Articles on Medical Diseases and Conditions

Category: Viral Infections

Viral Infections

  • Herpes Simplex (HSV)

    HSV infection is characterized by a primary infection, often asymptomatic, after which the virus remains dormant in the dorsal root ganglia of peripheral nerves interrupted in some patients by one or more episodes of recurrent disease. Primary infection usually requires a person without preexisting HSV antibody. However, a person can have antibody against one strain of HSV (from previous infection) and become infected by a different strain (reinfection as opposed to reactivation or recurrence of preexisting disease). Primary infection or reinfection is usually acquired from close contact with an infected person: the mouth in cases of nongenital infection, and sexual intercourse in cases of genital infection. However, transmission can occur through body secretions. Immunocompromised persons are at increased risk of primary HSV infection and reactivation. There are two closely related but distinct types of HSV that share some antigens but not others. Herpes simplex virus type 1 (HSV-1) typically produces nongenital infections of various types such as lesions of the mouth, blisters on the mucous membrane border of the lips (“canker sores,” or “cold sores”), keratitis (corneal ulcers of the eye), focal lesions of the fingers (“Whitlow”), and encephalitis (most frequently involving the temporal lobes of the brain). Occasional immunocompromised patients develop disseminated HSV-1 disease. At one time about 90% of the population was found to have antibodies against HSV-1, but more recently this incidence is said to have fallen to about 25%-50%. About 20%-45% of patients with HSV-1 oral lesions eventually develop recurrence. Herpes simplex virus type 2 (HSV-2) produces blistering lesions (vesicles) on the genitalia of males and females and is considered a venereal disease. Reports indicate that HSV-2 causes 20%-50% of genital ulcerations in U.S. sexually transmitted disease clinics. About 5%-15% of patients with genital herpes have HSV-1 isolated rather than HSV-2, and one report indicates that up to 20% of labial or facial lesions are due to HSV-2 rather than HSV-1. About 85% of persons with HSV-2 with genital lesions have recurrences.

    In primary HSV-2 genital infection that is symptomatic, the incubation period is about 5-7 days (range, 1-45 days). About one half of the patients (range, 39%-68%) develop systemic symptoms (e.g., fever, malaise, myalgia, and headache), including a subset of about 25% (range, 13%-36%) of all patients who experience a mild self-limited episode of aseptic meningitis (which has a marked difference in severity and prognosis from the severe brain infection of HSV-1). Extragenital lesions on skin or mucous membranes occur in about 25% of patients (range, 10%-30%), most often in the general area of the groin. A few patients develop lesions on one or more fingers, and herpes ocular keratitis sometimes occurs. About 20% are reported to show evidence of pharyngeal involvement, and about 50% have urethral involvement. Herpes simplex virus can be cultured from the cervix in 80%-90% of female patients. About 80% of all patients develop tender inguinal adenopathy in the second or third week.

    Recurrent infection differs considerably from primary infection. Only about 5%-10% of patients experience systemic symptoms. Extragenital lesions appear in about 5%. Cervical culture is reported to detect HSV in less than 15%.

    Neonatal HSV infection is usually due to HSV-2 associated with active maternal HSV-2 genital infection and is usually (but not always) acquired during birth rather than by transmission through the placenta. About 1% of pregnant women are estimated to have either overt or nonapparent HSV-2 infection. However, asymptomatic cervical or vulvar infection has itself been reported in about 1% (range, 0.5%-8.0%) of women. In genital HSV-2 infection during pregnancy only about 40% of infected women have typical lesions, and about 40% do not have visible lesions. It is estimated that if primary maternal HSV infection is present at delivery, there is a 40%-60% chance of symptomatic neonatal HSV-2 infection. If this occurs, there is serious or even fatal disease in about 50% of those infants. Delivery-infected infants do not develop symptoms until several days to 4 weeks after delivery. Symptoms may suggest sepsis or meningitis. If recurrent maternal HSV is present, there is only about a 5%-8% chance of infant infection. It is also reported that 70%-80% of neonatally infected infants are born to mothers who are asymptomatic at the time of delivery.

    Diagnosis of herpes simplex infection culture.

    Culture is still considered the gold standard HSV diagnosis. Material for culture must be inoculated into special transport media. Although some authorities advocate freezing the specimen, others report that refrigerator temperature is better for virus in transport media. Culture sensitivity depends on several factors. Some types of cells used for culture give better results than others. Specimens taken from vesicular lesions are considerably (50%-100%) more sensitive than material taken from ulcerative lesions, which, in turn, provide better results than crusted lesions. The earlier a lesion is cultured after it appears, the more likely it will yield a positive result (in one study, culture was positive in 70% of lesions less than 24 hours old, 50% in those 24-48 hours old, and 35% in those over 5 days old). A lesion from a primary infection is more likely to be positive than a lesion due to reinfection or recurrence. When urine or other secretions are cultured, the results from patients with primary infections are much more likely to be positive, since they shed virus much longer than patients with reinfection or recurrent disease. Culture in asymptomatic patients is much less likely to be positive than in patients with lesions. In addition to problems with sensitivity, specimens (in most hospitals) must be sent to a reference laboratory.

    Antigen detection. Several methods are available to detect HSV antigen; most differentiate between HSV-1 and HSV-2 or claim to be specific for one or the other. Most have the advantage of same-day or overnight results. Some depend on abbreviated culture followed by use of specific antibody to HSV. Others (such as fluorescent immunoassay or latex agglutination) employ specific antibody on material from clinical specimens. To date, all methods have failed to consistently detect 95% or more patients who have positive results by standard culture and, in general, independent evaluations have not consistently upheld manufacturer’s claims. Some have achieved sensitivity in the 85%-95% range compared to culture; others have not. In general, whether the lesion is from primary or recurrent infection, the type of lesion and the number of days after the lesion appears before the specimen was obtained affects methods that detect HSV antigen similarly to culture. Sensitivity of direct antigen methods tends to be better in material from mucocutaneous vesicles than from genital lesions. Also, there have been problems in cross-reaction between HSV-1 and HSV-2, especially in fluorescent antibody methods. Some nucleic acid probe methods with PCR amplification have been reported to be equal to or better than culture in tissue or CSF.

