can be used to detect both virus and = can be used to detect both virus and antibody to it antibody to it

= antibody detected by testing patient serum = antibody detected by testing patient serum against antigen applied to a slide, against antigen applied to a slide,

incubated, incubated, washed and a fluorescent washed and a fluorescent antibody addedantibody added

= virus is detected by fixing patient cells to = virus is detected by fixing patient cells to slide, slide, incubating with antibody incubating with antibody


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection6. Detection of p24 HIV antigen6. Detection of p24 HIV antigen

= p24 antigen only present for short time, disappears when = p24 antigen only present for short time, disappears when antibody to p24 appears antibody to p24 appears

= anti-HIV-1 bound to membrane, incubated with patient = anti-HIV-1 bound to membrane, incubated with patient serum, serum, second anti-HIV-1 antibody attached to second anti-HIV-1 antibody attached to enzyme label is added enzyme label is added (sandwich technique), color (sandwich technique), color change occurschange occurs= optical density measured, standard curve prepared to = optical density measured, standard curve prepared to

quantitate results quantitate results= positive confirmed by neutralizing reaction, preincubate = positive confirmed by neutralizing reaction, preincubate

patient sample with anti-HIV, retest, if p24 patient sample with anti-HIV, retest, if p24 present immune present immune complexes form preventing binding to complexes form preventing binding to HIV antibody on HIV antibody on membrane added membrane added


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection6. Detection of p24 HIV antigen6. Detection of p24 HIV antigen

= test not recommended for routine screening as = test not recommended for routine screening as appearance and rate of rise are unpredictable appearance and rate of rise are unpredictable

= sensitivity lower than ELISA= sensitivity lower than ELISA= most useful for the following= most useful for the following

a. early infection suspected in seronegative a. early infection suspected in seronegative patientpatient b. newbornsb. newborns

c. CSFc. CSF d. monitoring disease progressd. monitoring disease progress


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection7. Polymerase Chain Reaction (PCR)7. Polymerase Chain Reaction (PCR)

= looks for HIV DNA in the WBC’s of a person= looks for HIV DNA in the WBC’s of a person= amplifies tiny quantities of the HIV DNA present, each = amplifies tiny quantities of the HIV DNA present, each cycle of cycle of PCR results in doubling of the DNA PCR results in doubling of the DNA sequences presentsequences present= the DNA is detected by using radioactive or biotiny = the DNA is detected by using radioactive or biotiny lated lated probes probes= once DNA is amplified it is placed on nitrocellulose = once DNA is amplified it is placed on nitrocellulose paper and paper and allowed to react with a radio-labeled probe, allowed to react with a radio-labeled probe, a single stranded a single stranded DNA fragment unique to HIV, which DNA fragment unique to HIV, which will hybridize with the will hybridize with the patient’s HIV DNA if present patient’s HIV DNA if present= radioactivity is determined= radioactivity is determined


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection8. Virus isolation8. Virus isolation

= definitively diagnose HIV= definitively diagnose HIV= best sample is peripheral blood, but can use = best sample is peripheral blood, but can use CSF, CSF, saliva, cervical secretions, semen, tears saliva, cervical secretions, semen, tears or material or material from organ biopsy from organ biopsy= cell growth in culture is stimulated, amplifies = cell growth in culture is stimulated, amplifies

number of cells releasing virus number of cells releasing virus= cultures incubated one month, infection = cultures incubated one month, infection confirmed confirmed by detecting reverse transcriptase by detecting reverse transcriptase or p24 antigen in or p24 antigen in supernatant supernatant


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection9. Viral Load Tests9. Viral Load Tests

= viral load or viral burden is the quantity of HIV-RNA that = viral load or viral burden is the quantity of HIV-RNA that is in the blood is in the blood

= measures the amount of HIV-RNA in one milliliter of = measures the amount of HIV-RNA in one milliliter of bloodblood

take 2 measurements 2-3 weeks apart to take 2 measurements 2-3 weeks apart to determine baseline determine baseline repeat every 3-6 months in conjunction with repeat every 3-6 months in conjunction with

CD4 CD4 counts to monitor viral load and T-cell counts to monitor viral load and T-cell countcount

repeat 4-6 weeks after starting or changing repeat 4-6 weeks after starting or changing antiretroviral therapy to determine effect antiretroviral therapy to determine effect

on viral loadon viral load


• Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection10. Testing of neonates10. Testing of neonates

= difficult due to presence of maternal IgG = difficult due to presence of maternal IgG antibodiesantibodies

