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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. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES. SEROLOGY The scientific study of blood sera and their effects

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Lecture on serological diagnosis of infectious diseases and tumor markers

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


Serological diagnosis of infectious diseases
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases1
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases2
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases3
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases4
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases5
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases6
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases7
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases8
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases9
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • METHODS OF DETECTION OF ANTIBODIES

    7. Radioimmunoassay

    = high sensitivity


Serological diagnosis of infectious diseases10
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases11
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases12
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • Molecular Biology

    • Nucleic acid amplification

    • DNA sequencing and typing

    • Direct molecular probe (in situ hybridization)

    • Nucleic acid quantitation


Serological diagnosis of infectious diseases13
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases14
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases15
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases16
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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)


Serological diagnosis of infectious diseases17
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases18
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases19
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases20
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases21
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases22
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases23
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases24
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • 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


Serological diagnosis of infectious diseases25
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES TUMOR MARKERS

  • SYPHILIS - - DIAGNOSIS

    • Laboratory Testing

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


Serological diagnosis of infectious diseases syphilis
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = TUMOR MARKERSSYPHILIS

  • NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN TEST

    • Venereal Disease Research Laboratory=VDRL

      = 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


Serological diagnosis of infectious diseases syphilis1
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = TUMOR MARKERSSYPHILIS

  • 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


Serological diagnosis of infectious diseases syphilis2
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = TUMOR MARKERSSYPHILIS

  • 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


Serological diagnosis of infectious diseases syphilis3
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = TUMOR MARKERSSYPHILIS

  • 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.


Serological diagnosis of infectious diseases syphilis4
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = TUMOR MARKERSSYPHILIS

  • SPECIFIC TREPONEMAL TESTS

    2. Treponema pallidum Hemagglutination – TPHA

    = adapted to microtechniques (MHA-TP)

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

    = positive result is agglutination of RBC’s


Serological diagnosis of infectious diseases syphilis5
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = TUMOR MARKERSSYPHILIS

  • SPECIFIC TREPONEMAL TESTS

    3. Fluorescent treponemal antibody absorption test (FTA-ABS)

    = one of the most used confirmatory test

    = diluted, heat inactivated serum added to Reiter’s strain of T. pallidum to remove cross reactivity due to other Treponemes

    = slides are coated with Nichol’s 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


Serological diagnosis of infectious diseases syphilis6
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = TUMOR MARKERSSYPHILIS

  • 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





Serological diagnosis of infectious diseases syphilis7
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases syphilis8
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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)


Serological diagnosis of infectious diseases syphilis9
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases syphilis10
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases syphilis11
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases26
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES Syphilis at Different Stages

  • LYME’S 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


Serological diagnosis of infectious diseases lyme s disease
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages LYME’S 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  Bell’s palsy, peripheral neuropathy, aseptic meningitis

      = cardiac include carditis and arrythmia

      3. Chronic disseminated

      = characterized by chronic arthritis

      = affects the large joints, especially the knee


Serological diagnosis of infectious diseases lyme s disease1
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages LYME’S 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


Serological diagnosis of infectious diseases lyme s disease2
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages LYME’S 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


Serological diagnosis of infectious diseases streptococcal infection
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases streptococcal infection1
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases streptococcal infection2
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases streptococcal infection3
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases streptococcal infection4
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases streptococcal infection5
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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, <125 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


Serological diagnosis of infectious diseases streptococcal infection6
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases streptococcal infection7
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases streptococcal infection8
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages STREPTOCOCCAL INFECTION

  • LABORATORY TESTING

    4. Streptozyme Testing

    = hemagglutination procedure to detect antibodies to numerous Streptococcal antigens

    = sheep RBC’s are coated with Streptolysin, Streptokinase, Hyaluronidase, DNase, and NADase

    = patient serum diluted 1 : 100, mixed with sheep RBC’s and observed for agglutination

    = rapid and simple to perform, more false positive and negative results occur


Serological diagnosis of infectious diseases27
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES Syphilis at Different Stages

  • 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


Serological diagnosis of infectious diseases viral hepatitis
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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%


Serological diagnosis of infectious diseases viral hepatitis1
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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



Serological diagnosis of infectious diseases viral hepatitis2
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = Syphilis at Different Stages 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


