Immunodiagnostic Studies



Immunodiagnostic Studies





OVERVIEW OF IMMUNODIAGNOSTIC STUDIES

Immunodiagnostic or serodiagnostic testing studies antigen-antibody reactions for diagnosis of infectious disease, autoimmune disorders, immune allergies, and neoplastic disease. These modalities also test for blood groups and types, tissue and graft transplant matching, and cellular immunology. Blood serum is tested for antibodies against particular antigens—hence, the term blood serology testing.

Antigens are substances that stimulate and subsequently react with the products of an immune response. They may be enzymes, toxins, microorganisms (e.g., bacterial, viral, parasitic, fungal), tumors, or autoimmune factors. Antibodies are proteins produced by the body’s immune system in response to an antigen or antigens. The antigen-antibody response is the body’s natural defense against invading organisms. Red blood cell groups contain almost 400 antigens. Immune reactions to these antigens result in a wide variety of clinical disorders, which can be tested (e.g., Coombs’ test).


Pathologically, autoimmune disorders are produced by autoantibodies—that is, antibodies against self. Examples include systemic rheumatic diseases (SRDs), such as rheumatoid arthritis (RA) and lupus erythematosus.

Immunodeficiency diseases exhibit a lack of one or more basic components of the immune system, which includes B lymphocytes, T lymphocytes, phagocytic cells, and the complement system. These diseases are classified as primary (e.g., congenital, DiGeorge syndrome) and secondary (e.g., AIDS).

Hypersensitivity reactions are documented using immediate hypersensitivity tests and are defined as abnormally increased immune responses to some allergens (e.g., allergic reaction to bee stings or pollens). Delayed hypersensitivity skin tests are commonly used to evaluate cellmediated immunity. Histocompatibility antigens (transplantation antigens) and tests for human leukocyte antigen (HLA) are important diagnostic tools to detect and prevent immune rejection in transplantation.


Types of Tests

Many methods of varying sophistication are used for immunodiagnostic studies (Table 8.1).


Collection of Serum for Immunologic Tests

Specific antibodies can be detected in serum and other body fluids (e.g., synovial fluid, cerebrospinal fluid [CSF]).



  • Procure samples. For diagnosis of infectious disease, a blood sample (serum preferred) using a 7-mL red-topped tube should be obtained at illness onset (acute phase), and the other sample should be drawn 3 to 4 weeks later (convalescent phase). In general, serologic test usefulness depends on a titer increase in the time interval between the acute and the convalescent phase. For some serologic tests, one serum sample may be adequate if the antibody presence indicates an abnormal condition or the antibody titer is unusually high. See Appendix A for standard precautions.


  • Perform the serologic test before doing skin testing. Skin testing often induces antibody production and could interfere with serologic test results.


  • Label the specimen with the patient’s name, date, and tests ordered and place in a biohazard bag. Send samples to the laboratory promptly. Hemolyzed samples cannot yield accurate results. Hemoglobin in the serum sample can interfere with complement-fixing antibody values.


Interpreting Results of Immunologic Tests

The following factors affect test results:



  • History of previous infection by the same organism


  • Previous vaccination (determine time frame)


  • Anamnestic reactions caused by heterologous antigens: An anamnestic reaction is the appearance of antibodies in the blood after administration of an antigen to which the patient has previously developed a primary immune response.


  • Cross-reactivity: Antibodies produced by one species of an organism can react with an entirely different species (e.g., Tularemia antibodies may agglutinate Brucella, and vice versa, rickettsial infections may produce antibodies reactive with Proteus OX19).


  • Presence of other serious illness states (e.g., lack of immunologic response in agammaglobulinemia, cancer treatment with immunosuppressant drugs)


  • Seroconversion: the detection of specific antibody in the serum of an individual when this antibody was previously undetectable









TABLE 8.1 Some Tests That Determine Antigen-Antibody Reactions






































































Name of Test


Observable Reaction


Visible Change


Tests for


Agglutination, hemagglutination (HA), immune hemagglutination assay (IHA)


Particulate antigen reacts with corresponding antibody; antigen may be in form of red blood cells (hemagglutination, latex, or charcoal coated with antigen).


Clumping


Treponemal, heterophile, and cold agglutinin antibodies


Precipitation (e.g., immunodiffusion [ID], counterimmunoelectrophoresis [CIE])


Soluble antigen reacts with corresponding antibody by ID or count.


