Week 12: Diagnosis of infectious disease TUMOR MARKERS Thursday, October 8, 2015 Biochemistry, Microbiology and Immunology.

Week 12:

Diagnosis of

infectious disease TUMOR MARKERS

Friday, April 21, 2023

Biochemistry, Microbiology and Immunology

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


• 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


• 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.


• 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


• 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• Determination of virulence of antimicrobial

resistance genes


• 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


• 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


• SYPHILIS - - Stages of the Disease1. Primary stage

= primary lesion is chancre (is a painless ulceration (sore) most commonly formed during the primary stage of syphilis. This infectious lesion forms approximately 21 days after initiation of infection)= 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


• SYPHILIS - - Stages of the Disease2. 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


• SYPHILIS - - Stages of the Disease3. 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


• SYPHILIS - - Stages of the Disease4. Tertiary Stage

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

= gummatous syphilis: A gumma is a soft, non-

cancerous growth resulting from the tertiary stage of syphilis.

= cardiovascular syphilis

= neurosyphilis

http://en.wikipedia.org/wiki/Syphilis


• 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 (pus-containing discharge from the nose)= skin eruptions (rash) especially around mouth, palms of hands and soles of feet= general lymphadenopathy, hepatosplenomegaly, jaundice, anemia, painful limbs & bone abnormality


• SYPHILIS - - DIAGNOSIS• Evaluation based on 3 factors

» Clinical findings» Demonstration of spirochetes bacteria in clinical

specimen» Present of antibodies in blood or CSF


• SYPHILIS - - DIAGNOSIS• Laboratory Testing

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


• LYME’S DISEASE= Disease first recognized in 1977 in Lyme, Connecticut= Causative organism is Borrelia burgdorferi: (bacteria)= Organism has been isolated from blood, CSF, skin lesions and joint fluid= Can be transmitted perinatally, causing intra-uterine death= Vector of transmission is the tick= Must remain attached a minimum of 24-48 hours

for transmission to occur

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = LYME’S DISEASE

• STAGES OF THE DISEASE1. Localized rash 2. 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: an idiopathic unilateral facial nerve paralysis , peripheral neuropathy, aseptic meningitis: bacteria do not grow in cultures of the fluid around the brain and spinal cord (cerebrospinal fluid).

= cardiac include carditis and arrythmia3. spread widely to other organs

= characterized by chronic arthritis= affects the large joints, especially the knee

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = LYME’S DISEASE

• Diagnostic criteria• Isolation of organism from clinical specimen or• Diagnostic titers of IgG and IgM in serum or CSF

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = STREPTOCOCCAL INFECTION

• STREPTOCOCCAL SEROLOGY• Streptococci are gram (+),

• Divided into groups or serotypes based on cell wall components

• Culture and rapid screening tests detect early infection


• GROUP A STREPTOCOCCAL INFECTION

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

• Upper respiratory tract = sore throat, tonsils• Skin • Suppurative complications =scarlet fever, septic

arthritis, meningitis


• GROUP A STREPTOCOCCAL INFECTIONA. Rheumatic Fever

= only certain serotypes of S. pyogenes is involved= develops 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 one= 30-60% of patients may suffer permanent disability


• GROUP A STREPTOCOCCAL INFECTIONB. 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 = one theory is that the damage caused by antigen-antibody complexes depositing in kidneys


• LABORATORY TESTING• Most reliable test is culture and identification

of the organism from infected site

• Rapid streptococcal screening tests from the throat exudates

• Detection of Streptococcal antibodies

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


• SEROLOGY OF VIRAL INFECTIONSA. 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

= initial infection may be clinically silent= chronic carrier state may develop and may result to liver

failure due to cirrhosis (malfunction of liver), hepatocellular carcinoma, or fulminant hepatitis: a rare and frequently fatal form of acute hepatitis B in which the patient's condition rapidly deteriorates, with hepatic encephalopathy, necrosis of the hepatic.

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 (right upper quadrant) , dark urine

and pale stool» Recovery 2-4 weeks, no carrier state» Mortality 0-1%

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 recovery and may remain detectable for life

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = VIRAL

HEPATITIS• Hepatitis B virus (HBV)

• 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


HEPATITIS• Hepatitis D virus (HDV) = Serological

marker• IgM anti-D and total anti-HD (IgM and IgG)

detected during acute phase


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


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• Pregnant women with HEV may develop liver

failure and death• No distinctive markers, diagnosis based on

symptoms for exposed individuals in endemic countries


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.

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: a viral infection causing fever, sore throat, and swollen lymph glands, especially in the neck

• Other diseases – malignant lymphoma , nasopharyngeal carcinoma, B-cell lymphoma


VIRUS GROUP• Epstein-Barr Virus (EBV)

– Characteristics of infection• 4-7 week incubation, acute self limiting • Enlarged LN (lymph nodes) 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


VIRUS GROUP• Cytomegalovirus

• Transmission occurs from person to person

• 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

Rubella Virus

• Rubella, also known as German measles or three-day measles, is a disease caused by the rubella virus. The name "rubella" is derived from Latin, meaning little red. Rubella is also known as German measles because the disease was first described by German physicians in the mid-eighteenth century. This disease is often mild and attacks often pass unnoticed. The disease can last one to three days.

http://en.wikipedia.org/wiki/Disease

http://en.wikipedia.org/wiki/Rubella_virus

Rubella

• Children recover more quickly than adults. Infection of the mother by Rubella virus during pregnancy can be serious; if the mother is infected within the first 20 weeks of pregnancy, the child may be born with congenital rubella syndrome (CRS), which entails a range of serious incurable illnesses. Spontaneous abortion occurs in up to 20% of cases.

http://en.wikipedia.org/wiki/Congenital_rubella_syndrome

Rubella

• It is transmitted via airborne droplet emission from the upper respiratory tract of active cases (can be passed along by the breath of people sick from Rubella). The virus may also be present in the urine, feces and on the skin. The disease has an incubation period of 2 to 3 weeks.

http://en.wikipedia.org/wiki/Incubation_period

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

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• 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.