    Direct smear methods. The most rapid diagnosis is made through stained smears from scrapings obtained from a lesion. A sterile scalpel blade is used to unroof a vesicle and material from gentle scraping of the base of the lesion is smeared gently on a slide. Giemsa, Wright’s, or Papanicolaou stains can be used. For Giemsa or Wright’s stain, the smear is air-dried or fixed in methanol. For Papanicolaou, the smear is immediately fixed in cytology fixative. The slide preparation is sometimes called a Tzanck test. The technologist looks for multi-nucleated epithelial cells with enlarged atypical nuclei. The same findings are seen in varicella-zoster lesions. Pap stain also can show intranuclear inclusions. Sensitivity of the Tzanck test is reported to be 30%-91%, with average sensitivity probably about 45%-50%. It is probably less with persons who are inexperienced in obtaining specimens and interpreting the smears. Sensitivity is higher from vesicle scrapings than from other specimens. Fluorescent antibody tests have been applied to the smears, which increases positive results to about two thirds of cases.

    Serologic tests for antibody. Most current methods are ELISA or fluorescent immunoassay plus a few LA kits. Antibody detection has also been somewhat disappointing. Acute and convalescent specimens must be obtained 2 weeks apart. A fourfold rise in titer is needed to prove recent onset of infection; this is most likely to be found in HSV-2 disease and during the time of primary infection (60%-70% of cases). Only about 5% of patients with recurrent HSV demonstrate a fourfold rise in titer. There may also be problems with interpretation due to the high rate of positive results in the general population and because of cross-reaction between HSV-1 and HSV-2 antibodies.

    Other tests. In culture-proved HSV-1 encephalitis, one study reported that radionuclide brain scan revealed a focal lesion or lesions in the temporal lobe in 50% of cases, computerized tomography scan displayed some type of abnormality in 59%, and electroencephalogram (EEG) was abnormal in 81%. However, these procedures or their results cannot prove that the etiology is herpes infection. Spinal fluid tests show elevated CSF protein levels in about 80% of cases, increased WBC count in 97% (with about 75% of all cases between 50 and 500 WBCs/mm3), and normal glucose levels in 95% of cases. Another study found a normal cell count and protein level in 10% of cases on first spinal tap. Increase in WBC count is predominantly lymphocytic, although segmented neutrophils may be present in varying percentage (occasionally substantial) in the early stages. CSF immunofluorescent IgG antibody tests are about 25% sensitive by 10 days after onset of symptoms and about 85% after 15 days. At present, brain biopsy with culture of the specimen is the most accurate method of diagnosis. However, there is controversy about biopsy of such a vital organ. Culture of brain biopsy specimens is said to detect up to 95% of patients with HSV–1 encephalitis. Microscopic examination of the biopsy specimens can demonstrate encephalitis in about 85% of cases, but detects the intranuclear inclusions necessary to diagnosis HSV in only about 50% of cases. Use of immunofluorescent staining methods increases diagnosis to about 70%. Nucleic acid probe with PCR amplification was reported to detect over 95% of patients with HSV encephalitis testing CSF. However, homemade reagents were used. Clinical assessment alone is not sufficiently accurate: in one series of patients who underwent brain biopsy for suspected herpes, about 45% did not disclose herpes and about 10% were found to have treatable diseases other than herpes. CSF culture was positive in only about 5% of patients whose brain biopsy results were culture positive. In one large series, serologic tests suggested that 30% of patients had primary HSV infection and 70% had recurrent infection.

  • Human T-Cell Lymphotropic Virus I and II (HTLV-I and HTLV-II)

    These are closely related retroviruses somewhat distantly related to HIV-1. Transmission is similar to that of HIV-1 (contaminated blood products, less frequently by sexual intercourse or breast feeding). HTLV-I is found predominantly in Southern Japan, some of the Caribbean islands, parts of Central and South America, and sub-Saharan Africa. HTLV-I has been detected in U.S. intravenous drug abusers (20%-25%; range 7%-49%) and female prostitutes (7%; range, 0-25%) and in Native Americans in the United States (1%-13%) and Central and South America (8%-33%). HTLV-I is associated with adult T-cell leukemia (also called T-cell leukemia/lymphoma), involving peripheral blood and lymph nodes with large malignant cells having a multilobated (monocyte-shaped) nucleus and having a short clinical course. HTLV-I is less frequently associated with a neurologic condition called tropical spastic paraparesis. HTLV-II currently has no definite disease association, although several have been suggested.

    Serologic tests for HTLV-I antibody are mostly ELISA methods based on whole virus antigen. In general, these tests also detect most patients with HTLV-II. However, some reports indicate a significant number of HTLV-II patients are missed. Western blot methods are used to confirm and differentiate positive test results, and these procedures also have shown inconsistent results. Several new ELISA tests are based on several recombinant viral proteins and are said to reliably detect and differentiate the two viruses. At present, nucleic acid probe with PCR enhancement is the most sensitive and reliable way to differentiate HTLV-I and II.

    Idiopathic CD4 T-cell lymphocytopenia (ICL)

    This syndrome is being defined as CD4 T-cell counts below 300/mm3 (µL) or less than 20% of the total number of lymphocytes, no serologic evidence of HIV or HTLV infection, and no other known cause for CD4 depression. The main clinical findings are infection and other conditions associated with immunosuppression. Only a few cases have been reported as of 1994. Thus far, there has not been any strong evidence of blood-borne or sexual transmission. Retrovirus etiology has been suspected but not proven (to date).

  • Human Immunodeficiency Virus 2 (HIV-2)

    HIV-2 is closely related to, but not identical, to HIV-1. HIV-2 is found predominantly in West Africa, where in some areas it is the predominant HIV infection. In other areas it may occur with less frequency than HIV-1. It has also been found in low frequency in Central, East, and Southern Africa. It is spread through sexual intercourse. A few cases have been reported in various Western countries, including the United States, thus far almost entirely in immigrants from West Africa or a few persons who traveled or lived temporarily in that region. Clinically, HIV-2 resembles HIV-1, although in general HIV-2 appears to be somewhat slower to progress to AIDS. Antibodies to HIV-2 cross-react to some extent with standard serologic tests for antibody to HIV-1; the frequency of cross-reaction has been variable (8%-91%). Also, cross-reactivity to HIV-1 tests decreases as severity of HIV-2 infection increases. The typical HIV-2 reaction pattern with HIV-1 tests is a reactive HIV-1 screening test result plus an “indeterminant” Western blot result.