Precipitates


Fungal antibodies, food poisoning


Complement fixation (CF)


Competition between two antigen-antibody systems (test and indicator systems)


Complement activation, hemolysis


Viral antibodies


Immunofluorescence (e.g., indirect fluorescent antibody [IFA])


Fluorescent-tagged antibody reacts with antigen-antibody complex in the presence of ultraviolet light.


Visible microscopic fluorescence


Antinuclear antibodies (ANAs); antimitochondrial antibodies (AMAs)


Enzyme immunoassay (EIA)


Enzymes are used to label induced antigen-antibody reactions.


Chromogenic fluorescent or luminescent change in substrate


Hepatitis and HIV (screening)


Enzyme-linked immunosorbent assay (ELISA)


Indirect EIA for quantification of an antigen or antibody enzyme and substrate


Color change indicates enzyme substrate reaction.


Lyme disease, Epstein-Barr virus, extractable nuclear antibodies (connective tissue/systemic rheumatic disease)


Immunoblot (e.g., Western blot [WB])


Electrophoresis separation of antigen subspecies


Detection of antibodies of specific mobility


Confirms HIV-1


Polymerase chain reaction (PCR)


Amplifies low levels of specific DNA sequences; each cycle doubles the amount of specific DNA sequence.


Exponential accumulation of DNA fragment being amplified; defects in DNA appear as mutations.


Slightest trace of infection can be detected; more accurate than traditional tests for chlamydia; genetic disorders


Rate nephelometry


Measures either antigen or antibody in solution through the scattering of a light beam; antibody reagent used to detect antigen IgA, IgG, IgM; concurrent controls are run to establish amount of background scatter in reagents and test samples.


Light scatter proportionately increases as numbered size of immune complexes increases.


Quantitative immunoglobulins IgA, IgM, C-reactive protein, anti-streptolysin O recorded in mg/dL or IU/mL


Flow cytometry


Blood cell types are identified with monoclonal antibodies (mABs) specific for cell markers by means of a flow cytometer with an argon laser beam; as the cells pass the beam, they scatter the light; light energy is converted into electrical energy cells and stained with green (fluorescence) or orange (phycoerythrin).


Light scatter identifies cell size and granularity of lymphocytes, monocytes, and granulocytes; color fluorochromes tagged to monoclonal antibodies bind to specific surface antigens for simultaneous detection of lymphocyte subsets.


Lymphocyte immunophenocytology differentiates B cells from T cells and T-helper cells from T-suppressor cells.


Restriction fragment length polymorphism (RFLP)


DNA-based typing technique



Epidemiology of nosocomial and community-acquired infections


Complementary DNA (cDNA) probes


Uses cDNA probes directed against ribosomal RNA


Amplifies nucleic acid to identify presence of bacterial or viral load


Infectious disease such as tuberculosis, hepatitis C virus, and HIV


IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M.





Serologic Versus Microbiologic Methods

Serologic testing for microbial immunology evaluates the presence of antibodies produced by antigens of bacteria, viruses, fungi, and parasites. The best means of establishing infectious disease etiology is by isolation and confirmation of the involved pathogen. Serologic methods can assist or confirm microbiologic analysis when the patient is tested late in the disease course, antimicrobial therapy has suppressed organism growth, or culture methods cannot verify a causative agent.


BACTERIAL TESTS


• Syphilis Detection Tests

Syphilis is a venereal disease caused by Treponema pallidum, a spirochete with closely wound coils approximately 8 to 15 µm long. Untreated, the disease progresses through three stages that can extend over many years.

Antibodies to syphilis begin to appear in the blood 4 to 6 weeks after infection (Table 8.2). Nontreponemal tests determine the presence of reagin, which is a nontreponemal autoantibody directed against cardiolipin antigens. These tests include the rapid plasma reagin (RPR) and Venereal Disease Research Laboratory (VDRL) tests. The U.S. Centers for Disease Control and Prevention (CDC) recommend these tests for syphilis screening; however, they may show negative results in some cases of late syphilis. Biologic false-positive results can also occur (Table 8.3).

Conversely, treponemal (i.e., specific) tests detect antibodies to T. pallidum. These tests include the passive particle agglutination T. pallidum test (TP-PA) and the fluorescent treponemal antibody absorption test (FTA-ABS). These tests confirm syphilis when a positive nontreponemal test result is obtained. Because these tests are more complex, they are not used for screening. Certain states require automatic confirmation for all reactive screening tests by using a treponemal test such as the TP-PA or FTA-ABS.


