LECTURE ON SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES AND TUMOR MARKERS

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LECTURE ON SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES AND TUMOR MARKERS

• ROBERTO D. PADUA JR., MD, DPSP
• DEPARTMENT OF PATHOLOGY AND LABORATORY DIAGNOSIS
• FATIMA COLLEGE OF MEDICINE

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SEROLOGY
• The scientific study of blood sera and their
  effects
• Subdivision of immunology concerned with in-vitro
  Ag-Ab reaction
• Concerned with the laboratory study of the
  activities of the components of serum that
  contribute to immunity

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• IMMUNOLOGY
• The study of the molecules, cells, organs and
  systems responsible for the recognition and
  disposal of foreign (non-self) material
• The study of how the body components respond and
  interact
• The desirable and undesirable consequences of
  immune interactions
• The ways in which the immune system can be
  manipulated to protect or treat disease

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• IMMUNITY
• The ability of an organism to resist infection by
  means of the presence of circulating antibodies
  and white blood cells
• Distinctive characteristics of the immune system
• Specificity
• Memory
• Mobility
• Replicability
• cooperativity

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• METHODS OF DETECTION OF ANTIBODIES
• Immuno-precipitation Assays
• detect antibodies in solution
• qualitative indication of the presence of
  antibodies
• end-point is visual flocculation of the
  antigen and antibody in suspension

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• METHODS OF DETECTION OF ANTIBODIES
• 2. Complement Fixation
• based on the activation or fixation of
  complement following binding of complement
  factors to Ag-Ab immune complexes

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• METHODS OF DETECTION OF ANTIBODIES
• 3. Neutralization
• the ineffectivity of an organism or the
  activity of toxin is neutralized by specific
  antibody
• rarely used for diagnostic purposes
• mainly used to detect antibody formation
  after vaccination

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• METHODS OF DETECTION OF ANTIBODIES
• 4. Particle Agglutination
• relatively simple and fast
• capable of detecting lower concentration of
  antibodies
• designed to detect antibodies to viruses,
  subsequent to interaction or vaccination
• utilize Ag coated latex particles, coal
  particles, bentonite particles or erythrocytes
• direct and indirect methods

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• METHODS OF DETECTION OF ANTIBODIES
• 5. Immunofluorescence
• requires use of microscope equipped to
  provide ultraviolet illumination or an
  instrument capable of irradiating the assay
  with UV light and detecting the resultant
  fluorescence with a fluorometer

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• METHODS OF DETECTION OF ANTIBODIES
• 6. Enzyme Immunoassay
• the most sensitive
• usually indirect assay that depends on the use
  of an antihuman IgG or IgM antibody conjugate
• the antibody conjugate (if present) is made to
  attach to enzyme which catalyzes conversion of
  the substrate to a colored product which will
  then be read with the use of a spectrophotometer

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• METHODS OF DETECTION OF ANTIBODIES
• 7. Radioimmunoassay
• high sensitivity

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• Microbial antigen detection provides direct
  evidence of infection, and is preferred for
  diagnosis of infection over antibody detection
  (indirect evidence of infection)
• However, not all infectious agents have available
  antigen assays or culture techniques making the
  detection of specific antibodies diagnostically
  useful

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• Infectious Disease Indicators, Non-specific
• Acute phase reactants
• Limulus lysate assay
• Detects trace amounts of endotoxin from all gram
  (-) bacteria
• Presence in CSF gram (-) bacterial meningitis
• Rapid clearance from blood makes serum test
  unreliable

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• Molecular Biology
• Nucleic acid amplification
• DNA sequencing and typing
• Direct molecular probe (in situ hybridization)
• Nucleic acid quantitation

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• Molecular Biology
• Uses
• Cases requiring increased sensitivity and
  specificity of identification
• Cases requiring faster report turnaround time
• Confirmation of culture
• Identification of organisms that are non-viable
  or cannot be cultured
• Identification of fastidious, slow growing
  organisms
• Identification of organisms that are dangerous to
  culture
• Identification of organisms in small numbers or
  in small volume specimens

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• Molecular Biology
• Uses
• Density of amplifiable DNA correlates with
  microbial density
• Monitoring of disease progression or initiation
  or modification of therapy
• Drug susceptibility testing
• Differentiation of antigenically similar
  organisms
• Molecular epidemiology and infection control
• Disease diagnosis by characterization of genetic
  materials without direct identification of
  infectious agent
• Determination of virulence of antimicrobial
  resistance genes