= use tests to detect IgM or IgA antibodies, IgM = use tests to detect IgM or IgA antibodies, IgM lacks lacks sensitivity, IgA more promising sensitivity, IgA more promising

= measurement of p24 antigen= measurement of p24 antigen

= PCR testing maybe helpful but still not = PCR testing maybe helpful but still not detecting detecting antigen soon enough : 38 days to antigen soon enough : 38 days to 6 months to be 6 months to be positive positive

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES =INFECTIOUS DISEASES = DENGUEDENGUE

• Dengue feverDengue fever•Transmitted by mosquitoesTransmitted by mosquitoes

•There are 4 known distinct serotypes There are 4 known distinct serotypes ( dengue virus 1, 2, 3 and 4)( dengue virus 1, 2, 3 and 4)

• In children , infection is often sub-clinical or In children , infection is often sub-clinical or causes a self-limited febrile diseasecauses a self-limited febrile disease

•Secondarily infected with a different Secondarily infected with a different serotype, dengue hemorrhagic fever or serotype, dengue hemorrhagic fever or dengue shock syndromedengue shock syndrome

Algorithm for Serologic Testing for Algorithm for Serologic Testing for AIDSAIDS

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES =INFECTIOUS DISEASES = DENGUEDENGUE

• Dengue feverDengue fever•Dengue IgG/IgM Rapid Test is a solid phase Dengue IgG/IgM Rapid Test is a solid phase

immunochromatographic assay for the rapid immunochromatographic assay for the rapid qualitative and differential detection of IgG qualitative and differential detection of IgG and IgM antibodies to dengue virus in and IgM antibodies to dengue virus in human serum, plasma or whole blood. This human serum, plasma or whole blood. This test can also detect all 4 Dengue serotypes test can also detect all 4 Dengue serotypes by using a mixture of recombinant Dengue by using a mixture of recombinant Dengue envelop proteinsenvelop proteins

Rapid Test for DengueRapid Test for Dengue

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES =INFECTIOUS DISEASES =DENGUEDENGUE

• Dengue feverDengue fever– Interpretation of the testInterpretation of the test

• IgG and IgM positive = indicative of a late primary or IgG and IgM positive = indicative of a late primary or early secondary dengue infectionearly secondary dengue infection

• IgM positive = indicative of primary Dengue infectionIgM positive = indicative of primary Dengue infection• IgG positive = indicative of secondary or past dengue IgG positive = indicative of secondary or past dengue

infectioninfection• Negative = retest in 3-5 days if Dengue infection is Negative = retest in 3-5 days if Dengue infection is

suspectedsuspected• Invalid = insufficient specimen volume or incorrect Invalid = insufficient specimen volume or incorrect

procedural technique. Repeat the test using a new procedural technique. Repeat the test using a new test devicetest device

SEROLOGICAL DIAGNOSIS OF SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES =INFECTIOUS DISEASES = Typhoid Typhoid

FeverFever• Typhoid FeverTyphoid Fever

• Caused by Salmonella typhiCaused by Salmonella typhi

• Rapid detection is now available in the marketRapid detection is now available in the market Typhidot = a qualitative detection test against a Typhidot = a qualitative detection test against a

specific antigen of Salmonella typhi. It can detect specific antigen of Salmonella typhi. It can detect both IgG and IgM separately and simultaneously. both IgG and IgM separately and simultaneously. Thus, indicating the status of acute infection, Thus, indicating the status of acute infection, convalescence or previous exposureconvalescence or previous exposure

Salmonella typhi IgG/IgM Rapid test = an Salmonella typhi IgG/IgM Rapid test = an immunochromatographic assay for rapid, immunochromatographic assay for rapid, qualitative and differential detection of IgG and IgM qualitative and differential detection of IgG and IgM antibodies to Salmonella typhi in human serum, antibodies to Salmonella typhi in human serum, plasma or whole bloodplasma or whole blood

TyphidotTyphidot

Typhoid Fever Rapid testTyphoid Fever Rapid test

Typhoid Fever Rapid TestTyphoid Fever Rapid Test

H. Pylori Rapid testH. Pylori Rapid test

Malaria Ab Rapid testMalaria Ab Rapid test

Rapid test for TBRapid test for TB

Rapid test for ChlamydiaRapid test for Chlamydia

Rotavirus/Adenovirus Rapid Rotavirus/Adenovirus Rapid testtest

Rapid test for RubellaRapid test for Rubella

Rapid test for RSVRapid test for RSV

Rapid test for TetanusRapid test for Tetanus

Rapid test for LegionellaRapid test for Legionella

Rapid test Rapid test

TUMOR MARKERSTUMOR MARKERS


• What are they?What are they?• Are substances usually proteins, that are produced Are substances usually proteins, that are produced

by the body in response to cancer growth or by the by the body in response to cancer growth or by the cancer tissue itself and certain benign cancer tissue itself and certain benign (noncancerous) conditions(noncancerous) conditions