Normal Findings

Nonreactive, negative for syphilis








TABLE 8.2 Sensitivity of Commonly Used Serologic Tests for Syphilis










































Stage


Test


Primary (%)


Secondary (%)


Late (%)


Nontreponemal (Reagin) Tests


Venereal Disease Research Laboratory (VDRL) test


70


99


1a


Rapid plasma reagin (RPR) card test; automated reagin test (ART)


80


99


0


Specific Treponemal Tests


Fluorescent treponemal antibody absorption test (FTA-ABS)


85


100


98


Treponema pallidum particle agglutination (TP-PA)


65


100


95


(This new procedure has sensitivity similar to MHA-TP.)


a Treated late syphilis.


Modified from Tramont EC: Treponema pallidum (syphilis). In Mandell GL, Douglas RE, Bennett JE (eds): Principles and Practice of Infectious Diseases, 7th ed., Vol. 2, Philadelphia, Elsevier Health Services, 2010. Also product insert Serodia TP-PA, Fujirebio, Inc., Tokyo, Japan, 2000.










TABLE 8.3 Nonsyphilitic Conditions Giving Biologic False-Positive Results Using Venereal Disease Research Laboratory and Rapid Plasma Reagin Tests

























































































Disease


Approximate Percentage BFPs


Malaria


100


Leprosy


60


Relapsing fever


30


Active immunization in children


20


Infectious mononucleosis


20


Lupus erythematosus


20


Lymphogranuloma venereum


20


Pneumonia, atypical


20


Rat-bite fever


20


Typhus fever


20


Vaccinia


20


Infectious hepatitis


10


Leptospirosis (Weil’s disease)


10


Periarteritis nodosa


10


Trypanosomiasis


10


Chancroid


5


Chickenpox


5


Measles


5


Rheumatoid arthritis


5-7


Rheumatic fever


5-6


Scarlet fever


5


Subacute bacterial endocarditis


5


Pneumonia, pneumococcal


3-5


Tuberculosis, advanced pulmonary


3-5


Blood loss, repeated


? (low)


Common cold


? (low)


Pregnancy


? (low)


BFPs, biologic false-positive results.



Sensitivity of FTA-ABS

Primary syphilis: 84%

Secondary syphilis: 100%

Latent syphilis: 100%

Late syphilis: 96%


Sensitivity of TP-PA

Primary syphilis: 86%

Secondary syphilis: 100%

Latent syphilis: 100%





False-Positive and False-Negative Reactions

A positive reaction is not conclusive for syphilis. Several conditions produce biologic false-positive results for syphilis. Biologic false-positive reactions are by no means “false.” They may reveal the presence of other serious diseases. It is theorized that reagin (reaction) is an antibody against tissue lipids. Lipids are presumed to be liberated from body tissue in the normal course of activity. These liberated lipids may then induce antibody formation. Nontreponemal biologic false-positive reactions can occur in the presence of drug abuse, lupus erythematosus, mononucleosis, malaria, leprosy, viral pneumonia, recent immunization, or, on rare occasions, pregnancy. False-negative reactions may occur early in the disease course or during inactive or later stages of disease.


Interfering Factors



  • Hemolysis can cause false-positive results.


  • Hepatitis can result in a false-positive test.


  • Testing too soon after exposure can result in a false-negative test.



• Lyme Disease Tests

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi. It is transmitted by the bite of tiny deer ticks, which reside on deer and other wild animals. Lyme disease is present worldwide, but certain geographic areas show higher incidences. Transmission to humans is highest during the spring, summer, and early fall months. The tick bite usually produces a characteristic rash, termed erythema chronicum migrans. If untreated, sequelae lead to serious joint, cardiac, and CNS symptoms.

Serologic testing for antibodies to Lyme disease includes enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Antibody formation takes place in the following manner: Immunoglobulin M (IgM) is detected 3 to 4 weeks after Lyme disease onset, peaks at 6 to 8 weeks after onset, and then gradually disappears. Immunoglobulin G (IgG) is detected 2 to 3 months after infection and may remain elevated for years. Current CDC recommendations for the serologic diagnosis of Lyme disease are to screen with a polyvalent ELISA (IgG and IgM) and to perform supplemental testing (Western blot) on all equivocal and positive ELISA results.

Western blot assays for antibodies to B. burgdorferi are supplemental rather than confirmatory because their specificity is less than optimal, particularly for detecting IgM. Two-step positive results provide supportive evidence of exposure to B. burgdorferi, which could support a clinical diagnosis of Lyme disease but should not be used as a criterion for diagnosis.