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS
• The most commonly acquired spirochete disease in
  the U.S.
• A complex sexually transmitted disease that has a
  highly variable clinical course
• Over 50,000 cases reported in 1990 in the U.S.
• Causative agent is Treponema pallidum
• No natural reservoir in the environment, requires
  living host
• Spiral shaped and motile due to peri-plasmic
  flagella
• Variable length

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS
• Three other pathogens in the group Treponema
  which are morphologically and anti-genetically
  similar to T. pallidum, differences are in
  characteristics of lesions, amount of systemic
  involvement and course of the disease
• T. pertenue (Yaws)
• T. endemicum (non-venereal syphilis)
• T. carateum (pinta)
• T. cuniculi (rabbit syphilis)

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS
• Mode of Transmission
• Organism is very fragile, destroyed rapidly by
  heat, cold and drying
• Sexual transmission most common, occurs when
  abraded skin or mucous membranes come in contact
  with open lesion
• Can be transmitted to fetus
• Rare transmission from needle stick and blood
  transfusion

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS - - Stages of the Disease
• Primary stage
• primary lesion is chancre
• the lesion heals spontaneously after 1-5 weeks
• swab of chancre smeared on slide, examined
  under dark-field microscope, spirochetes will be
  present
• 30 become serologically positive one week
  after appearance of chancre, 90 positive after
  three weeks

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS - - Stages of the Disease
• 2. Secondary Stage
• occurs 6-8 weeks after initial chancre,
  becomes systemic, patient highly infectious
• characterized by localized or diffuse
  mucocutaneous lesions, often with generalized
  lymphadenopathy
• primary chancre may still be present
• secondary lesions subside in about 2-6 weeks
• serology tests nearly 100 positive

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS - - Stages of the Disease
• 3. Latent Stage
• stage of infection in which organisms persists
  in the body of the infected person without
  causing symptoms or signs
• this stage may last for years
• one-third of untreated latent stage
  individuals develop signs of tertiary syphilis
• after 4 years it is rarely communicable
  sexually but can be passed from mother to fetus

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS - - Stages of the Disease
• 4. Tertiary Stage
• occurs anywhere from months to years after
  secondary stage, typically between 10 to 30 years
• gummatous syphilis
• cardiovascular syphilis
• neurosyphilis

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS
• Congenital Syphilis
• Transmitted from mother to fetus
• Fetus affected during the second or third
  trimester
• 40 result in syphilitic stillbirth
• Live-born infants show no signs during first few
  weeks
• 60-90 develop clear or hemorrhagic rhinitis
• skin eruptions (rash) especially around mouth,
  palms of hands and soles of feet
• general lymphadenopathy, hepatosplenomegaly,
  jaundice, anemia, painful limbs bone abnormality

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS - - DIAGNOSIS
• Evaluation based on 3 factors
• Clinical findings
• Demonstration of spirochetes in clinical specimen
• Present of antibodies in blood or CSF
• more than one test should be performed
• no serological test can distinguish between
  other treponemal infections

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS - - DIAGNOSIS
• Laboratory Testing
• Direct examination of clinical specimen by
  dark-field microscopy or fluorescent antibody
  testing of sample
• Non-specific or non-treponemal serological test
  to detect reagin, utilized as screening test
  only, not diagnostic
• Reagin is an antibody formed against
  cardiolipin
• Found in sera of patients with syphilis as
  well as other diseases
• Non-treponemal tests become positive 1-4 weeks
  after appearance of primary chancre, in
  secondary stage may have false positive due
  to prozone, in tertiary 25 are negative,
  after successful treatment will become
  non-reactive after 1 to 2 years

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

• SYPHILIS - - DIAGNOSIS
• Laboratory Testing
• C. Specific Treponemal antibody tests are used as
  a confirmatory test for a positive reagin test

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• NON-TREPONEMAL SEROLOGICAL TESTS REAGIN TEST
• Venereal Disease Research LaboratoryVDRL
• Flocculation test, antigen consists of very
  fine particles that precipitate out in the
  presence of reagin
• Utilizes antigen consists of cardiolipin,
  cholesterol and lecithin
• serum must be heated to 56 C for 30 minnutes
  to remove anti-complimentary activity which may
  cause false positive
• reported as Non-reactive, weakly reactive and
  reactive
• used primarily to screen CSF