• Laboratory diagnosis of HIV infection1. Methods utilized to detect

• Antibody• Antigen• Viral nucleic acid• Virus in culture


• Laboratory diagnosis of HIV infectionViral 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 = 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

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = 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

Typhoid Fever Rapid test

Typhoid Fever Rapid Test

H. Pylori Rapid test

Malaria Ab Rapid test

Rapid test for TB

Rapid test for Chlamydia

Rotavirus/Adenovirus Rapid test

Rapid test for Rubella

Rapid test for RSV

Rapid test for Tetanus

TUMOR MARKERS

• 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 MARKERS

• 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 MARKERS• 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

TUMOR MARKERS

• 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 MARKERS

• 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 MARKERS

• 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

Renal disease

TUMOR MARKERS

• 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 (gastrointestinal tract) 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 MARKERS

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

• 2nd and 3rd trimesters of pregnancy

• Cirrhosis

• Acute and chronic hepatitis

• Hepatic necrosis

TUMOR MARKERS

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

aside from hepatoma• Teratocarcinoma : cancer made of cysts that

contain one or more of the three layers of cells found in a developing (70%)

• Carcinoma of the pancreas (23%)• Carcinoma of the stomach (18%)• Carcinoma of the lung (7%)• Carcinoma of the colon (5%)

TUMOR MARKERS

• HUMAN CHORIONIC GONADOTROPIN (HCG)

• 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 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 MARKERS

• CALCITONIN• A peptide hormone

• 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 carcinoma of the thyroid

TUMOR MARKERS• CATECHOLAMINE METABOLITES

• Most useful in diagnosing and monitoring patients with NEUROBLASTOMA (malignant (cancerous) tumor that develops from nerve tissue. It usually occurs in infants and children).

TUMOR MARKERS

• PROSTATIC ACID PHOSPHATASE: PAP• 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• Levels can be elevated in some benign conditions

— osteoporosis, hypoparathyroidism, hyperthyroidism, prostatic surgical treatment, and benign prostatic hypertrophy

TUMOR MARKERS

• ADRENOCORTICOTROPHIC HORMONE (ACTH)

• Most frequently observed ectopic hormone produced by neoplasms

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

TUMOR MARKERS• ANTIDIURETIC HORMONE (ADH)

• Malignant diseases with ectopic secretion of ADH – carcinoma of pancreas, bronchial tumors, carcinoma of the adrenal cortex, carcinoma of the bladder and prostate

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

TUMOR MARKERS

• 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 MARKERS

• 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 MARKERS

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

secretions but not in other tissues• It is a glycoprotein• Highly sensitive for the presence of prostatic

cancer• Elevation correlated with stage and tumor

volume• Predictive of recurrence and response to

treatment

COMMON TUMOR MARKERS CURRENTLY IN USE

Tumor Markers Cancers What else? Sample

AFP (Alpha-fetoprotein)

Liver, germ cell cancers of ovaries or testes

Also elevated during pregnancy

blood

CA 15-3 Breast and others including lung and ovaries

Also elevated in benign breast conditions;

blood

CA 19-9 Pancreatic, sometimes colorectal and bile ducts

Also elevated in pancreatitis and inflammatory bowel disease

blood

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

blood


Tumor markers Cancers What else? Sample

CEA (Carcino-embryonic antigen

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

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

blood

Estrogen Receptors

breast Increased in hormone dependent cancer

tissue

hCG (human chorionic gonadotropin)

Testicular and trophoblastic

Elevated in pregnancy, testicular failure

Blood, urine

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

tissue


Tumor markers Cancer What else? Sample

Monoclonal Immunoglobulins

Multiple Myeloma and Waldenstrom’s macroglobulinemia

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

Blood, tissue

Progesterone Receptors

breast Increased in hormone dependent cancer

tissue

PSA, total and free

prostate Elevated in BPH, prostatitis and with age

blood

LESS COMMON TUMOR MARKERS

Tumor Markers

Cancers What else? Sample

B2M (Beta-2 microglobulin

Multiple myeloma, lymphomas

Crohn’s disease, hepatitis

Blood

BTA (Bladder tumor antigen

Bladder Gaining acceptance

Urine

CA 72-4 (Cancer antigen 72-4

Ovarian No evidence that is better than CA 125

Blood



Calcitonin Thyroid Medullary carcinoma

Also elevated in pernicious anemia and thyroidits

Blood

NSE (Neuron-specific enolase

Neuroblastoma, lung cancer

Blood

NMP22 Bladder Not widely used Urine

Prostate-specific membrane antigen (PSMA)

Prostate Not widely used, levels increase normally with age

Blood



Prostatic acid phosphatase (PAP

Metastatic prostate cancer, myeloma, lung cancer

Not widely used anymore, elevated in prostatitis and other conditions

Blood

S-100 Metastatic melanoma

Not widely used Blood

TA-90 Metastatic melanoma

Not widely used, being studied

Blood

Thyroglobulin Thyroid Used after thyroid is removed to evaluate treatment

Blood

Week 12: Diagnosis of infectious disease TUMOR MARKERS Thursday, October 8, 2015 Biochemistry, Microbiology and Immunology.

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