    Specific ELISA tests for HIV-2 antibody are not available, and a Western blot technique can be used to verify HIV-2 infection. In addition, commercial tests are now available designed specifically to detect both HIV-1 and HIV-2. These tests are being used predominantly in blood banks.

  • Human Immunodeficiency Virus 1 (HIV-1)

    The HIVs are retroviruses; their genetic information (genome) is composed of RNA rather than the usual DNA. To reproduce, the virus uses an enzyme known as reverse transcriptase to produce a DNA copy of its genetic RNA and incorporates this material into the host cell genetic material. Some of the copied viral genome also exists in the host cell without being incorporated into host chromosomes. Thus, the host cell nucleus reproduces the virus as well as itself. The HIVs have an unusual property, similar to the herpesvirus group, that active viral reproduction and infection can coexist with presence of antibodies against the virus. In most other virus infections, appearance of specific antibody marks the end of the infection and confers partial or complete protection against subsequent infection by that virus. The HIVs attack a subgroup of T-lymphocytes known as helper (inducer) T-cells (CD4 cells). Helper T-cells are important in cell-mediated immunity (delayed hypersensitivity), which is the immunologic mechanism classically defending against chronic lower virulence infections such as tuberculosis, fungus, and parasites. Monocytes, macrophages, and possibly Langerhans cells also become infected.

    The first HIV to be discovered was isolated from patients with the acquired immunodeficiency syndrome (AIDS) by three different groups of investigators who each gave the virus a different name (human T-cell lymphotropic virus type III, or HTLV-III; lymphadenopathy-associated virus, or LAV; and AIDS-associated retrovirus, or ARV). The current terminology for this virus is human immunodeficiency virus type 1 (HIV-1). This virus is present endemically in Central Africa. A related virus that produces a syndrome similar to AIDS is found in West Africa and has been named HIV-2 (originally called HTLV-IV). The HIV viruses are related to a similar virus found in African green monkeys. They are also related, but less closely, to certain animal viruses called lenteviruses (“slow viruses”), of which the most well known is the visna virus of sheep. Besides the HIV virus group that injures or destroys helper T-cells, there is another group of viruses that affects T-cells but that causes excessive T-cell proliferation rather than destruction. This group has retained the name of HTLV and includes HTLV-I (which causes human T-cell leukemia) and HTLV-II (which may be associated with hairy cell leukemia). Similar to the HIV viruses, the HTLV virus group is related to a monkey T-cell leukemia virus and more distantly to a T-cell leukemia virus of cattle.

    Clinical findings

    HIV-1 can be isolated from many body fluids (including blood or blood products, semen, cervical secretions, saliva, tears, cerebrospinal fluid, breast milk, urine, and various tissues including the cornea). However, urine and saliva appear to have relatively little infectious capacity. HIV-1 is predominantly transmitted in three ways: by sexual intercourse (heterosexual or male homosexual), by transfusion or inoculation of infected blood or blood products, and by mother to fetus through the placenta. After exposure, there is an incubation period that typically lasts 2-6 weeks (range, 6 days-8 weeks, but sometimes lasting several months or years). In about 50% of patients (range, 4%-70%) this is followed by an acute viral type of illness (sometimes called the “acute seroconversion” or “acute HIV syndrome”) resembling infectious mononucleosis or CMV infection that usually lasts 2-3 weeks (range, 3 days-several weeks). Symptoms usually include fever, sore throat, and lymphadenopathy; often include skin rash, myalgias, diarrhea, vomiting, and aseptic meningitis; and sometimes thrombocytopenia. Some patients never develop the initial acute febrile illness or any clinical infection; they may recover completely from the initial exposure (although this is probably uncommon) or may become an asymptomatic carrier. Those who develop the initial acute illness exhibit a wide spectrum of possible outcomes. After recovery they may become asymptomatic carriers; may have defective immunologic responses without clinical disease; may develop persistent generalized lymphadenopathy (PGL); may develop a variety of non-life-threatening fungal, bacterial, or viral infections (e.g., oral Candida) as part of the so-called AIDS-related complex (ARC); or may develop the AIDS syndrome.

    AIDS is the most severe manifestation of HIV-1 infection, defined briefly as serologic evidence of HIV antigen or antibody plus certain opportunistic infections or Kaposi’s sarcoma (a malignant tumor of fibroblasts and capillary-sized blood vessels) in a patient who is immunocompromised without known cause. The most frequent opportunistic organism producing active infection in AIDS is pneumocystis carinii (about 60% of cases; range, 35%-80%); other common agents include Cryptococcus neoformans (4%-13% of cases), Candida albicans esophagitis (14%-25%), “atypical” mycobacteria of the Mycobacterium avium-intracellulare complex (22%-30%), and protozoans such as Toxoplasma(3%-12%) and Cryptosporidium (4%-13%). Other organisms with evidence of frequent past or recent infection include CMV (66%94%) and HSV (4%-98%, the lower figures being active infection). Incidence of Kaposi’s sarcoma varies according to risk group; in male homosexuals with AIDS the incidence is about 35% (range, 25%-50%) in clinical studies and 30%-75% in autopsy studies; but in drug abusers and hemophiliacs it is found in less than 5%. Some 50%-80% of patients with AIDS develop various types of neurologic disorders with or without dementia, which may precede other evidence of AIDS in 25% of patients. In one series, 66% of these patients had elevated CSF protein levels (42-189 mg/100 ml; 0.42-1.89g/L); 20% had a small degree of mononuclear cell count elevation (4-51 WBCs); and three of seven patients tested had oligoclonal bands detected in their CSF. Cerebral abnormalities were found in two thirds of the patients with AIDS who were autopsied. There is an increased incidence of B-cell lymphoma, especially primary CNS lymphoma (2%-6% of patients).