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• NON-TREPONEMAL SEROLOGICAL TESTS REAGIN TEST
• 2. Rapid Plasma Reagin RPR
• general screening test
• can not be performed on CSF
• the VDRL cardiolipin antigen is modified with
  choline chloride to make it more stable and is
  attached to charcoal particles to allow
  macroscopic reading, the antigen comes prepared
  and is very stable
• serum or plasma may be used for testing, serum
  is not heated
• results are read macroscopically
• appears to be more sensitive than the VDRL

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• NON-TREPONEMAL SEROLOGICAL TESTS REAGIN TEST
• 3. Other tests which use modified VDRL Ag
• A. USR unheated serum reagin test
• modified VDRL Ag, uses choline
  chloride/EDTA
• microscopic flocculation test
• B. RST reagin screen test
• modified VDRL Ag with Sudan Black
• Sudan Black makes flocculation reaction
  macroscopically visible

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• SPECIFIC TREPONEMAL TESTS
• Treponema pallidum Immobilization Test TPI
• live T. pallidum become immobilized by
  antibody in serum of infected persons
• cumbersome and expensive, no longer used in
  U.S.

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• SPECIFIC TREPONEMAL TESTS
• 2. Treponema pallidum Hemagglutination TPHA
• adapted to microtechniques (MHA-TP)
• tanned sheep RBCs are coated with T.
  pallidum antigen from Nichols strain
• positive result is agglutination of RBCs

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• SPECIFIC TREPONEMAL TESTS
• 3. Fluorescent treponemal antibody absorption
  test (FTA-ABS)
• one of the most used confirmatory test
• diluted, heat inactivated serum added to
  Reiters strain of T. pallidum to remove cross
  reactivity due to other Treponemes
• slides are coated with Nichols strain of T.
  pallidum and add absorbed patient serum
• slides are washed and incubated with Ab bound
  to a fluorescent tag
• after washing again the slides are examined
  for fluorescence
• requires experienced personnel to read
• highly sensitive and specific, but time
  consuming to perform

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• SPECIFIC TREPONEMAL TESTS
• 4. ELISA
• tubes coated with T. pallidum antigen
• antibody in serum attaches to antigen
• following washing, add an anti-antibody
  tagged with enzyme alkaline phosphatase
• detectable color changes occur

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Sensitivity and Specificity of Serologic Tests
for Untreated Syphilis at Different Stages
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Serologic Test for Syphilis in Various Conditions
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Algorithm for Positive Serologic Test for
Syphilis
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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• PROBLEM AREAS
• Biologic False Positives (BFP)
• A. Collagen diseases such as arthritis, LE,
  etc., sometimes result in increased amount of
  reagin
• B. Certain infections IM, malaria, leprosy
• C. Other treponemal infections

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• PROBLEM AREAS
• 2. False negatives
• A. Very early in disease or latent, inactive
  stage
• B. Immunosuppressed patients
• C. Consumption of alcohol prior to testing
  (temporary)

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• PROBLEM AREAS
• 3. Congenital syphilis
• A. Non-treponemal tests on cord blood or baby
  serum detect IgG antibody, maybe of maternal
  origin
• B. Detection of IgM lacks sensitivity
• C. Western blot has demonstrated high
  sensitivity and specificity
• D. Recommended that all mothers be tested

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• PROBLEM AREAS
• 4. Cerebrospinal Fluid tests
• A. Used to determine if Treponemes have invaded
  the CNS
• B. VDRL utilized to confirm neurosyphilis
• C. Lacks sensitivity

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
SYPHILIS

• CORRELATION OF TREATMENT WITH TEST RESULTS
• Treatment at the primary stage, serology tests
  become non-reactive after 6 months
• Treatment at secondary stage, tests usually
  non-reactive after 12-18 months
• If treatment is not initiated until 10 or more
  years, the reagin tests probably positive for life

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

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

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
LYMES DISEASE

• STAGES OF THE DISEASE
• Localized rash erythema chronicum migrans
• Dissemination to multiple organ system
• occurs by way of the bloodstream
• may occur weeks to months after infection
• migratory pain may occur in the joints,
  tendons and bones
• neurologic ? Bells palsy, peripheral
  neuropathy, aseptic meningitis
• cardiac include carditis and arrythmia
• 3. Chronic disseminated
• characterized by chronic arthritis
• affects the large joints, especially the knee

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
LYMES DISEASE

• Diagnostic criteria
• Isolation of organism from clinical specimen or
• Diagnostic titers of IgG and IgM in serum or CSF
  or
• Significant change in serum titers of IgG or IgM
  in paired acute and convalescent sera