Normal Findings

Negative for both IgG and IgM Lyme antibodies by ELISA and Western blot




Interfering Factors



  • False-positive results may occur with high levels of rheumatoid factors or in the presence of other spirochete infections, such as syphilis (cross-reactivity).


  • Asymptomatic individuals who spend time in endemic areas may have already produced antibodies to B. burgdorferi.



• Legionnaires’ Disease Antibody Test

Legionnaires’ disease is a respiratory condition caused by Legionella pneumophila. It is best diagnosed by organism culture; however, the organism is difficult to grow.

Detection of L. pneumophila in respiratory specimens by means of direct fluorescent antibody (DFA) technique is useful for rapid diagnosis but lacks sensitivity when only small numbers of organisms are available. Serologic tests should be used only if specimens for culture are not available or if culture and DFA produce negative results.


Normal Findings

Negative for Legionnaires’ disease by indirect fluorescent antibody (IFA) test or ELISA





• Chlamydia Nucleic Acid Amplification Test (NAAT)

Chlamydia is caused by a genus of bacteria (Chlamydia spp.) that require living cells for growth and are classified as obligate cell parasites. Recognized species include Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis. C. psittaci causes psittacosis in birds and humans, and C. pneumoniae is responsible for approximately 10% of cases of community-acquired pneumonia. C. trachomatis is grouped into three serotypes. One group causes lymphogranuloma venereum (LGV), a venereal disease. Another group causes trachoma, an eye disease. The third group causes genital tract infections different from LGV. Culture of the organism is definitive for chlamydiae. C. trachomatis infection is the most common reportable sexually transmitted infection (STI) in the United States, and reporting has been required in all 50 states and the District of Columbia since 2000. The national infection rate for C. trachomatis has increased from 2000 to 2015, with the exception of a slight decrease during 2011 to 2013. In 2015, there were 478.8 reported cases per 100,000 people.

Screening for chlamydia is particularly important because it is such a common sexually transmitted disease (STD), which is often asymptomatic, but which can cause serious complications if left untreated. Annual screening for chlamydia is recommended for all sexually active women under the age of 25, for women older than 25 if they have new or multiple sex partners or a sex partner with a diagnosed STD, and for men who have sex with other men. Pregnant women who are at risk should also be screened for chlamydia early in pregnancy.

Although methods such as culture, direct fluorescent antibody stain and DNA probe are available to test for chlamydia, the nucleic acid amplification test (NAAT) (which detects the genetic material of chlamydia trachomatis), is recommended because it is more sensitive and specific and can be done without a pelvic exam for women.



Normal Findings

Negative for chlamydia DNA





• Streptococcal Antibody Tests: Antistreptolysin O Titer (ASO), Streptozyme, Antideoxyribonuclease-B Titer (Anti-DNase-B, ADNase-B) (ADB, Streptodornase)

Group A β-hemolytic streptococci are associated with streptococcal infections or illness.

These tests detect antibodies to enzymes produced by organisms. Group A β-hemolytic streptococci produce several enzymes, including streptolysin O, hyaluronidase, and DNase B. Serologic tests that detect these enzyme antibodies include antistreptolysin O titer (ASO), which detects streptolysin O; streptozyme, which detects antibodies to multiple enzymes; and anti-DNase B (ADB), which detects DNase B. Serologic detection of streptococcal antibodies helps to establish prior infection but is of no value for diagnosing acute streptococcal infections. Acute infections should be diagnosed by direct streptococcal cultures or the presence of streptococcal antigens.

The ASO test aids in the diagnosis of several conditions associated with streptococcal infections, such as rheumatic fever, glomerulonephritis, endocarditis, and scarlet fever. Serial rising titers over several weeks are more significant than a single result. ADB antibodies may appear earlier than ASO in streptococcal pharyngitis, and this test is more sensitive for streptococcal pyoderma.



Normal Findings

ASO titer:

Adult: <160 Todd units/mL or <200 IU Child (5 to 12 years of age): 170 to 330 Todd units/mL

ADB: A negative test is normal.

Preschool-age children: <60 Todd units/mL

School-age children: <170 Todd units/mL

Adults: <85 Todd units/mL

Streptozyme: negative for streptococcal antibodies




Interfering Factors



  • An increased titer can occur in healthy carriers.


  • Antibiotic therapy suppresses streptococcal antibody response.


  • Increased B-lipoprotein levels inhibit streptolysin O and produce falsely high ASO titers.