    In the United States as of 1992, about 58% of AIDS patients were male homosexuals, usually those who had multiple sex partners. About 23% were intravenous drug abusers; about 6% were persons infected heterosexually; and about 4% were of undetermined etiology. However, incidence of heterosexual infection (as opposed to current incidence of AIDS, a late-stage development of HIV-1 infection) is becoming more frequent. Infection has been reported after a single heterosexual encounter, although more commonly it takes more than one episode. After an infected person develops detectable antibody, the current approximate calculated progression to AIDS per year is about 2.5% for asymptomatic patients, 3.5% for PGL patients, and 8.5% for ARC patients. Progression to AIDS is highest among infected male homosexuals (4%-10%/year) and low among transfusion-infected hemophiliacs. About 20%-40% (range, 10%-73%) of infected mothers transmit the virus to the fetus during pregnancy. A few infants appear to become infected during delivery and some during breast feeding.

    Laboratory findings

    In the few small studies in AIDS patients that contain hematologic data, anemia was present in about 80% (range, 45%-95%), leukopenia in about 65% (range, 40%-76%), thrombocytopenia in about 25%-30% (range, 3%-59%; about 5%-10%, range 3%-15% in HIV-infected non-AIDS patients) and pancytopenia in 17%-41%. Lymphocytopenia was reported in about 70%-80% (range, 30%-83%).

    Diagnosis of HIV-1 infection

    Culture. HIV-1 can be isolated from concentrated peripheral blood lymphocytes and less frequently from body fluids. Isolation rates in already seropositive patients average about 50%-60% (range, 8%-100%; more likely positive just before or during the first half of the acute HIV syndrome). Culture is difficult, is expensive, takes several days, is available only at a relatively few laboratories, and is positive more often in early stages of infection than in later stages. Culture may be the only method that can confirm infection in the first 2-3 weeks after exposure. Culture detects only about 50% of neonates infected in utero in the newborn period up to the first month of life (range, 30%-50%) but a greater percentage at 3 and 6 months. Culture is positive in CSF from about 30% (range, 20%-65%) of seropositive adult patients whether or not CNS symptoms are present, but about 20% more often in more advanced states of disease.

    Antigen detection. Viral antigen may become detectable as soon as 2 weeks after infection (in one report it was detected 4 days after a transplant operation). Antigenemia (viremia) lasts roughly 3 months (range, 1 week-5 months). In several reports, antigen could be detected from a few days to as many as 6-9 months before first-generation ELISA antibody test results became positive. Several methods have been used, including RIA, fluorescent antibody, and ELISA. Until about 1990, sensitivity was usually less than 50% and varied considerably between kits of different manufacturers. It was discovered that varying amounts of the circulating p24 antigen were bound to immune complexes. Methods are now available that break up (dissociate) the bound complexes before testing. In one study this increased test sensitivity to 60%-65% in patients without symptoms and 80%-90% in patients with symptoms.

    Nucleic acid probe kits with PCR amplification (NA-PCR) have become available. These detect HIV antigen within infected peripheral blood lymphocytes. Sensitivity appears to be about 40%-60% in the first 1-2 weeks of life and up to 98% by age 3 months. NA-PCR detects about 96%-100% of seropositive pediatric patients over age 6 months or adult patients with CD4 counts over 800/mm3 and about 85%-97% of those with CD4 counts below 200/mm3. NA-PCR is more sensitive than culture in HIV-infected but seronegative patients and can detect HIV in CSF from about 60% of seropositive patients. As with all laboratory tests, all manufacturer’s NA-PCR probes are not identical in sensitivity.

    Antibody detection. Seroconversion occurs on the average about 6-10 weeks after infective exposure (range, 12 days-5 years), which corresponds to the last part of the acute HIV syndrome stage or up to several weeks afterward (in some degree depending on the sensitivity of the test). Two types of antibodies are produced, IgM and IgG. IgM antibodies are detectable first, and several studies report IgM antibody present in some patients 1-10 weeks before IgG antibody (using first-generation IgG ELISA methods). In general, IgM antibody becomes detectable about 1-2 weeks after onset of the “acute HIV syndrome” (about 5-6 weeks after infection), peaks about 2-3 weeks after first detection, and becomes nondetectable 2-4 months after first detection. IgG antibody becomes detectable 1-2 weeks after IgM antibody, peaks several weeks later, and persists for life (there is controversy over whether a few patients lose antibody shortly before death from AIDS). However, one recent study using a second-generation IgG ELISA found little difference. Commercial ELISA IgM, second-generation IgG, and rapid slide LA methods are now available. Many of these use antibody against one (sometimes more) selected protein components of HIV-1 obtained through recombinant DNA techniques (see Confirmatory Methods section). Test results in HIV-1 infection are shown in Fig. 17-10.

    Tests in HIV-1 infection

    Fig. 17-10 Tests in HIV-1 infection.

    The bulk of published test kit evaluations involve first-generation ELISA methods, which are based on crude extracts of the whole virus. There were a considerable number of such methods commercially available, but even the first was introduced in only mid-1985. These tests detect antibody in 94%-99.5% of patients with confirmed AIDS, depending on the particular assay and the investigator. Positive tests in random blood donors have averaged about 0.9% (range, 0.2%-2.56%). However, in some (not all) of these first-generation kits only about 25%-35% (range, 17%-44%) of initially positive ELISA test results on random blood donors remain reactive when retested with the same kit. Of those whose results were repeatedly positive, only about 20%-35% (range, 15%-62%) were positive on confirmatory tests. This means that only about 10%-15% (range, 3%-22%) of the initial positive results on random blood donors from these particular kits eventually were confirmed positive. Some manufacturer’s kits were shown to be more sensitive than others, and some produced more false positive results than others. Some of this discrepancy is explained on the basis of different appearance times or quantity present of different viral antigens being detected by the different kit antibodies being used. There is also controversy whether reactivity against only a single antigen or a certain type (e.g., the core proteins [“group-specific antigen” or gag] p24 and p55 or the envelope glycoproteins gp 120/160 and gp 41) is sufficient to consider the test truly reactive and thus indicative of HIV-1 infection in the absence of reactivity against any other important structural protein. When this happens, it is often considered a false positive or an “indeterminant” reaction, although its significance has not yet been definitely established. In addition to these controversies, definite false negative results and false positive results may occur. Previously mentioned have been false negative results due to variable time periods before antibody is produced and variation in sensitivity of different methods and different manufacturers kits. Also, in the late or terminal stages of AIDS, antibody may disappear from patient serum in about 2%-4% (range, 0%-7%) of those who previously had antibody.