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
LYMES DISEASE

• LABORATORY DIAGNOSIS
• Diagnosed clinically, confirmed serologically
• Antibodies to antigens of B. burgdorferi can be
  detected by latex agglutination, IFA, ELISA, and
  Western Blot
• Serological tests are often falsely negative
  during early weeks.
• Specific IgM Abs usually appear 2- 4 weeks after
  erythema migrans, peak after 3-6 weeks of
  illness, decline to normal after 4-6 months
• IgG titers appears more slowly (4-8 weeks after
  the rash), peak after 4-6 months, may remain high
  for months or years
• Western Blot is most sensitive
• IFA and ELISA are more commonly performed due to
  ease of procedure, but are subject to false
  positives due to either spirochete diseases and
  some autoimmune diseases

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
STREPTOCOCCAL INFECTION

• STREPTOCOCCAL SEROLOGY
• Streptococci are gram (), beta-hemolytic,
  spherical, ovoid, or lancet-shaped organisms
  which are catalase negative and seen in pairs or
  chains
• Divided into groups or serotypes based on cell
  wall components ? Streptococcus pyogenes belongs
  to Lancefield group A and it is believed the M
  protein is the chief virulent factor of this
  group
• Numerous exo-antigens are produced and excreted
  as the cell metabolizes (Streptolysin O, DNase,
  Hyaluronidase, Nicotinamide, Adenine
  dinucleotidase (NADase), Streptokinase)
• Culture and rapid screening tests detect early
  infection
• Sequelae include Rheumatic Fever and Acute GN

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
STREPTOCOCCAL INFECTION

• GROUP A STREPTOCOCCAL INFECTION
• Two major sites of infection upper respiratory
  tract and skin
• Upper respiratory tract sore throat, tonsillar
  exudate
• Skin pyoderma or impetigo
• Suppurative complications erysipelas, scarlet
  fever, septic arthritis, meningitis
• Non-suppurative complications RF or
  Post-streptococcal GN

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
STREPTOCOCCAL INFECTION

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

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
STREPTOCOCCAL INFECTION

• GROUP A STREPTOCOCCAL INFECTION
• B. Post-Streptococcal Glomerulonephritis
• follows Streptococcal infection of skin or
  pharynx
• occurs about 10 days following initial
  infection
• characterized by damage to glomeruli of the
  kidneys
• renal function impaired due to reduction in
  glomerular filtration rate, results in edema and
  HPN
• renal failure not typical
• one theory is damage caused by
  antigen-antibody complexes depositing in kidneys
• complement is activated resulting in low levels

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
STREPTOCOCCAL INFECTION

• LABORATORY TESTING
• Most reliable test is culture and identification
  of the organism from infected site
• Rapid streptococcal screening tests from the
  throat exudates have high specificity but low
  sensitivity, 60-85
• Detection of Streptococcal antibodies most useful
  in Streptococcal sequelae
• The most useful antibodies are ASO, anti-DNase
  B, anti-NADase, anti-Hyaluronidase
• Serological evidence of disease is based on
  elevated or rising titer of Streptococcal
  antibodies
• Four-fold (2 tube dilution) rise in titer is
  considered clinically significant

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
STREPTOCOCCAL INFECTION

• LABORATORY TESTING
• Anti-Streptolysin O Titer (ASO Titer)
• two of the toxins produced are Streptolysin S,
  which is oxygen stable, non-antigenic and
  Streptolysin O (SLO), which is oxygen labile and
  antigenic
• SLO is a hemolysin which is toxic to many
  tissues, including heart and kidneys
• evokes an antibody response (anti-SLO) which
  neutrolizes the hemolytic action of SLO
• the test is specific for ASO, it does not test
  for antibodies to any other Streptococcal
  exotoxins
• normal values will vary, lt125 Todd units for
  adults, 5-125 Todd units for children, recent
  Strep infections 250 Todd units for adults, 333
  Todd units for children
• a single titer is of little significance
  unless extremely elevated, titers performed over
  a period of time will give the most information

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
STREPTOCOCCAL INFECTION

• LABORATORY TESTING
• 2. Anti-DNase B Testing
• may appear earlier than ASO
• increased sensitivity for detection of
  glomerulonephritis preceded by streptococcal
  skin infection
• macro- and micro-titer, ELISA, and
  neutralization techniques are available
• Neutralization technique has advantage of
  stability of reagents