Helicobacter pylori (HPY) IgG Antibody Serum, Stool, and Breath (PY) Test

Helicobacter pylori (previously known as Campylobacter pylori) is a bacterium associated with gastritis, duodenal and gastric ulcers, and possibly gastric carcinoma. The healthcare provider orders this test when screening a patient for possible H. pylori infection. The organism is present in 95% to 98% of patients with duodenal ulcers and 60% to 90% of patients with gastric ulcers. A person with gastrointestinal symptoms with evidence of H. pylori colonization (e.g., presence of specific antibodies, positive breath test, positive culture, positive biopsy) is considered to be infected with H. pylori. A person without gastrointestinal symptoms having evidence of the presence of H. pylori is said to be colonized rather than infected.

This test detects H. pylori infection of the stomach. Traditionally, the presence of H. pylori has been detected through biopsy specimens obtained by endoscopy. As with any invasive procedure, there is risk and discomfort to the patient. Noninvasive methods of detection include the following:



  • Breath: measures isotopically labeled CO2 in breath specimens


  • Stool: H. pylori stool antigen test (HpSa)

The presence of H. pylori-specific IgG antibodies has been shown to be an accurate indicator of H. pylori colonization. ELISA testing relies on the presence of H. pylori IgG-specific antibody to bind to antigen on the solid phase, forming an antigen-antibody complex that undergoes further reactions to produce a color indicative of the presence of antibody and is quantified using a spectrophotometer or ELISA microweld plate reader. The sensitivity is 94% and specificity 78%, compared with an invasive procedure, such as biopsy, for which the sensitivity is 93% and specificity 99%.


Normal Findings

Negative for H. pylori by ELISA indicates no detectable IgG antibody in serum or stool. A positive result indicates the presence of detectable IgG antibody in serum or stool.


Breath

Negative: <50 disintegrations per minute (DPM) for H. pylori

50 to 199 DPM indeterminate for H. pylori

>200 DPM positive for H. pylori





VIRAL TESTS


• Epstein-Barr Virus (EBV) Antibody Tests: Infectious Mononucleosis (IM) Slide (Screening) Test, Heterophile Antibody Titer, Epstein-Barr Antibodies to Viral Capsid Antigen and Nuclear Antigen

Epstein-Barr virus (EBV) is a herpesvirus found throughout the world. The most common symptomatic manifestation of EBV infection is a disease known as infectious mononucleosis (IM). This disease induces formation of increased numbers of abnormal lymphocytes in the lymph nodes and stimulates increased heterophile antibody formation. IM occurs most often in young adults who have not been previously infected through contact with infectious oropharyngeal secretions. Symptoms include fever, pharyngitis, and lymphadenopathy. EBV is also thought to play a role in the etiology of Burkitt’s lymphoma, nasopharyngeal carcinoma, and chronic fatigue syndrome.


The most common test for EBV is the rapid slide test (Monospot) for heterophile antibody agglutination. The heterophile antibody agglutination test is not specific for EBV and therefore is not useful for evaluating chronic disease. If the heterophile test is negative in the presence of acute IM symptoms, specific EBV antibodies should be determined. These include antibodies to viral capsid antigen (anti-VCA) and antibodies to EBV nuclear antigen (EBNA) using IFA and ELISA tests.

Diagnosis of IM is based on the following criteria: clinical features compatible with IM, hematologic picture of relative and absolute lymphocytosis, and presence of heterophile antibodies.


Normal Findings

Negative for IM by latex agglutination (LA)

Negative for EBV antibodies by IFA or ELISA







• Hepatitis Tests: Hepatitis A (HAV), Hepatitis B (HBV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Hepatitis G (HGV)

Hepatitis can be caused by viruses and several other agents, including drugs and toxins. Approximately 95% of hepatitis cases are due to five major virus types: hepatitis A, B, C, D, and E (Table 8.4). Diagnosing the specific virus is difficult because the symptoms (e.g., chills, weight loss, fever, distaste for cigarettes and food, darker urine and lighter stool) presented by each viral type are similar. Additionally, some individuals may be asymptomatic or have very mild symptoms that are ascribed to the “flu.” Serologic tests for hepatitis virus markers have made it easier to define the specific type.

Hepatitis A virus (HAV), which is acquired through enteric transmission, infects the gastrointestinal tract and is eliminated through the feces. Serologically, the presence of the IgM antibody to HAV (IgM anti-HAV) and the total antibody to HAV (total anti-HAV) identifies the disease and determines previous exposure to or recovery from HAV.