    When HIV infection is acquired in utero, IgM (and IgA) antibody is slow to rise until 3-6 months after birth. In several reports, IgA was detectable in 6%-17% at one month of age, 57%-67% at 3 months, and 77%-94% at 6 months. IgG antibody was not helpful for the first 6 months of life (range, 3-18 months) because it may be acquired from the mother through the placenta. False negative and positive results can be due to technical and clerical errors. False positive results in some kits may be due to patient antibodies against certain HLA antigens (most often, DR4) in antigenic material from infected H9 cells used in the kits to capture the patient antibodies. Antigenic material from different cell lines or synthetic (recombinant) antigen does not have this problem. Some kits, but not others, have an increased incidence of false positive results in active alcoholic cirrhosis, renal failure, and autoimmune diseases. Gamma globulin used for HBV prophylaxis may contain antibody against HIV-1, although the gamma globulin is noninfectious due to certain steps in its manufacture. This passively transferred antibody may be detectable for as long as 3 months. Antibody detection methods for urine have been developed with sensitivity reported to be comparable to serum tests.

    Confirmatory antibody detection methods. Until 1988 these tests consisted of Western blot and immunofluorescent methods. Western blot is an immunochromatographic technique in which virus material from cell culture is separated into its major component proteins by electrophoresis, transferred (“blotted”) onto a chromatography support medium, and exposed to patient serum. Antibody in patient serum, if present, attaches to whatever virus component proteins it recognizes. Then the preparation is stained to display visually what protein areas had reacted. Not all the virus proteins are considered specific for HIV-1. There is some controversy as to what specific proteins must be present for the results to be considered definitely positive. This affects sensitivity of the test. The two most specific proteins are the virus envelope glycoprotein gp41 and the group-specific antigen (gag) core protein p24 (the numbers refer to molecular weight). However, other proteins, particularly a precursor envelope protein called gp160 (from which gp41 is derived), often appears before either of the more specific proteins. Western blot in general has been shown to detect antibody earlier than most of the first-generation ELISA tests but not as early as IgM or antigen-detection methods (or “second-generation” IgG tests). Unfortunately, Western blot is time consuming, takes 2 days to complete, and reliable results are considerably technique dependent. False negative and false positive results have been reported, although the exact incidence is currently unknown, due to lack of quality control surveys. The test is currently available only in large medical centers and reference laboratories. Immunofluorescence procedures are also available and are claimed to produce results equivalent to Western blot. Immunofluorescence is easier to perform and produces same-day results. However, a minority of investigators found Western blot to be more reliable. Both of these techniques are generally considered suitable to confirm screening test results. The Western blot, however, is currently considered the gold standard. There is also a radioimmunoprecipitation (RIPA) technique that has also been used as a confirmatory procedure. This method is slightly more sensitive than Western blot. However, it is technically difficult and currently is used only in research laboratories.

    Recently, tests have become available based on genetically engineered HIV proteins, most often gp160, gp120, gp41, and p24. One or more of these are used in “second-generation” ELISA or LA tests. In general, these tests are somewhat more sensitive and specific than the “first-generation” tests. One kit (HIVAGEN) consists of separate ELISA tests for antibody against several of these antigens, thus becoming, in effect, a sort of ELISA version of the Western blot.

    Tests for immunologic status. As noted previously, HIV-1 selectively infects T-lymphocyte CD4 cells (also called helper/inducer, Leu3, or OKT4 cells; CD means cluster designation), which eventually leads to defective immune function. CD8 T-cells (suppressor/cytotoxic or OKT8 cells) are normal or become increased. The 1993 CDC revised classification system for HIV infection considers 500 CD4 T-cells/mm3 or more to be normal; 200-499, moderately decreased; and less than 200, severely decreased. CD4 absolute or relative counts are considered to be the best index of HIV disease severity. Eighty percent to 95% of AIDS patients have a decreased absolute number of helper T-cells (<400/mm3) and a reversed (inverted) helper T-cell/suppressor T-cell ratio, with a T4/T8 ratio less than 1.0. One possible cause of false T4 decrease is the recent report that about 20% of African Americans have helper T-cells that fail to react with the OKT4 antibody but do react with the Leu3 and certain other helper T-cell antibodies. A lesser but substantial number of AIDS patients display more nonspecific immune system abnormalities, such as lymphocytopenia (<1,500/mm3) and nonreactivity to skin test challenge by standard delayed sensitivity antigens such as Candida, mumps, or Trichophyton. Tests of immune function usually do not become abnormal until relatively late stages of HIV-1 infection. These tests are not tests for HIV infection, nor are they diagnostic of AIDS. CD4 cell levels are currently considered the best overall indicator of HIV-1 disease severity and prognosis.

    Beta-2 microglobulin. Beta-2 microglobulin (B2M) is a small polypeptide that forms the light chain of the class I histocompatibility complex antigen (HLA) molecules present on the surface of many nucleated cells, including lymphocytes. It is released into serum by cell destruction or membrane turnover and is filtered by the renal glomerulus, after which it is more than 99% reabsorbed and metabolized by the proximal renal tubules. About 50% of serum B2M is derived from lymphocytes. Therefore, B2M levels have been used as a nonspecific marker for lymphocyte proliferation or turnover, as seen in immunologic stimulation or lymphoproliferative disorders. Since it is excreted by the kidney, it has been used to estimate renal function. Since CD4 (T-helper) lymphocytes are affected in HIV infection, B2M is reported to be elevated in about 85%-90% (range, 68%-100%) of patients with AIDS or ARC, 45% of patients with PGL, in smaller numbers of other persons with HIV infection but few or no symptoms, and in varying numbers of clinically healthy male homosexuals (20%-44% in two studies). Some investigators have used B2M as a marker for progression to AIDS, because in general the degree of B2M elevation corresponds roughly with degree of HIV illness severity and inversely with CD4 count. B2M can be assayed by RIA, EIA, or immunodiffusion.

    B2M may also become elevated in persons with poor renal function; in various lymphomas and leukemias, especially (but not exclusively) those of B-cell origin, in myeloma, in nonlymphoid malignancies, in sarcoidosis, in infectious mononucleosis and certain other viral infections, in various chronic inflammatory diseases including active liver disease, and in autoimmune disorders.