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
STREPTOCOCCAL INFECTION

• LABORATORY TESTING
• 3. Anti-Hyaluronidase Testing
• test patient serum for antibodies which
  inhibit action of Hyaluronidase
• after performance of the test, a clot will
  form into the tubes where enzyme activity of
  Hyaluronidase has been neutralized by patient
  antibody
• Hyaluronidase produced by patients with throat
  or skin infections, ASO produced in response to
  throat infections only

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
STREPTOCOCCAL INFECTION

• LABORATORY TESTING
• 4. Streptozyme Testing
• hemagglutination procedure to detect
  antibodies to numerous Streptococcal antigens
• sheep RBCs are coated with Streptolysin,
  Streptokinase, Hyaluronidase, DNase, and NADase
• patient serum diluted 1 100, mixed with
  sheep RBCs and observed for agglutination
• rapid and simple to perform, more false
  positive and negative results occur

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

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

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis A virus (HAV)
• Transmitted by fecal oral route
• Occurs worldwide
• Most hepatitis epidemics are due to HAV
• Progress of infection
• Incubation of 2-7 weeks, may be asymptomatic or
  may include jaundice
• Clinical illness develop abruptly and include
  fever, anorexia, vomiting, fatigue and malaise
• Increase in serum transaminases
• RUQ pain, dark urine and pale stool
• Recovery 2-4 weeks, no carrier state
• Mortality 0-1

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis A virus (HAV)
• Antibody and antigen markers
• First and most clinically useful is IgM antibody
  to HAV
• IgM indicates acute infection, appears 4-5 weeks
  after exposure
• IgM disappears in 3-6 months, replaced by IgG
  anti-HAV
• IgG peaks during convalescence and may remain
  detectable for life

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Time course of Hepatitis A virus (HAV) infection
60
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis B virus (HBV)
• Old term serum hepatitis, incubation period of
  4-26 weeks
• Route of infection is usually parenteral, direct
  inoculation
• Incidence of infection is 140,000-320,000 cases
  per year resulting in 5-6,000 deaths per year
• Duration of acute infection ranges from 4-8 weeks
  with symptoms similar to HAV
• 10 progress to chronic
• One-third of chronic at risk of developing
  chronic active hepatitis, cirrhosis and/or
  hepatocellular carcinoma

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis B virus (HBV) Lab Diagnosis
• Involve the detection of three marker system
• Hepatitis B surface antigen (HBsAg) is the first
  to appear, appears 2-4 weeks during late
  incubation, marker of choice for recent infection
• Anti-Hepatitis B surface antigen (anti-HBs) is
  the last antibody to appear, may persist for life
• Between disappearance of HBsAg and appearance of
  anti-HBs is known as the core window

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis B virus (HBV) Lab Diagnosis
• IgM antibody to Hepatitis B core antigen
  (anti-HBc) may be the only detectable marker
  during the core window, differentiates recent
  infection from chronic carrier state
• Third marker is Hepatitis Be antigen (HBeAg),
  appearance of HBeAg and anti-HBe, closely
  coincide with HBsAg

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Hepatitis B viral genome
64
Spread of Hepatitis B virus (HBV) in the body
65
Symptoms of typical acute viral hepatitis B
infection correlated with the four clinical
periods of this disease
66
Clinical outcomes of Acute Hepatitis B infection
67
The serologic events associated with the typical
course of acute HBV infection
68
Interpretation of Serologic Markers of Hepatitis
B Virus Infection
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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis D virus (HDV)
• Requires infection with Hepatitis B
• Route of transmission the same as HBV
• Can occur as coinfection or superinfection

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Consequences of delta virus infection
71
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis D virus (HDV) Serological markers
• HDAg found early, disappears rapidly, not very
  useful
• IgM anti-D and total anti-HD (IgM and IgG)
  detected during acute phase
• Presence of IgM anti-D and HBsAg together with
  IgM anti-HBc indicates co-infection
• Absence of IgM anti-HBc indicates superinfection
• Presence of anti-HD indicates chronic infection

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis C virus (HCV)
• Clinically and epidemiologically similar to HBV
• 60-70 of HCV patients will develop chronic
  hepatitis, 10-20 cirrhosis and 15
  hepatocellular carcinoma
• HCV and HBV may be present as co-infections

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis C virus (HCV) Serological Markers
• Serological profile not fully developed
• Present of HCV antibodies only indicates present
  or past infection
• Can have false negative in some patients