Hepatitis B virus (HBV) demonstrates a central core containing the core antigen and a surrounding envelope containing the surface antigen: <0.01 pg/mL for viral load. Detection of hepatitis B core antigen (HBcAg), envelope antigen (HBeAg), and surface antigen (HBsAg) or their corresponding antibodies constitutes hepatitis B serologic or plasma assessment. Viral transmission occurs through exposure to contaminated blood or blood products through an open wound (e.g., needlesticks, lacerations). Hepatitis monitoring panel for serial testing includes four B markers: HBsAg, HBeAg, anti-HBe, and anti-HBs. Interpretation depends on clinical setting. Hepatitis B DNA ultrasensitive quantitative PCR is the most sensitive test available for hepatitis B viral load.








TABLE 8.4 Hepatitis Test Findings in Various Stages


































































Disease Stages


Viral Specific and Serologic Tests


HAV


HBV


HCV


HDV


HEV


Acute


IgM
anti-HAV


IgM anti-HBc, HBsAg


Anti-HCV


HDAg


IgM anti-HEV


Chronic


Fecal HAV 1-2 wk before symptoms


2%-10% of all persons >5 yr will progress to chronic infection


85% Anti-HCV


6% total anti-HDV


None


Infectivity


None


HBeAg, HBsAg, HBV-DNA


Anti-HCV


Total anti-HDV


None


Recovery


None


Anti-HBe, anti-HBs


None


None


None


Viral load (viral genome)



HBV DNA


HCV RNA


Carrier state


None


HBsAg


None


HBAg, anti-HDV


None


Immunity


Total anti-HAV


Anti-HBs, total anti-HBc


None


None


Uncertain


Acute viral panel


IgM anti-HAV


HBsAg


Anti-HCV, HIV test also


HAV, hepatitis A; anti-HBc, antibody to hepatitis B core antigen; HBeAg, hepatitis B envelope antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B; HCV, hepatitis C; HDV, hepatitis D; HEV, hepatitis E; IgM, immunoglobulin M.



Hepatitis C virus (HCV), formerly known as non-A, non-B hepatitis, is also transmitted parenterally. HCV infection is characterized by presence of antibodies to HCV (anti-HCV) and levels of alanine aminotransferase (ALT) that fluctuate between normal and markedly elevated. Levels of anti-HCV remain positive for many years; therefore, a reactive test indicates infection with HCV or a carrier state but not infectivity or immunity. PCR or reverse transcriptase PCR (RT-PCR) (viral load), which detects HCV RNA, should be used to confirm infection when acute hepatitis C is suspected. A negative hepatitis C antibody (recombinant immunoblot assay [RIBA]) does not exclude the possibility of HCV infection because seroconversion may not occur for up to 6 months after exposure.

Hepatitis D virus (HDV) is encapsulated by the HBsAg. Without the HBsAg coating, HDV cannot survive. Because HDV can cause infection only in the presence of active HBV infection, it is usually found where a high incidence of HBV occurs. Transmission is parenteral. Serologic HDV determination is made by detection of the hepatitis D antigen (HDAg) early in the course of the infection and by detection of anti-HDV antibody (anti-HDV) in the later stages of the disease.

Hepatitis E virus (HEV) is transmitted enterically and is associated with poor hygienic practices and unsafe water supplies, especially in developing countries. It is quite rare in the United States. Specific serologic tests include detection of IgM and IgG antibodies to HEV (anti-HEV).

Hepatitis G virus (HGV) is transmitted by contaminated blood supply and is seen when HCV and HBV are detected together. See Table 8.5 for a summary of the features of the different hepatitis agents.

The following terms are used:

ALT (alanine aminotransferase): an enzyme normally produced by the liver; blood levels may increase in cases of liver damage

Anti-HBc: antibody to hepatitis B core antigen

Anti-HBe: antibody to hepatitis B envelope antigen

Anti-HBs: antibody to hepatitis B surface antigen

Antibody: a Y-shaped protein molecule (immunoglobulin) in serum or body fluid that either neutralizes an antigen or tags it for attack by other cells or chemicals; acts by uniting with and firmly binding to an antigen. The prefix anti– followed by initials of a virus refers to specific antibody against the virus.