    Neopterin. Neopterin is an intermediate substance in the biopterin synthesis pathway. It is produced by macrophages when stimulated by gamma interferon released by activated T-cells (lymphocytes also produce neopterin but to a minor degree). Therefore, neopterin is an indirect indicator of increased T-cell activity. Neopterin is excreted by the kidney. Plasma or urine neopterin levels can be elevated in acute or active chronic infections or noninfectious inflammatory conditions, similar to B2M or C-reactive protein (CRP). The neopterin level is likely to be elevated in both viral and bacterial infection, whereas the CRP level is more likely to become elevated in bacterial than in viral infection. Also, the neopterin level is more likely than the CRP level to be elevated in immunologic graft rejection, whereas both become elevated in graft infection. In general, like B2M, the neopterin level becomes elevated in HIV infection; and the incidence and degree of elevation have a rough correlation to degree of HIV severity and inverse correlation to CD4 count. One reference states that the neopterin level is elevated in about 90% of seropositive but asymptomatic HIV-infected persons. Another found B2M elevated in 75% of these asymptomatic HIV patients and the neopterin level elevated in 60%; both were elevated in all ARC patients. B2M thus far seems to have aroused more interest than neopterin as a marker for severity in HIV infection.

    Summary of human immunodeficiency virus 1 tests. In summary, ELISA tests for HIV-1 antibody are used as a general screen to detect persons infected with HIV-1. Western blot (or equivalent) tests help establish presence of infection. Culture, tests for HIV-1 antigen, and possibly ELISA-IgM antibody tests, may detect infection earlier than the ELISA screening tests. Tests for decreased immunologic function (especially CD4 lymphocyte absolute counts) are useful to help confirm a clinical impression of advanced-stage HIV-1 and AIDS.

  • Cytomegalovirus (CMV)

    CMV is part of the herpesvirus group (herpes simplex, CMV, Epstein-Barr, and varicella-zoster). CMV infection is widespread, since serologic evidence of infection varies from about 30% to over 90% between different geographic areas and population groups. In general, there is lower incidence in Western European nations and many areas of the United States. The two major periods of infection appear to be fetal or early childhood and late adolescence or young adulthood. Certain population subgroups (e.g., male homosexuals, transplant patients, and patients with HIV-1 infection) have increased incidence or risk. Infections are acquired through contact with body secretions or urine, since CMV is present for variable (sometimes long) periods of time in saliva, blood, semen, cervical secretions, breast milk, and urine.

    The majority of persons with acute CMV illness remain totally or almost asymptomatic. Those who become symptomatic most often develop a 2-3 week illness resembling Epstein-Barr infectious mononucleosis both in clinical symptoms and signs and in laboratory test results (with the exception that the heterophil antibody test and specific tests for EBV are negative (see discussion of EBV in this chapter). Some patients, mostly immunocompromised, develop more severe disease.

    After infection there is an incubation period of 40-60 days (range, 4-12 weeks). During this time circulating CMV antigen can be detected at about day 30-60 and viremia can be demonstrated by sensitive culture methods during a restricted period from approximately days 55-85. Incubation leads to acute illness, manifested by shedding of virus into body secretions, a process that can last for months or years. IgM-type antibody rises early in the acute phase of illness, followed in about one week by IgG antibody.

    After the acute infection stage, there is usually a latent period during which viral shedding may continue but at reduced levels. The latent stage may last throughout life or there may be one or more episodes of reactivation.

    About 1%-2% (range, 0.7%-4.0%) of pregnant women develop primary CMV infection; of these, fetal infection is thought to occur in 30%-50% of cases, of which about 20% develop symptomatic disease. About 5%-15% of mothers have CMV reactivation during pregnancy, with fetal infection occurring in about 10%. There are some reports that congenital (in utero) infection is more likely to occur in the second and third trimesters but that severe injury to the fetus more likely (but not exclusively) occurs when infection takes place in the first or second trimester. Primary CMV maternal infection is much more dangerous to the fetus than a reactivated infection during pregnancy. Overall, congenital intrauterine CMV infection is reported in about 1% (range, 0.2%-2.2%) of infants, of which only about 5%-10% develop clinical symptoms. In the newborn, CMV disease may appear in two forms:

    1. A subacute form with predominantly cerebral symptoms, manifested by the picture of cerebral palsy or mental retardation. This is the classic form acquired in utero.
    2. An acute form with various combinations of hepatosplenomegaly, thrombocytopenia, hepatitis with jaundice, and cerebral symptoms such as convulsions. There usually is anemia, and there may be nucleated RBCs and an increase in immature neutrophils (predominantly bands) on peripheral blood smear.

    Noncongenital infection in the newborn may take place during or after birth. It has been reported that 3%-28% of pregnant women have cervical infection by CMV, this presumably being the source of infection during birth. The infant can also become infected through breast milk. The great majority of these infants are asymptomatic, but a few develop some aspects of the acute congenital CMV syndrome, which may include pneumonia. Infants, especially when premature or seriously ill, who acquire CMV infection through blood transfusion are more likely to have severe disease. In young nontransfused children less than age 6 months, some may develop pneumonia as the predominating or only manifestation of CMV infection. In young children in general, infection is common, with reported infection rates in the United States varying from 10%-15% by age 1 year and about 35% (range 20%-80%) by age 10 years in some populations and 36%-56% by age 1 year in other populations. After the neonatal period, infection is most commonly acquired from other children through contact with saliva or urine. Infection is especially common in day-care centers and similar institutions. The great majority of affected children are clinically asymptomatic; but in those with symptoms, probably the most common manifestation is a viruslike febrile illness (often mild), frequently accompanied by mildly abnormal liver function tests. In older children, incidence of infection is much less. In adults, many primary infections are thought to be related to sexual intercourse and many others are due to exposure to infected children. In older children and adults the majority are asymptomatic but those patients with symptoms usually have a 2-3 week illness resembling Epstein-Barr IM, discussed previously in this chapter, except for negative serologic tests for IM. Data from several studies indicate that about 65% (range, 33%-79%) of heterophil-negative IM-like illnesses are due to CMV. CMV infection is unusually frequent in kidney or other organ transplant patients (38%-96% of cases) with symptomatic cases ranging from 8%-39% (least common in renal transplants). Most serious CMV transplant infections occur in previously noninfected recipients who receive infected organs. It is also more frequent in immunosuppressed persons, patients on steroid therapy, and patients with leukemia or lymphoma. In these patients there is predominantly lung or liver involvement that usually is overshadowed by the preexisting nonviral disease. Cytomegalovirus is the predominant cause for the mononucleosis-like postperfusion (posttransfusion) syndrome that may occur 3-7 weeks after multiple-unit blood transfusion or bypass surgery. Studies have estimated that about 7% of single unit transfusions produce CMV infection and about 20% (range 3%-38%) of multiple-unit transfusions. More than 90% of homosexual males are said to have CMV antibody, and severe symptomatic infection occurs with increased frequency in advanced HIV-1 conditions including AIDS.