• Detected in higher than normal amounts in the blood, Detected in higher than normal amounts in the blood, urine, or body tissuesurine, or body tissues

• Some tumor markers are specific for one type of Some tumor markers are specific for one type of cancer, while others are seen in several cancer typescancer, while others are seen in several cancer types

• Measurements can be useful – when used along with Measurements can be useful – when used along with x-rays, or other tests in the detection and diagnosis x-rays, or other tests in the detection and diagnosis of some types of cancerof some types of cancer


• Measurements of tumor marker levels Measurements of tumor marker levels alone are not sufficient to diagnose cancer alone are not sufficient to diagnose cancer for the following reasons:for the following reasons:– Tumor marker levels can be elevated in people Tumor marker levels can be elevated in people

with benign conditionswith benign conditions– Tumor marker levels are not elevated in every Tumor marker levels are not elevated in every

person with cancer – especially in early stages person with cancer – especially in early stages of the diseaseof the disease

– Many tumor markers are not specific to a Many tumor markers are not specific to a particular type of cancer particular type of cancer


• Characteristics required of the “ideal” tumor Characteristics required of the “ideal” tumor markermarker– The following are desirableThe following are desirable

• 100% accuracy in differentiating between healthy 100% accuracy in differentiating between healthy individuals and tumor patientsindividuals and tumor patients

• Ability to detect all tumor patients, if possible at a Ability to detect all tumor patients, if possible at a very early stagevery early stage

• Organ specificity, so that information is provided on Organ specificity, so that information is provided on the localization of the tumorthe localization of the tumor

• Correlation between the concentration of the marker Correlation between the concentration of the marker freely circulating in serum and the individual tumor freely circulating in serum and the individual tumor stagesstages

• Ability to indicate all changes in tumor patients Ability to indicate all changes in tumor patients receiving treatmentreceiving treatment

• Prognostic value of the tumor marker concentrationPrognostic value of the tumor marker concentration


• Clinical Uses of Tumor MarkersClinical Uses of Tumor Markers•Early detection of the tumorEarly detection of the tumor

•Differentiating benign from malignant Differentiating benign from malignant conditionsconditions

•Evaluating the extent of the diseaseEvaluating the extent of the disease

•Monitoring the response of the tumor to Monitoring the response of the tumor to therapytherapy

•Predicting the recurrence of the tumorPredicting the recurrence of the tumor


• CARCINO-EMBRYONIC ANTIGEN (CEA)CARCINO-EMBRYONIC ANTIGEN (CEA)•A complex glycoprotein with a MW of A complex glycoprotein with a MW of

approximately 180,000 daltonsapproximately 180,000 daltons•First discovered in patients with First discovered in patients with

adenocarcinoma of the colon in 1965adenocarcinoma of the colon in 1965•Metabolized primarily by the liver with a Metabolized primarily by the liver with a

circulating half-life ranging from 1 to 8 dayscirculating half-life ranging from 1 to 8 days•Hepatic diseases, including extrahepatic biliary Hepatic diseases, including extrahepatic biliary

obstruction, intrahepatic cholestasis and obstruction, intrahepatic cholestasis and hepatocellular disease, may impede clearance hepatocellular disease, may impede clearance rates and increase serum concentrationsrates and increase serum concentrations


• CARCINO-EMBRYONIC ANTIGEN (CEA)CARCINO-EMBRYONIC ANTIGEN (CEA)•Normally, it is present in the fetal intestine, Normally, it is present in the fetal intestine,

pancreas and liver during the first 2 trimesters pancreas and liver during the first 2 trimesters of gestationof gestation

•Normal colonic mucosa and pleural and Normal colonic mucosa and pleural and lactating mammary tissue bind to anti-CEA lactating mammary tissue bind to anti-CEA antiserum; however, the quantity of CEA or antiserum; however, the quantity of CEA or CEA-like molecules expressed in these tissues CEA-like molecules expressed in these tissues is much less than that observed in malignant is much less than that observed in malignant tumorstumors

•Normal range is from 0 to 2.5 to 3.0 ng/ml as Normal range is from 0 to 2.5 to 3.0 ng/ml as determined by radioimmunoassaydetermined by radioimmunoassay