Chronic hepatitis: a condition in which symptoms or signs of hepatitis persist for >6 months

Cirrhosis: irreversible scarring of the liver that may occur after acute or chronic hepatitis

Delta agent: a unique RNA virus that causes acute or chronic hepatitis; requires HBV for replication and infects only patients who are HBsAg-positive; is composed of a delta antigen core and an HBsAg coat; also known as HDV

Endemic: present in a community at all times but occurring in a small number of cases

Enteric route: spread of organisms through the oral-intestinal-fecal cycle

Flavivirus: a family of small RNA viruses; HCV is similar to members of the Flavivirus family

Fulminant hepatitis: the most severe form of hepatitis; may lead to acute liver failure and death

HBcAg: hepatitis B core antigen

HBsAg: hepatitis B surface antigen

Hepatotropic: having an affinity for or exerting a specific effect on the liver

IgG: a form of immunoglobulin that occurs late in an infectious process

IgM: a form of immunoglobulin that occurs early in an infectious process

IgM anti-HAV: M-class immunoglobulin antibody to HAV

IgM anti-HBc: M-class immunoglobulin antibody to HBcAg

Immune globulin: a sterile solution of water-soluble proteins that contains those antibodies normally present in adult human blood; used as a passive immunizing agent against various viruses such as HAV









TABLE 8.5 Summary of Clinical and Epidemiologic Features of Viral Hepatitis Agents










































































































Features


Hepatitis A


Hepatitis B (HBV)


Hepatitis C (HCV)


Hepatitis D


Hepatitis E


Hepatitis G Scan with HBV + HCV


Incubation period


45-50 d


30-150 d


15-110 d


30-150 d


230-240 d


Questionable


Onset


Abrupt


Insidious


Insidious


Abrupt


Insidious


Unknown


Jaundice


Children: 10%; adults: 70%-80%


25%


25%


Varies


Unknown


No


Asymptomatic patients


Most children


Most children; adults: 50%


About 75%


Rare


Rare


Contaminated blood


Routes of transmission



Fecal-oral


Yes


No


No


No


Yes


No



Parenteral


Rare


Yes


Yes


Yes


No


Blood supply



Sexual


No


Yes


Possible


Yes


No


No



Perinatal


No


Yes


Possible


Possible


No


No



Water/food


Yes


No


No


No


Yes


Yes


Chronic state


No


Adults: 6%-10%; children: 25%-50%; infants: 70%-90%


50%


10%-15%


No


Yes


Case fatality rate


0.6%


1.4% Liver cancer


1%-2% Liver cancer with HBV and HCV


30%


1%-2% Pregnant women: 20%


Unknown



Negative-sense RNA virus: a virus in which the viral proteins are encoded by messenger RNA molecules that are complementary to the viral genome

New viruses—GBV-A, GBV-B, and GBV-C: may be causative agents in non-A through E hepatitis

Non-A, non-B hepatitis: viral hepatitis caused by viruses other than A, B, or D (e.g., C, E)

Parenteral: entering the body subcutaneously, intramuscularly, or intravenously or other means whereby the organisms reach the bloodstream directly

Positive-sense RNA virus: a virus in which the parenteral (or genomic) RNA serves as the messenger RNA for protein synthesis

Recombinant antigen: an antigen that results from the recombination of genetic components, which then are artificially introduced into a cell, leading to synthesis of a new protein

Viral load: the amount or concentration of virus in the circulation

These measurements are used for differential diagnosis of viral hepatitis, viral load.

Serodiagnosis of previous exposure and recovery of viral hepatitis is complex because of the number of serum or plasma markers necessary to determine the stage of illness. Testing methods include ELISA, microparticle enzyme immunoassay (MEIA), PCR, and RT-PCR and tests for viral genome (viral load).


Indications for Hepatitis A Vaccine


Pre-exposure Protection



  • Children should be vaccinated between 12 and 23 months of age.


  • Communities with existing vaccination programs for children 2 to 18 years of age should maintain their programs.


  • In areas without vaccination programs, catch-up vaccination of unvaccinated children 2 to 18 years of age can be considered.


Individuals at Increased Risk



  • Persons traveling to or working in countries that have high or intermediate endemicity


  • Users of injection and noninjection illicit drugs


  • Persons with clotting-factor disorders who have received solvent-detergent, treated high-purity factor VIII concentrates


  • Homosexual men


  • Individuals working with nonhuman HAV-infected primates


  • Food handlers


  • Persons employed in child care centers


  • Healthcare workers


  • Susceptible individuals with chronic liver disease


Indications for Hepatitis B Vaccine



  • Family members of adoptees from foreign countries who are HBsAg-positive


  • Healthcare workers and trainees in healthcare fields


  • Hemodialysis patients or patients with early renal failure


  • Household or sexual contacts of persons chronically infected with hepatitis B


  • Immigrants from Africa or Southeast Asia; recommended for children <11 years old and all susceptible household contacts of persons chronically infected with hepatitis B


  • Injection drug users


  • Inmates of long-term correctional facilities


  • Clients and staff of institutions for the developmentally disabled


  • International travelers to countries of high or intermediate HBV endemicity


  • Laboratory workers



  • Public safety workers (e.g., police, fire fighters)


  • Recipients of clotting factors. Use a fine needle (<23 gauge) and firm pressure at injection site for >2 minutes.