    Laboratory abnormalities in CMV infection. In symptomatic adult infection, splenomegaly is reported in about 35% of cases (range, 22%-40%) and lymphadenopathy in about 15% (range, 5%-28%). Hematologic and biochemical results are summarized in Table 17-4. In general, abnormal enzyme levels display only about one half the degree of elevation seen in patients with IM (which themselves are only mild to moderate), but there is a considerable degree of overlap. Peak elevations are reported to occur about 4-5 days after onset of illness for bilirubin, AST, and ALP and between 7-21 days for GGT. Enzyme elevations usually return to normal by 90 days after onset of clinical illness. GGT abnormality is often the last to disappear and occasionally may persist to some degree for several months.

    Laboratory test results in cytomegalovirus infection

    Table 17-4 Laboratory test results in cytomegalovirus infection

    Laboratory diagnosis of cytomegalovirus infection. The most definitive method for diagnosis of CMV infection is virus culture, but serologic tests are the most widely used procedures. In the newborn with congenital CMV brain disease, periventricular cerebral calcification is demonstrable by x-ray film in about 25%; this is highly suggestive, although the same pattern may be found in congenital toxoplasmosis.

    Cytomegalovirus inclusion body cytology. In newborns or young children, characteristic CMV inclusion bodies may be demonstrated within renal epithelial cells on stained smears of the urinary sediment in about 60% of cases; this may be an intermittent finding and may require specimens on several days. A fresh specimen is preferable to a 24-hour collection, since the cells tend to disintegrate on standing. Virus culture is unquestionably better than search for urine cells with cytomegalic inclusion bodies. In older children and adults the kidney is not often severely affected, so urine specimens for CMV inclusion bodies usually are not helpful. However, in tissue biopsies, presence of intranuclear cytomegalic inclusion bodies correlates better with CMV actual disease than detection of virus by other means.

    Virus culture. Classic culture methods have been replaced by the newer, faster, and more sensitive shell vial technique. Urine, sputum, or mouth swab culture for the virus is the method of choice. Fresh specimens are essential. For urine, an early morning specimen is preferable. For best results, any specimen must reach the virus culture laboratory within 1-2 days. The specimen should not be frozen, because freezing progressively inactivates the virus. This is in contrast to most other viruses, for which quick freezing is the procedure of choice for preserving specimens. The specimen should be refrigerated without actual freezing. In this way it may be preserved up to 1 week. It should be sent to the virus laboratory packed in ordinary ice (not dry ice) and, if possible, in an insulated container. Isolation of the CMV now takes 3-7 days (in contrast to conventional culture, which took several weeks). Both urine and throat swab specimen results may be positive for CMV several weeks or months after the end of acute illness. CMV culture cannot differentiate between active infection, reinfection, or reactivation of latent infection, with three exceptions: a positive culture of peripheral blood lymphocytes demonstrates the short-lived (2-3 weeks) viremic phase of primary acute infection; a positive fetal amniotic fluid culture or positive urine culture from newborns or neonates means congenital CMV infection; and a positive urine culture in previously seronegative transplant patients strongly suggests newly acquired infection.

    Detection of CMV antibody. Conversion of a negative to a significantly reactive test or a fourfold rising titer in specimens taken 1-2 weeks apart is one way to demonstrate primary infection. Current methods are immunofluorescence, ELISA, indirect hemagglutination, and LA. Most of these tests detect IgG antibody. Since CMV antibody is common in the general population and since IgG antibody levels persist for years, if only a single specimen is obtained, a negative result cannot guarantee that virus is not present in the latent stage at low titer; while a positive result can only show exposure to CMV with possible partial immunity. Acute and convalescent IgG specimens are necessary to demonstrate acute-stage infection. One difficulty with IgG tests applied to neonatal specimens is maternal IgG antibody to CMV, which may appear in fetal or newborn serum.

    Procedures are available that detect IgM antibody alone. IgM antibody persists in the blood for only a relatively short time (1-6 months, occasionally as long as 1 year). In adults, CMV-IgM by EIA has been reported in 90%-100% of patients in symptomatic phase of primary infection and in about 40% of reactivated infections. In maternal CMV primary infections, maternal IgM does not cross the placenta. Theoretically, the presence of IgM antibody should mean primary acute or recent infection. However, besides acute infection, reinfection by another CMV strain and reactivation can also induce a CMV-IgM response. Other sources of IgM such as rheumatoid factor can produce false abnormality in CMV-IgM tests unless there is some way to remove or counteract these interfering substances. Rheumatoid factor has been reported in 27%-60% of patients with CMV infection, both neonates and adults. In addition, acute EBV infection (which resembles CMV infection clinically) also produces IgM antibody that may react in the CMV-IgM tests. Finally, it is reported that 10%-50% of infants and 10%-30% of adults with acute CMV infection have no detectable CMV-IgM antibody. Immunocompromised patients and some patients with AIDS also fail to produce detectable amounts of IgM antibody.

    Detection of CMV antigen. Three antigens, called early, intermediate-early, and late have been cloned from the core portion of the CMV and can be detected by monoclonal antibodies using immunofluorescence or ELISA methods, or tissue cell stains on smears or biopsies. The most useful have been immunofluorescent or tissue immunologic stains on bronchioalveolar lavage or biopsy specimens, and on peripheral blood leukocyte preparations to detect early antigens for demonstration of acute infection antigenemia. This is reported to be more sensitive than culture with faster results and earlier detection of acute-phase CMV infection. Nucleic acid (DNA) probe methods (now commercially available) also have been used to detect CMV virus in bronchoalveolar lavage, urine, and peripheral blood leukocytes. When amplified by PCR, the probes have shown greater sensitivity than culture. DNA probes can also be used on biopsy specimens.