• CARCINO-EMBRYONIC ANTIGEN (CEA)CARCINO-EMBRYONIC ANTIGEN (CEA)– Benign conditions that cause elevated CEABenign conditions that cause elevated CEA

•Cigarette smokingCigarette smoking BronchitisBronchitis

•EmphysemaEmphysema GastritisGastritis

•Gastric ulcerGastric ulcer Hepatic diseaseHepatic disease

•PancreatitisPancreatitis Polyps of colon & Polyps of colon & rectumrectum

•DiverticulitisDiverticulitis Crohn’s diseaseCrohn’s disease

•BPHBPH Renal diseaseRenal disease


• CARCINO-EMBRYONIC ANTIGEN (CEA)CARCINO-EMBRYONIC ANTIGEN (CEA)– Malignant conditionsMalignant conditions causing elevation of CEA in causing elevation of CEA in

addition to adenocarcinoma of colon & rectum --- addition to adenocarcinoma of colon & rectum --- Ca of the pancreas, lung, breast, stomach, Ca of the pancreas, lung, breast, stomach, thyroid gland and female reproductive tractthyroid gland and female reproductive tract

– Of these non-colonic CA, levels of CEA are most Of these non-colonic CA, levels of CEA are most commonly elevated in CA of the pancreas (65-commonly elevated in CA of the pancreas (65-90%) and lung (52-77%)90%) and lung (52-77%)

– The magnitude of elevation of levels of CEA The magnitude of elevation of levels of CEA correlates with stage of disease to a lesser correlates with stage of disease to a lesser extentextent


• Alpha-FETOPROTEIN (AFP)Alpha-FETOPROTEIN (AFP)•An oncofetal protein that was first discovered in An oncofetal protein that was first discovered in

1963 in the serum of mice with hepatoma1963 in the serum of mice with hepatoma•Normal fetal protein synthesized by the liver, Normal fetal protein synthesized by the liver,

yolk sac, and GIT that shares sequence yolk sac, and GIT that shares sequence homology with albuminhomology with albumin

•A major component of fetal plasma, reaching a A major component of fetal plasma, reaching a peak concentration of 3mg/ml at 12 weeks of peak concentration of 3mg/ml at 12 weeks of gestation -- following birth, it clears rapidly gestation -- following birth, it clears rapidly from the circulation, having a half-life of 3.5 from the circulation, having a half-life of 3.5 days days

•Concentration in adult serum <20ng/mlConcentration in adult serum <20ng/ml


• Alpha-FETOPROTEIN (AFP)Alpha-FETOPROTEIN (AFP)– Benign conditions causing elevation of Benign conditions causing elevation of

AFPAFP•22ndnd and 3 and 3rdrd trimesters of pregnancy trimesters of pregnancy

•CirrhosisCirrhosis

•Acute and chronic hepatitisAcute and chronic hepatitis

•Hepatic necrosisHepatic necrosis


• Alpha-FETOPROTEIN (AFP)Alpha-FETOPROTEIN (AFP)– Malignant conditionsMalignant conditions causing elevation of AFP causing elevation of AFP

aside from hepatomaaside from hepatoma• Teratocarcinoma of the testis and embryonal Ca (70%)Teratocarcinoma of the testis and embryonal Ca (70%)

• Carcinoma of the pancreas (23%)Carcinoma of the pancreas (23%)

• Carcinoma of the stomach (18%)Carcinoma of the stomach (18%)

• Carcinoma of the lung (7%)Carcinoma of the lung (7%)

• Carcinoma of the colon (5%)Carcinoma of the colon (5%)

*** In patients with hepatoma, the incidence of elevation *** In patients with hepatoma, the incidence of elevation of levels of AFP correlates with tumor burdenof levels of AFP correlates with tumor burden


• HUMAN CHORIONIC GONADOTROPIN HUMAN CHORIONIC GONADOTROPIN (HCG)(HCG)

•A glycoprotein hormone with a MW of 45,000 A glycoprotein hormone with a MW of 45,000 daltonsdaltons

•Composed of 2 polypeptide chain – alpha and Composed of 2 polypeptide chain – alpha and betabeta

Alpha-chain is common to several glycoprotein Alpha-chain is common to several glycoprotein hormones secreted by the anterior pituitaryhormones secreted by the anterior pituitary

Beta- chain is unique and confers structural and Beta- chain is unique and confers structural and functional identity to these hormones. Homology functional identity to these hormones. Homology exists with human luteinizing hormone and may exists with human luteinizing hormone and may cause immunologic cross-reactivity. Basis of det’n.cause immunologic cross-reactivity. Basis of det’n.