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Outcomes of Hepatitis C infection
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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis E virus (HEV)
• Similar to HAV in transmission and clinical
  course
• Found primarily in developing countries, Africa
  and Asia
• Results in acute hepatitis, no risk of chronic
  hepatitis
• Pregnant women with HEV may develop fulminant
  liver failure and death
• No distinctive markers, diagnosis based on
  symptoms for exposed individuals in endemic
  countries

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
VIRAL HEPATITIS

• Hepatitis G virus
• Independently discovered 1995-1996 by 2 separate
  research groups
• RNA virus
• Transmissible by blood-borne route
• Found in patients with acute or chronic liver
  dse.
• Exact clinical significance needs to be further
  defined
• ELISA and Western Blot methods have been developed

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS

• B. HERPES VIRUS GROUP
• includes EBV, CMV, Herpes simplex virus type
  I and II, Varicella-zoster virus
• DNA viruses that remain within nucleus while
  completing life cycle
• most infections are subclinical and result in
  latent stage

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Epstein-Barr Virus (EBV)
• Spread through oral transmission of infective
  saliva and is the cause of infectious
  mononucleosis
• Other diseases Burkitts lymphoma,
  nasopharyngeal carcinoma, B-cell lymphoma
• Virus may become reactivated and is the suggested
  cause of chronic fatigue syndrome

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Epstein-Barr Virus (EBV)
• Characteristics of infection
• 4-7 week incubation, acute self limiting
• Enlarged LN in the neck, sore throat, fever, rash
• Malaise, lethargy, extreme tiredness
• Liver and spleen involvement and enlargement
• Hematology high WBC, over 20 atypical reactive
  lymphocytes

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Epstein-Barr Virus (EBV)
• Serological testing may involve screening tests
  to detect heterophile antibodies
• Heterophile antigens are a group of similar
  antigens found in unrelated animals
• Heterophile antibodies produced against
  heterophile antigens of one species will cross
  react with others
• Forssman antigen is an example of a heterophile
  antigen and is found on the RBCs of many species
• Forssman antibodies formed against Forssman
  antigens will agglutinate sheep RBCs

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Epstein-Barr Virus (EBV)
• Infectious Mononucleosis slide tests
• Horse RBCs possess antigens which react with the
  antibody associated with IM
• Patient serum mixed with horse RBCs,
  agglutination is positive
• Not diagnostic, must look at total clinical
  picture

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Epstein-Barr Virus (EBV)
• EBV specific antibodies may be measured
• Must know pattern of appearance of EBV antigens
• Most valuable is IgM antibody to viral capsid
  antigen (VCA), indicates a current infection
  (best marker), lasts about 12 weeks
• Can also detect anti-early antigen (EA), recent
  infection and anti-EB nuclear antigen (EBNA),
  older infection
• ELISA and immunofluorescence techniques most
  commonly used

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Cytomegalovirus
• Transmission occurs from person to person
• Symptoms resemble IM but has negative test for
  EBV
• In babies may cause life-threatening illness
  resulting in CNS involvement, hearing loss, and
  mental retardation
• Seen in patients with deficient immune system,
  AIDS, transplantation

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Cytomegalovirus
• Immunologic response
• For best diagnostic results, lab tests for CMV
  antibody should be performed by using paired
  serum samples
• One blood sample should be taken upon suspicion
  of CMV, and another one taken within 2 weeks. A
  virus culture can be performed at any time the
  pt. is symptomatic
• IgM antibodies produced against early and
  intermediate-early (IE) CMV antigens, last for 3
  to 4 months
• IgG appear shortly after and peak at 2 to 3 months

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Cytomegalovirus
• Laboratory Diagnosis
• Range from culture and cytologic techniques to
  DNA probes, PCR and serologic techniques
• Detection of antibodies indicator of recent
  infection
• Viral culture lack sensitivity and are time
  consuming and expensive
• Microscopic examination of biopsy specimens,
  urine sediment or peripheral blood may reveal the
  typical cytomegalic cell with owls eye
  inclusion

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Cytomegalovirus
• Laboratory Diagnosis
• Detection of CMV Ag in cells more appropriately
  detected by immunofluorescent techniques using
  monoclonal antibodies
• ELISA is the most commonly available serologic
  test for measuring antibody to CMV
• The result can be used to determine if acute
  infection, prior infection, or passively acquired
  maternal antibody in an infant is present
• Other tests include various fluorescence assays,
  indirect hemagglutination, and latex
  agglutination
• Screening tests using coated latex particles
  compare favorably to more complex tests for
  antibody detection
• False positives can occur RA and Ebstein-Barr
  antibodies