  • Persons with STIs or multiple sexual partners in previous 6 months, prostitutes, homosexual and bisexual men


  • Postvaccination blood testing is recommended for sexual contacts of HBsAg-positive persons; healthcare workers, recipients of clotting factors, and those who are HBsAg-positive are at high risk.


  • Persons in nonresidential day care programs should be vaccinated if an HBsAg-positive classmate behaves aggressively or has special medical problems that increase the risk for exposure to blood. Staff in nonresidential day care programs should be vaccinated if a client is HBsAgpositive.



    • Observe enteric and standard precautions for 7 days after onset of symptoms or jaundice with hepatitis B. Hepatitis A is most contagious before symptoms or jaundice appears.


    • Use standard blood and body fluid precautions for type B hepatitis and B antigen carriers. Precautions apply until the patient is HBsAg-negative and the anti-HBs appears. Avoid “sharps” (e.g., needles, scalpel blades) injuries. Should accidental injury occur, encourage some bleeding and wash area well with a germicidal soap. Report injury to proper department, and follow up with necessary interventions. Put on gown when blood splattering is anticipated. A private hospital room and bathroom may be indicated.


  • Persons with a history of receiving blood transfusion should not donate blood for 6 months. Transfusion-acquired hepatitis may not show up for 6 months after transfusion. Persons who test positive for HBsAg should never donate blood or plasma.


  • Persons who have sexual contact with hepatitis B-infected individuals run a greater risk for acquiring that same infection. HBsAg appears in most body fluids, including saliva, semen, and cervical secretions.


  • Observe standard precautions in all cases of suspected hepatitis until the diagnosis and hepatitis type are confirmed.


Normal Findings



  • Negative (nonreactive) for hepatitis A, B, C, D, or E by ELISA, MEIA, PCR, RIBA, or RT-PCR


  • Negative or undetected viral load (not used for primary infection, only to monitor). PCR requires a separate specimen collection.


  • Hepatitis B viral DNA (HBV-DNA) negative or nonreactive viral load (<0.01 pg/mL) in an infected individual before treatment





• HIV-1/2 Tests

Infection with HIV-1 is most prevalent in the United States and Western Europe. HIV-1 p24 antigen is a viral protein that is present in high amounts in individuals who are newly infected. A combination immunoassay is used to detect HIV-1 and HIV-2 antibodies, as well as HIV-1 p24 antigen (referred to as 4th generation testing). If a specimen is reactive on the initial immunoassay, then additional testing will take place to differentiate between HIV-1 and HIV-2 antibodies. If the results of the additional testing are either nonreactive or indeterminate, then HIV-1 nucleic acid testing (NAT) will be done. This test process provides the foundation for identifying acute and established HIV-1 infections, providing more accurate diagnosis of HIV-2 infection and timely reporting of test results to public health entities. After identification and differentiation of HIV-1 and HIV-2 antibodies, additional tests such as HIV-1 viral load, CD4+T-lymphocyte, and antiretroviral resistance assay are necessary to confirm infection, determine the stage of HIV disease and tailor treatment (Table 8.9). Most cases associated with HIV-2 are reported in West Africa.


Normal Findings

Negative for HIV antibodies and antigens




Interfering Factors



  • Test kits require the use of specific specimens. Refer to test kit instructions to verify that the proper specimen type is being used; otherwise, the kit may produce inaccurate results. Timing of appearance of viral and serologic markers varies (see Fig. 8.1)







FIGURE 8.1. Time course for appearance of viral and serologic markers during primary HIV infection. (Modified from Busch MP, Satten GA: Time course of viremia and antibody seroconversion following human immunodeficiency virus exposure. Am J Med 102(5B):117-124, 1997. Reproduced with permission of EXCERPTA MEDICA, INC. via Copyright Clearance Center.)

Sep 25, 2018 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Immunodiagnostic Studies
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