• HUMAN CHORIONIC GONADOTROPIN (HCG)HUMAN CHORIONIC GONADOTROPIN (HCG)• Circulating half-life is 12 to 20 hoursCirculating half-life is 12 to 20 hours• Normally secreted by placental tissue with highest Normally secreted by placental tissue with highest

circulating levels occurring at 60 days of gestationcirculating levels occurring at 60 days of gestation• Significant elevation occurs during pregnancy and in Significant elevation occurs during pregnancy and in

patients with trophoblastic neoplasms or patients with trophoblastic neoplasms or nonseminomatous germ cell tumorsnonseminomatous germ cell tumors

• It maybe secreted in small amounts by the testis, It maybe secreted in small amounts by the testis, pituitary gland and GITpituitary gland and GIT

• Maybe elevated in some benign conditions – peptic Maybe elevated in some benign conditions – peptic ulcer disease, inflammatory intestinal disease and ulcer disease, inflammatory intestinal disease and cirrhosiscirrhosis

• In patients with trophoblastic disease, levels of HCG In patients with trophoblastic disease, levels of HCG correlate with tumor burden, prognosis of patient and correlate with tumor burden, prognosis of patient and response to therapyresponse to therapy


• CALCITONINCALCITONIN• A peptide hormone composed of 32 amino acids with A peptide hormone composed of 32 amino acids with

a MW of 3,149 daltonsa MW of 3,149 daltons• A hypocalcemic factor secreted by C cells of the A hypocalcemic factor secreted by C cells of the

thyroid glandthyroid gland• Serum half-life is 12 minutes and normal levels are Serum half-life is 12 minutes and normal levels are

<0.1 nanogram/ml using radioimmunoassay<0.1 nanogram/ml using radioimmunoassay• Marked elevations are observed in medullary Marked elevations are observed in medullary

carcinoma of the thyroidcarcinoma of the thyroid• Primary clinical application is to detect familial Primary clinical application is to detect familial

medullary carcinoma of the thyroid which is medullary carcinoma of the thyroid which is transmitted as an autosomal dominant patterntransmitted as an autosomal dominant pattern

Secretion normally fluctuates in these patients, Secretion normally fluctuates in these patients, provocative tests (pentagastrin stimulation or provocative tests (pentagastrin stimulation or calcium infusion) greatly increased the sensitivity calcium infusion) greatly increased the sensitivity of this test to detect MCTof this test to detect MCT


• CALCITONINCALCITONIN– Other neoplasms less frequently Other neoplasms less frequently

associated with associated with increased levelsincreased levels•Small cell carcinoma of the lungSmall cell carcinoma of the lung•Carcinoma of the breastCarcinoma of the breast•CarcinoidCarcinoid•HepatomaHepatoma•Renal cell carcinomaRenal cell carcinoma•Zollinger-Ellison syndromeZollinger-Ellison syndrome


• CALCITONINCALCITONIN– Benign conditions associated with Benign conditions associated with

increased levelincreased level•PancreatitisPancreatitis

•Hyperparathyroidism (primary and Hyperparathyroidism (primary and secondary)secondary)

•Paget’s disease of bonePaget’s disease of bone

•Pulmonary diseasePulmonary disease


• CATECHOLAMINE METABOLITESCATECHOLAMINE METABOLITES• Most commonly assayed catecholamine metabolites Most commonly assayed catecholamine metabolites

are vanillylmandelic acid (VMA) and homovanillic acid are vanillylmandelic acid (VMA) and homovanillic acid (HVA), which are metabolites of norepinephrine and (HVA), which are metabolites of norepinephrine and dopamine, respectivelydopamine, respectively

• Urinary levels of this metabolites can be accurately Urinary levels of this metabolites can be accurately measured from a single urine specimen using gas measured from a single urine specimen using gas chromatographic techniques – requires avoidance of chromatographic techniques – requires avoidance of tea, coffee, fruit and vanilla from the diet 72 hours tea, coffee, fruit and vanilla from the diet 72 hours before urinary samplingbefore urinary sampling

• Most useful in diagnosing and monitoring patients Most useful in diagnosing and monitoring patients with NEUROBLASTOMAwith NEUROBLASTOMA


• CATECHOLAMINE METABOLITESCATECHOLAMINE METABOLITES•Neuroblastoma is a malignant lesion of the Neuroblastoma is a malignant lesion of the

neural crest tissue, which most commonly neural crest tissue, which most commonly occurs in childrenoccurs in children

•Elevated urinary levels of VMA and HVA are Elevated urinary levels of VMA and HVA are observed in 75 to 95% of patientsobserved in 75 to 95% of patients