Serological diagnosis of infectious diseases viral hepatitis3
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


Serological diagnosis of infectious diseases viral hepatitis4
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = HEPATITISVIRAL 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




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


Clinical outcomes of acute hepatitis b infection
Clinical outcomes of Acute Hepatitis B infection correlated with the four clinical periods of this disease




Serological diagnosis of infectious diseases viral hepatitis5
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionVIRAL HEPATITIS

  • Hepatitis D virus (HDV)

    • Requires infection with Hepatitis B

    • Route of transmission the same as HBV

    • Can occur as coinfection or superinfection



Serological diagnosis of infectious diseases viral hepatitis6
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionVIRAL 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


Serological diagnosis of infectious diseases viral hepatitis7
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionVIRAL 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


Serological diagnosis of infectious diseases viral hepatitis8
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionVIRAL 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



Serological diagnosis of infectious diseases viral hepatitis9
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionVIRAL 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


Serological diagnosis of infectious diseases viral hepatitis10
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionVIRAL 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


Serological diagnosis of infectious diseases herpes virus
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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


Serological diagnosis of infectious diseases herpes virus group
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES VIRUS GROUP

  • Epstein-Barr Virus (EBV)

    • Spread through oral transmission of infective saliva and is the cause of infectious mononucleosis

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

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


Serological diagnosis of infectious diseases herpes virus group1
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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


Serological diagnosis of infectious diseases herpes virus group2
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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 RBC’s of many species

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


Serological diagnosis of infectious diseases herpes virus group3
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES VIRUS GROUP

  • Epstein-Barr Virus (EBV)

    • Infectious Mononucleosis slide tests

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

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

      • Not diagnostic, must look at total clinical picture


Serological diagnosis of infectious diseases herpes virus group4
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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


Serological diagnosis of infectious diseases herpes virus group5
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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


Serological diagnosis of infectious diseases herpes virus group6
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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


Serological diagnosis of infectious diseases herpes virus group7
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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 “owl’s eye” inclusion


Serological diagnosis of infectious diseases herpes virus group8
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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


Serological diagnosis of infectious diseases herpes virus group9
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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


Serological diagnosis of infectious diseases herpes virus group10
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHERPES 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


Serological diagnosis of infectious diseases german measles
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionGERMAN 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


Serological diagnosis of infectious diseases measles
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionMEASLES

  • 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


Serological diagnosis of infectious diseases mumps
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionMUMPS

  • 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


Serological diagnosis of infectious diseases hiv
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv1
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv2
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv3
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv4
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • Laboratory diagnosis of HIV infection

    1. Methods utilized to detect

    • Antibody

    • Antigen

    • Viral nucleic acid

    • Virus in culture


Serological diagnosis of infectious diseases hiv5
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv6
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv7
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv8
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv9
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv10
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv11
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv12
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • Laboratory diagnosis of HIV infection

    7. Polymerase Chain Reaction (PCR)

    = looks for HIV DNA in the WBC’s 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 patient’s HIV DNA if present

    = radioactivity is determined


Serological diagnosis of infectious diseases hiv13
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv14
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases hiv15
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionHIV

  • 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


Serological diagnosis of infectious diseases dengue
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionDENGUE

  • 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



Serological diagnosis of infectious diseases dengue1
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionDENGUE

  • Dengue fever

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




Serological diagnosis of infectious diseases dengue2
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionDENGUE

  • Dengue fever

    • Interpretation of the test

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

      • IgM positive = indicative of primary Dengue infection

      • IgG positive = indicative of secondary or past dengue infection

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

      • Invalid = insufficient specimen volume or incorrect procedural technique. Repeat the test using a new test device


Serological diagnosis of infectious diseases typhoid fever
SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = InfectionTyphoid Fever

  • Typhoid Fever

    • Caused by Salmonella typhi

    • Rapid detection is now available in the market

      • Typhidot = a qualitative detection test against a specific antigen of Salmonella typhi. It can detect both IgG and IgM separately and simultaneously. Thus, indicating the status of acute infection, convalescence or previous exposure

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


Typhidot
Typhidot Infection













Rapid test
Rapid test Infection


Tumor markers
TUMOR MARKERS Infection


Tumor markers1
TUMOR MARKERS Infection

  • What are they?