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Herpes Simplex Virus (HSV)
• Laboratory testing
• Recovery of the virus in cell culture is
  considered the gold standard for detection of
  this virus from sources other than CSF, culture
  helpful in differentiating types of HSV
• Direct examination using immunofluorescence or
  immunoperoxidase staining of cells from lesion
• DNA probes, ELISA, latex agglutination, RIA and
  indirect immunofluorescence
• Serology is not very useful because there is a
  high prevalence of antibody in the normal
  population

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
HERPES VIRUS GROUP

• Varicella-Zoster Virus
• Laboratory testing important to distinguish VZV
  from other infections, selection of antiviral
  drugs, or determining immune status of
  individuals
• PCR is now the routine testing method for VZV
• Direct fluorescent antibody staining and viral
  culture techniques may be used for the detection
  of VZV in most specimen types
• IgG and IgM antibody tests by ELISA may be used

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
GERMAN MEASLES

• Rubella Virus
• Laboratory testing
• Performed primarily for diagnosis of acquired
  infections and to determine immune status of
  pregnant patients
• Some tests detect IgG antibodies, other IgM
• Methods include hemagglutination inhibition,
  passive hemagglutination, neutralization,
  hemolysis in gel, complement fixation,
  fluorescent immunoassay, RIA, ELISA and latex
  agglutination
• Method depends on volume of testing, turn around
  time, complexity, expense and whether a
  qualitative or quantitative test is needed

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
MEASLES

• Rubeola
• Serology testing provides best means of
  confirming a measles diagnosis
• Methods to detect Rubeola antibodies include
  hemagglutination inhibition, endpoint
  neutralization, complement fixation, IFA and
  ELISA
• In addition to signs and symptoms, diagnosis
  confirmed by presence of Rubeola specific IgM
  antibodies or four-fold rise in IgG antibody
  titer in paired samples taken after rash to 10 to
  30 days later
• IgM test highly depended on time of sample
  collection with 3-11 days after rash being optimal

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
MUMPS

• Mumps
• Methods to detect mump antibodies include
  complement fixation, hemagglutination inhibition,
  hemolysis-in-gel, neutralization assays, IFA and
  ELISA
• Current or recent infections indicated by
  presence of specific IgM antibody in single
  sample which can be detected within 5 days of
  illness
• Fourfold rise in specific IgG antibody in 2
  samples collected during acute and convalescent
  phases
• Fluorescent antibody staining for mumps antigens
  developed but not widely used
• Cross-reactivity between antibodies to mumps and
  parainfluenza viruses has been reported in test
  for IgG

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Human Immunodeficiency Virus (HIV)
• Etiologic agent of AIDS
• Discovered independently by Luc Montagnier of
  France and Robert Gallo of the US in 1983-1984
• Former names of the virus include
• Human T cell Lymphotrophic virus (HTLV-III)
• Lymphadenopathy associated virus (LAV)
• AIDS associated retrovirus (ARV)
• HIV-2 discovered in 1986, antigenically distinct
  virus endemic in West Africa
• One million people infected in US, 30 Million
  worldwide are infected
• Leading cause of death of men aged 25-44 and 4th
  leading cause of death of women in this age group
  in the US

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Structural genes
• Gag is p55 from which three core proteins (p15,
  p17 and p24) are formed
• Env gene codes for envelope proteins gp160, gp120
  and gp41
• Pol codes for p66 and p51 subunits of reverse
  transcriptase and p31 an endonuclease

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Immunologic Manifestations
• Early stage slight depression of CD4 count, few
  symptoms, temporary
• Window of up to 6 weeks before antibody is
  detected, by 6 months 95 positive
• During window p24 antigen present, acute viremia
  and antigenemia
• Antibodies produced to all major antigens
• First antibodies detected produced against gag
  proteins p24 and p55
• Followed by antibody to p51, p120 and gp41
• As disease progresses, antibody levels decreases

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Immunologic Manifestations
• Immune abnormalities associated with increased
  viral replication
• Decrease in CD4 cells
• B cells have decreased response to antigen
• CD8 cells initially increase and may remain
  elevated
• As HIV infection progresses, CD4 T cells drop
  resulting in immunosuppression and susceptibility
  of patient to opportunistic infections
• Death comes due to immuno-incompetence

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 1. Methods utilized to detect
• Antibody
• Antigen
• Viral nucleic acid
• Virus in culture