• Improved survival time was reported in Improved survival time was reported in patients with a ratio of urinary VMA to HVA patients with a ratio of urinary VMA to HVA of ≥1.5of ≥1.5


• PROSTATIC ACID PHOSPHATASEPROSTATIC ACID PHOSPHATASE• First proposed as a marker of advanced carcinoma of First proposed as a marker of advanced carcinoma of

the prostate in 1938the prostate in 1938

• Acid phosphatases are group of enzymes that are Acid phosphatases are group of enzymes that are also present in lower concentrations in the bone, also present in lower concentrations in the bone, kidney, liver, spleen, and intestinekidney, liver, spleen, and intestine

• PAP is a glycoprotein with a MW of 100,000 daltons, PAP is a glycoprotein with a MW of 100,000 daltons, which consists of two identical subunitswhich consists of two identical subunits

• Levels can be elevated in some benign conditions— Levels can be elevated in some benign conditions— osteoporosis, hypoparathyroidism, hyperthyroidism, osteoporosis, hypoparathyroidism, hyperthyroidism, prostatic surgical treatment, catheterization of the prostatic surgical treatment, catheterization of the urinary tract and benign prostatic hypertrophyurinary tract and benign prostatic hypertrophy


• PROSTATIC ACID PHOSPHATASEPROSTATIC ACID PHOSPHATASE• Other malignant conditions with elevated PAP – Other malignant conditions with elevated PAP –

multiple myeloma, osteogenic sarcoma and bony multiple myeloma, osteogenic sarcoma and bony metastasesmetastases

• Can be measured by biochemical or immunologic Can be measured by biochemical or immunologic methods; radioimmunoassay is much more sensitive methods; radioimmunoassay is much more sensitive than chemical determinationthan chemical determination

• In one study, a direct correlation was observed In one study, a direct correlation was observed between reduced levels of serum acid phosphatase between reduced levels of serum acid phosphatase and a 50% reduction in the mass of the tumor after and a 50% reduction in the mass of the tumor after therapy, thus, PAP has definite limitations as a tumor therapy, thus, PAP has definite limitations as a tumor marker for carcinoma for prostatemarker for carcinoma for prostate


• ADRENOCORTICOTROPHIC HORMONE (ACTH)ADRENOCORTICOTROPHIC HORMONE (ACTH)• Most frequently observed ectopic hormone produced by Most frequently observed ectopic hormone produced by

neoplasmsneoplasms

• First reported in 1928 with small cell carcinoma of the First reported in 1928 with small cell carcinoma of the lunglung

• Associated with other malignant diseases – Associated with other malignant diseases – adenocarcinoma and squamous cell carcinoma of the adenocarcinoma and squamous cell carcinoma of the lung, carcinoid, pancreatic islet cell tumor, carcinoma of lung, carcinoid, pancreatic islet cell tumor, carcinoma of the breast, carcinoma of the colon, pheochromocytoma, the breast, carcinoma of the colon, pheochromocytoma, thymoma, medullary thyroid carcinoma and carcinoma thymoma, medullary thyroid carcinoma and carcinoma of the ovariesof the ovaries

• Benign conditions – COPD, obesity, HPN, DMBenign conditions – COPD, obesity, HPN, DM


• ADRENOCORTICOTROPHIC HORMONE (ACTH)ADRENOCORTICOTROPHIC HORMONE (ACTH)• Ectopic secretion of ACTH can be differentiated from Ectopic secretion of ACTH can be differentiated from

ACTH that originates in the pituitary gland by the ACTH that originates in the pituitary gland by the dexamethasone suppression test; failure to suppress dexamethasone suppression test; failure to suppress plasma cortisol levels with high dose dexamethasone plasma cortisol levels with high dose dexamethasone suggests ectopic secretion of ACTHsuggests ectopic secretion of ACTH

• It has no value in screening for carcinoma and It has no value in screening for carcinoma and pretreatment levels demonstrate no correlation to patient pretreatment levels demonstrate no correlation to patient survival time or stage of diseasesurvival time or stage of disease

• It lacks the sensitivity and specificity to be clinically useful It lacks the sensitivity and specificity to be clinically useful for screening, staging, or predicting response to therapyfor screening, staging, or predicting response to therapy