    • Are substances usually proteins, that are produced by the body in response to cancer growth or by the cancer tissue itself and certain benign (noncancerous) conditions

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

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

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


Tumor markers2
TUMOR MARKERS Infection

  • Measurements of tumor marker levels alone are not sufficient to diagnose cancer for the following reasons:

    • Tumor marker levels can be elevated in people with benign conditions

    • Tumor marker levels are not elevated in every person with cancer – especially in early stages of the disease

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


Tumor markers3
TUMOR MARKERS Infection

  • Characteristics required of the “ideal” tumor marker

    • The following are desirable

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

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

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

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

      • Ability to indicate all changes in tumor patients receiving treatment

      • Prognostic value of the tumor marker concentration


Tumor markers4
TUMOR MARKERS Infection

  • Clinical Uses of Tumor Markers

    • Early detection of the tumor

    • Differentiating benign from malignant conditions

    • Evaluating the extent of the disease

    • Monitoring the response of the tumor to therapy

    • Predicting the recurrence of the tumor


Tumor markers5
TUMOR MARKERS Infection

  • CARCINO-EMBRYONIC ANTIGEN (CEA)

    • A complex glycoprotein with a MW of approximately 180,000 daltons

    • First discovered in patients with adenocarcinoma of the colon in 1965

    • Metabolized primarily by the liver with a circulating half-life ranging from 1 to 8 days

    • Hepatic diseases, including extrahepatic biliary obstruction, intrahepatic cholestasis and hepatocellular disease, may impede clearance rates and increase serum concentrations


Tumor markers6
TUMOR MARKERS Infection

  • CARCINO-EMBRYONIC ANTIGEN (CEA)

    • Normally, it is present in the fetal intestine, pancreas and liver during the first 2 trimesters of gestation

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

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


Tumor markers7
TUMOR MARKERS Infection

  • CARCINO-EMBRYONIC ANTIGEN (CEA)

    • Benign conditions that cause elevated CEA

      • Cigarette smoking Bronchitis

      • Emphysema Gastritis

      • Gastric ulcer Hepatic disease

      • Pancreatitis Polyps of colon & rectum

      • Diverticulitis Crohn’s disease

      • BPH Renal disease


Tumor markers8
TUMOR MARKERS Infection

  • CARCINO-EMBRYONIC ANTIGEN (CEA)

    • Malignant conditions causing elevation of CEA in addition to adenocarcinoma of colon & rectum --- Ca of the pancreas, lung, breast, stomach, thyroid gland and female reproductive tract

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

    • The magnitude of elevation of levels of CEA correlates with stage of disease to a lesser extent


Tumor markers9
TUMOR MARKERS Infection

  • Alpha-FETOPROTEIN (AFP)

    • An oncofetal protein that was first discovered in 1963 in the serum of mice with hepatoma

    • Normal fetal protein synthesized by the liver, yolk sac, and GIT that shares sequence homology with albumin

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

    • Concentration in adult serum <20ng/ml


Tumor markers10
TUMOR MARKERS Infection

  • Alpha-FETOPROTEIN (AFP)

    • Benign conditions causing elevation of AFP

      • 2nd and 3rd trimesters of pregnancy

      • Cirrhosis

      • Acute and chronic hepatitis

      • Hepatic necrosis


Tumor markers11
TUMOR MARKERS Infection

  • Alpha-FETOPROTEIN (AFP)

    • Malignant conditions causing elevation of AFP aside from hepatoma

      • Teratocarcinoma of the testis and embryonal Ca (70%)

      • Carcinoma of the pancreas (23%)

      • Carcinoma of the stomach (18%)

      • Carcinoma of the lung (7%)

      • Carcinoma of the colon (5%)

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


Tumor markers12
TUMOR MARKERS Infection

  • HUMAN CHORIONIC GONADOTROPIN (HCG)

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

    • Composed of 2 polypeptide chain – alpha and beta

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

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


Tumor markers13
TUMOR MARKERS Infection

  • HUMAN CHORIONIC GONADOTROPIN (HCG)

    • Circulating half-life is 12 to 20 hours

    • Normally secreted by placental tissue with highest circulating levels occurring at 60 days of gestation

    • Significant elevation occurs during pregnancy and in patients with trophoblastic neoplasms or nonseminomatous germ cell tumors