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 2. ELISA Testing
• first serological test developed to detect
  HIV infection
• antibodies detected include those directed
  against p24, gp120, gp160 and gp41, detected
  first in infection and appear in most individuals
• used for screening only, false positives do
  occur

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 4. Western Blot Testing
• most popular confirmatory test
• antibodies to p24 and p55 appear earliest
  but decrease or become undetectable
• antibodies to gp31, gp41, gp120, and
  gp160 appear later but are present throughout
  all stages of the disease

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 4. Western Blot Testing interpretation of
  result
• ? no bands, negative
• ? in order to be interpreted as positive a
  minimun of 3 bands directed against the
  following antigens must be present p24,
  p31, gp41 or gp120/160
• ? CDC criteria require 2 bands of the following
  p24, gp41 or gp120/160

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 4. Western Blot Testing interpretation of
  result
• ? indeterminate results are those samples that
  produce bands but not enough to be positive,
  may be due to the following
• 1. prior blood transfusions, even with
  non-HIV-1 infected blood
• 2. prior or current infection with syphilis
• 3. prior or current infection with malaria
• 4. autoimmune diseases
• 5. infection with other human retroviruses
• 6. second or subsequent pregnancies in women
• run an alternate HIV confirmatory assay

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 5. Indirect immunofluorescence assay
• can be used to detect both virus and
  antibody to it
• antibody detected by testing patient serum
  against antigen applied to a slide, incubated,
  washed and a fluorescent antibody added
• virus is detected by fixing patient cells to
  slide, incubating with antibody

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 6. Detection of p24 HIV antigen
• p24 antigen only present for short time,
  disappears when antibody to p24 appears
• anti-HIV-1 bound to membrane, incubated with
  patient serum, second anti-HIV-1 antibody
  attached to enzyme label is added (sandwich
  technique), color change occurs
• optical density measured, standard curve
  prepared to quantitate results
• positive confirmed by neutralizing reaction,
  preincubate patient sample with anti-HIV,
  retest, if p24 present immune complexes form
  preventing binding to HIV antibody on
  membrane added

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 6. Detection of p24 HIV antigen
• test not recommended for routine screening as
  appearance and rate of rise are unpredictable
• sensitivity lower than ELISA
• most useful for the following
• a. early infection suspected in seronegative
  patient
• b. newborns
• c. CSF
• d. monitoring disease progress

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 7. Polymerase Chain Reaction (PCR)
• looks for HIV DNA in the WBCs of a person
• amplifies tiny quantities of the HIV DNA
  present, each cycle of PCR results in doubling
  of the DNA sequences present
• the DNA is detected by using radioactive or
  biotiny lated probes
• once DNA is amplified it is placed on
  nitrocellulose paper and allowed to react with
  a radio-labeled probe, a single stranded DNA
  fragment unique to HIV, which will hybridize with
  the patients HIV DNA if present
• radioactivity is determined

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 8. Virus isolation
• definitively diagnose HIV
• best sample is peripheral blood, but can use
  CSF, saliva, cervical secretions, semen, tears
  or material from organ biopsy
• cell growth in culture is stimulated,
  amplifies number of cells releasing virus
• cultures incubated one month, infection
  confirmed by detecting reverse transcriptase or
  p24 antigen in supernatant

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 9. Viral Load Tests
• viral load or viral burden is the quantity of
  HIV-RNA that is in the blood
• measures the amount of HIV-RNA in one
  milliliter of blood
• ? take 2 measurements 2-3 weeks apart to
  determine baseline
• ? repeat every 3-6 months in conjunction with
  CD4 counts to monitor viral load and
  T-cell count
• ? repeat 4-6 weeks after starting or changing
  antiretroviral therapy to determine
  effect on viral load

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES HIV

• Laboratory diagnosis of HIV infection
• 10. Testing of neonates
• difficult due to presence of maternal IgG
  antibodies
• use tests to detect IgM or IgA antibodies, IgM
  lacks sensitivity, IgA more promising
• measurement of p24 antigen
• PCR testing maybe helpful but still not
  detecting antigen soon enough 38 days to 6
  months to be positive

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SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
DENGUE

• Dengue fever
• Transmitted by mosquitoes
• There are 4 known distinct serotypes ( dengue
  virus 1, 2, 3 and 4)
• In children , infection is often sub-clinical or
  causes a self-limited febrile disease
• Secondarily infected with a different serotype,
  dengue hemorrhagic fever or dengue shock syndrome

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Algorithm for Serologic Testing for AIDS
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