• ANTIDIURETIC HORMONE (ADH)ANTIDIURETIC HORMONE (ADH)• Small cell carcinoma is most commonly associated Small cell carcinoma is most commonly associated

with ectopic secretion of ADHwith ectopic secretion of ADH• Secretion of ADH may be detected biochemically or Secretion of ADH may be detected biochemically or

may present clinically as SIADHmay present clinically as SIADH• Other malignant diseases with ectopic secretion – Other malignant diseases with ectopic secretion –

carcinoma of pancreas, bronchial carcinoid tumors, carcinoma of pancreas, bronchial carcinoid tumors, carcinoma of the adrenal cortex, thymomas, carcinoma of the adrenal cortex, thymomas, carcinoma of the bladderand prostatecarcinoma of the bladderand prostate

• Benign conditions – pulmonary disease, disorders of Benign conditions – pulmonary disease, disorders of the CNS, anesthetics, and ingestion of drugsthe CNS, anesthetics, and ingestion of drugs

• Not a useful marker for screening of carcinoma, Not a useful marker for screening of carcinoma, staging or monitoring response to therapystaging or monitoring response to therapy


• CA 125CA 125•An antigen present on 80% of nonmucinous An antigen present on 80% of nonmucinous

ovarian carcinomasovarian carcinomas•Defined by a monoclonal antibody (OC125) Defined by a monoclonal antibody (OC125)

that was generated by immunizing that was generated by immunizing laboratory mice with a cell line established laboratory mice with a cell line established from human ovarian carcinomafrom human ovarian carcinoma

•Elevated in other cancers – endometrial, Elevated in other cancers – endometrial, pancreatic, lung, breast, and colonpancreatic, lung, breast, and colon

•Elevated in benign conditions – Elevated in benign conditions – menstruation, pregnancy, endometriosismenstruation, pregnancy, endometriosis


• CA 19-9CA 19-9•A monoclonal antibody generated against a A monoclonal antibody generated against a

colon carcinoma cell line to detect a colon carcinoma cell line to detect a monosialoganglioside found in patients with monosialoganglioside found in patients with gastrointestinal adenocarcinomagastrointestinal adenocarcinoma

•Elevated in gastric cancer (21-42%), colon Elevated in gastric cancer (21-42%), colon cancer (20-40%), pancreatic cancer (71-cancer (20-40%), pancreatic cancer (71-93%) 93%)


• PROSTATE-SPECIFIC ANTIGEN (PSA)PROSTATE-SPECIFIC ANTIGEN (PSA)• Found in normal prostatic epithelium and secretions Found in normal prostatic epithelium and secretions

but not in other tissuesbut not in other tissues

• It is a glycoprotein whose function may be to lyse the It is a glycoprotein whose function may be to lyse the seminal clotseminal clot

• Highly sensitive for the presence of prostatic cancerHighly sensitive for the presence of prostatic cancer

• Elevation correlated with stage and tumor volumeElevation correlated with stage and tumor volume

• Predictive of recurrence and response to treatmentPredictive of recurrence and response to treatment

• Has prognostic value in patients with very high Has prognostic value in patients with very high values prior to surgery are likely to relapsevalues prior to surgery are likely to relapse


• PROSTATE-SPECIFIC ANTIGEN (PSA)PROSTATE-SPECIFIC ANTIGEN (PSA)•Present in low concentrations in the blood of Present in low concentrations in the blood of

adult males adult males

• It is produced by both normal and abnormal It is produced by both normal and abnormal prostate cellsprostate cells

•Benign elevationsBenign elevations – prostatitis and BPH – prostatitis and BPH

COMMON TUMOR MARKERS CURRENTLY IN COMMON TUMOR MARKERS CURRENTLY IN USEUSETumor MarkersTumor Markers CancersCancers What else? What else? SampleSample

AFP (Alpha-AFP (Alpha-fetoprotein)fetoprotein)

Liver, germ Liver, germ cell cancers of cell cancers of ovaries or ovaries or testestestes

Also elevated Also elevated during during pregnancypregnancy

bloodblood

CA 15-3CA 15-3 Breast and Breast and others others including lung including lung and ovariesand ovaries

Also elevated in Also elevated in benign breast benign breast conditions; conditions;

bloodblood

CA 19-9CA 19-9 Pancreatic, Pancreatic, sometimes sometimes colorectal and colorectal and bile ductsbile ducts

Also elevated in Also elevated in pancreatitis and pancreatitis and inflammatory inflammatory bowel diseasebowel disease

bloodblood

CA 125CA 125 ovarianovarian Also elevated Also elevated with with endometriosis, endometriosis, some other some other diseases and diseases and benign benign conditions; not conditions; not recommended recommended as a general as a general screen screen

bloodblood

COMMON TUMOR MARKERS CURRENTLY IN COMMON TUMOR MARKERS CURRENTLY IN USEUSE

Tumor Tumor markersmarkers

CancersCancers What else?What else? SampleSample

CEA (Carcino-CEA (Carcino-embryonic embryonic antigenantigen

Colorectal, lung, Colorectal, lung, breast, thyroid, breast, thyroid, pancreatic, pancreatic, liver, cervix, liver, cervix, and bladderand bladder