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

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

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


Tumor markers14
TUMOR MARKERS Infection

  • CALCITONIN

    • A peptide hormone composed of 32 amino acids with a MW of 3,149 daltons

    • A hypocalcemic factor secreted by C cells of the thyroid gland

    • Serum half-life is 12 minutes and normal levels are <0.1 nanogram/ml using radioimmunoassay

    • Marked elevations are observed in medullary carcinoma of the thyroid

    • Primary clinical application is to detect familial medullary carcinoma of the thyroid which is transmitted as an autosomal dominant pattern

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


Tumor markers15
TUMOR MARKERS Infection

  • CALCITONIN

    • Other neoplasms less frequently associated with increased levels

      • Small cell carcinoma of the lung

      • Carcinoma of the breast

      • Carcinoid

      • Hepatoma

      • Renal cell carcinoma

      • Zollinger-Ellison syndrome


Tumor markers16
TUMOR MARKERS Infection

  • CALCITONIN

    • Benign conditions associated with increased level

      • Pancreatitis

      • Hyperparathyroidism (primary and secondary)

      • Paget’s disease of bone

      • Pulmonary disease


Tumor markers17
TUMOR MARKERS Infection

  • CATECHOLAMINE METABOLITES

    • Most commonly assayed catecholamine metabolites are vanillylmandelic acid (VMA) and homovanillic acid (HVA), which are metabolites of norepinephrine and dopamine, respectively

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

    • Most useful in diagnosing and monitoring patients with NEUROBLASTOMA


Tumor markers18
TUMOR MARKERS Infection

  • CATECHOLAMINE METABOLITES

    • Neuroblastoma is a malignant lesion of the neural crest tissue, which most commonly occurs in children

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

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


Tumor markers19
TUMOR MARKERS Infection

  • PROSTATIC ACID PHOSPHATASE

    • First proposed as a marker of advanced carcinoma of the prostate in 1938

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

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

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


Tumor markers20
TUMOR MARKERS Infection

  • PROSTATIC ACID PHOSPHATASE

    • Other malignant conditions with elevated PAP – multiple myeloma, osteogenic sarcoma and bony metastases

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

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


Tumor markers21
TUMOR MARKERS Infection

  • ADRENOCORTICOTROPHIC HORMONE (ACTH)

    • Most frequently observed ectopic hormone produced by neoplasms

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

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

    • Benign conditions – COPD, obesity, HPN, DM


Tumor markers22
TUMOR MARKERS Infection

  • ADRENOCORTICOTROPHIC HORMONE (ACTH)

    • Ectopic secretion of ACTH can be differentiated from ACTH that originates in the pituitary gland by the dexamethasone suppression test; failure to suppress plasma cortisol levels with high dose dexamethasone suggests ectopic secretion of ACTH

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

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


Tumor markers23
TUMOR MARKERS Infection

  • ANTIDIURETIC HORMONE (ADH)

    • Small cell carcinoma is most commonly associated with ectopic secretion of ADH

    • Secretion of ADH may be detected biochemically or may present clinically as SIADH

    • Other malignant diseases with ectopic secretion – carcinoma of pancreas, bronchial carcinoid tumors, carcinoma of the adrenal cortex, thymomas, carcinoma of the bladderand prostate

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

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


Tumor markers24
TUMOR MARKERS Infection

  • CA 125

    • An antigen present on 80% of nonmucinous ovarian carcinomas

    • Defined by a monoclonal antibody (OC125) that was generated by immunizing laboratory mice with a cell line established from human ovarian carcinoma

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

    • Elevated in benign conditions – menstruation, pregnancy, endometriosis


Tumor markers25
TUMOR MARKERS Infection

  • CA 19-9

    • A monoclonal antibody generated against a colon carcinoma cell line to detect a monosialoganglioside found in patients with gastrointestinal adenocarcinoma

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


Tumor markers26
TUMOR MARKERS Infection

  • PROSTATE-SPECIFIC ANTIGEN (PSA)

    • Found in normal prostatic epithelium and secretions but not in other tissues

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

    • Highly sensitive for the presence of prostatic cancer

    • Elevation correlated with stage and tumor volume

    • Predictive of recurrence and response to treatment

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


Tumor markers27
TUMOR MARKERS Infection

  • PROSTATE-SPECIFIC ANTIGEN (PSA)

    • Present in low concentrations in the blood of adult males

    • It is produced by both normal and abnormal prostate cells

    • Benign elevations – prostatitis and BPH








THANK YOU! Infection


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