Elevated in Elevated in other conditions other conditions such as such as hepatitis, COPD, hepatitis, COPD, colitis, colitis, pancreatitis and pancreatitis and in cigarette in cigarette smokerssmokers

bloodblood

Estrogen Estrogen ReceptorsReceptors

breastbreast Increased in Increased in hormone hormone dependent dependent cancercancer

tissuetissue

hCG (human hCG (human chorionic chorionic gonadotropin)gonadotropin)

Testicular and Testicular and trophoblastictrophoblastic

Elevated in Elevated in pregnancy, pregnancy, testicular failuretesticular failure

Blood, urineBlood, urine

Her-2/neuHer-2/neu breastbreast Oncogene that Oncogene that is present in is present in multiple copies multiple copies in 20-30% of in 20-30% of invasive breast invasive breast cancercancer

tissuetissue

COMMON TUMOR MARKERS CURRENTLY IN COMMON TUMOR MARKERS CURRENTLY IN USEUSE

Tumor Tumor markersmarkers

CancerCancer What else?What else? SampleSample

Monoclonal Monoclonal ImmunoglobulinImmunoglobulinss

Multiple Multiple Myeloma and Myeloma and Waldenstrom’s Waldenstrom’s macroglobulinemacroglobulinemiamia

Overproduction Overproduction of an Ig or Ab, of an Ig or Ab, usually usually detected by detected by protein protein electrophoresiselectrophoresis

Blood, tissueBlood, tissue

Progesterone Progesterone ReceptorsReceptors

breastbreast Increased in Increased in hormone hormone dependent dependent cancercancer

tissuetissue

PSA, total and PSA, total and freefree

prostateprostate Elevated in Elevated in BPH, prostatitis BPH, prostatitis and with ageand with age

bloodblood

LESS COMMON TUMOR MARKERSLESS COMMON TUMOR MARKERS

Tumor Tumor MarkersMarkers

CancersCancers What What else?else?

SampleSample

B2M (Beta-B2M (Beta-2 2 microglobulmicroglobulinin

Multiple Multiple myeloma, myeloma, lymphomaslymphomas

Crohn’s Crohn’s disease, disease, hepatitishepatitis

Blood Blood

BTA BTA (Bladder (Bladder tumor tumor antigenantigen

BladderBladder Gaining Gaining acceptanceacceptance

UrineUrine

CA 72-4 CA 72-4 (Cancer (Cancer antigen 72-antigen 72-44

OvarianOvarian No No evidence evidence that is that is better than better than CA 125CA 125

BloodBlood




Calcitonin Calcitonin Thyroid Thyroid Medullary Medullary carcinomacarcinoma

Also elevated Also elevated in pernicious in pernicious anemia and anemia and thyroiditsthyroidits

BloodBlood

NSE (Neuron-NSE (Neuron-specific specific enolaseenolase

NeuroblastomNeuroblastoma, small lung a, small lung cancercancer

May be better May be better than CEA for than CEA for ff. this kind of ff. this kind of lung cancerlung cancer

BloodBlood

NMP22NMP22 BladderBladder Not widely Not widely usedused

Urine Urine

Prostate-Prostate-specific specific membrane membrane antigen antigen (PSMA)(PSMA)

ProstateProstate Not widely Not widely used, levels used, levels increase increase normally with normally with ageage

BloodBlood




Prostatic acid Prostatic acid phosphatase phosphatase (PAP(PAP

Metastatic Metastatic prostate prostate cancer, cancer, myeloma, myeloma, lung cancerlung cancer

Not widely Not widely used used anymore, anymore, elevated in elevated in prostatitis and prostatitis and other other conditionsconditions

BloodBlood

S-100S-100 Metastatic Metastatic melanomamelanoma

Not widely Not widely usedused

BloodBlood

TA-90TA-90 Metastatic Metastatic melanomamelanoma

Not widely Not widely used, being used, being studiedstudied

BloodBlood

ThyroglobulinThyroglobulin ThyroidThyroid Used after Used after thyroid is thyroid is removed to removed to evaluate evaluate treatmenttreatment

BloodBlood

Lecture on Serological Diagnosis of Infectious Diseases And

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Transcript of Lecture on Serological Diagnosis of Infectious Diseases And