  • Epstein-Barr Virus (EBV)

    The Epstein-Barr virus is a member of the herpesvirus group and is reported to infect 80% or more of the U.S. population. It is thought to be spread from person to person, most likely through saliva, with the majority of infections occurring in childhood, adolescents, and young adults. The EBV infects B-lymphocytes. In common with the other herpesviruses, once infection (with or without symptoms) takes place the virus eventually becomes dormant but can be reactivated later into clinical disease. Reactivation is said to occur in 15%-20% of healthy persons and in up to 85% in some groups of immunosuppressed patients. Epstein-Barr virus infection in young children is usually asymptomatic. Primary infection by EBV in older children, adolescents, or young adults produces the infectious mononucleosis syndrome in up to 50% of cases. The EBV is also strongly associated with Burkitt’s lymphoma in Africa and nasopharyngeal carcinoma in southern China.

    Infectious mononucleosis (infectious mono; IM)

    Infectious mononucleosis (IM) patients are most often adolescents and young adults, but a significant number are older children and middle-aged or even older adults. When IM is part of a primary infection, the incubation period is 3-7 weeks (range, 2-8 weeks). The acute phase of illness in those patients who are symptomatic lasts about 2-3 weeks (range, 0-7 weeks). Convalescence takes about 4-8 weeks. The most common features of the acute illness are fever, pharyngitis, and adenopathy, with lymph node enlargement occurring in 80%-90% of patients. The posterior cervical nodes are the ones most commonly enlarged. Soft palate petechiae are found in 10%-30% of cases. Jaundice, usually mild, is found in about 5%-10% (range, 4%-45%) of patients in large series. The spleen is mildly enlarged in about 50% of patients (range, 40%-75%) and hepatomegaly is present in about 10% (range, 6%-25%).

    Laboratory findings. Patients usually have normal hemoglobin values. Mild thrombocytopenia is reported in 25%-50% of patients (range, 15%-50%). Leukocytosis between 10,000 and 20,000/ mm3 (10 Ч 109-20 Ч 109/L) occurs in 50%-60% of patients (range, 40%-70%) by the second week of illness. About 10% (range, 5%-15%) of patients develop a leukocytosis over 25,000/mm3 (25 Ч 109/L). However, during the first week there may be leucopenia. About 85%-90% (range, 80%-100%) of patients with IM have laboratory evidence of hepatic involvement (Table 17-2). Peak values are reported to occur 5-14 days after onset of illness for aspartate aminotransferase (AST), bilirubin, and alkaline phosphatase (ALP); and between 7 and 21 days for gamma-glutamyltransferase (GGT). The AST and ALP levels return to normal in nearly all patients by 90 days, but occasionally there may be some degree of GGT elevation persisting between 3-12 months. Total LDH is elevated in about 95% of patients. LDH isoenzyme fractionation by electrophoresis can show three patterns: elevation of all five fractions; elevation of LDH 3, 4, and 5; or elevation of LDH-5 only.

    Liver function tests in EBV-induced infectious mononucleosis

    Table 17-2 Liver function tests in EBV-induced infectious mononucleosis

    Peripheral blood smear. The first of three classic findings is a lymphocytosis, with lymphocytes making up more than 50% of the total white blood cells (WBCs). Lymphocytosis is said to be present in 80%-90% of patients (range, 62%-100%), peaks during the second or third week, and lasts for an additional 2-6 weeks. The second classic criterion is the presence of a “significant number” of atypical lymphocytes on Wright-stained peripheral blood smear. There is disagreement as to whether greater than 10% or greater than 20% must be atypical. These atypical lymphocytes are of three main types (Downey types). Type I has vacuolated or foamy blue cytoplasm and a rounded nucleus. Type II has an elongated flattened nucleus and large amounts of pale cytoplasm with sharply defined borders and often some “washed-out” blue cytoplasm coloring at the outer edge of the cytoplasm. Type III has an irregularly shaped nucleus or one that may be immature and even may have a nucleolus and resemble a blast. All three types are larger than normal mature lymphocytes, and their nuclei are somewhat less dense. Most of the atypical lymphocytes are activated T-lymphocytes of the CD-8 cytotoxic-suppressor type. Some of the Downey III lymphocytes may be EBV-transformed B lymphocytes, but this is controversial. These atypical lymphocytes are not specific for IM, and may be found in small to moderate numbers in a variety of diseases, especially cytomegalovirus and hepatitis virus acute infections. In addition, an appearance similar to that of the type II variety may be created artificially by crushing and flattening normal lymphocytes near the thin edge of the blood smear. IM cells are sometimes confused with those of acute leukemia or disseminated lymphoma, although in the majority of cases there is no problem.

    Although most reports state or imply that nearly all patients with IM satisfy the criteria for lymphocytosis and percent atypical lymphocytes, one study found only 55% of patients had a lymphocytosis and only 45% had more than 10% atypical lymphocytes on peripheral smear when the patients were first seen. Two studies found that only about 40% of patients with IM satisfied both criteria.

    Serologic tests. The third criterion is a positive serologic test for IM either based on heterophil antibodies or specific anti-EBV antibodies. The classic procedure is the heterophil agglutination tube test (Paul-Bunnell test). Rapid heterophil antibody slide agglutination tests have also been devised. Slide tests now are the usual procedure done in most laboratories. However, since the basic principles, interpretation, and drawbacks of the slide tests are the same as those of the older Paul-Bunnell tube test, there are some advantages in discussing the Paul-Bunnell procedure in detail.

    Serologic tests based on heterophil antibodies.

    Paul-Bunnell antibody is an IgM-type antibody of uncertain origin that is not specific for EBV infection but is seldom found in other disorders (there are other heterophil antibodies that are not associated with EBV infection). Paul-Bunnell antibodies begin to appear in the first week of clinical illness (about 50% of patients detectable; range, 38%-70%), reaching a peak in the second week (60%-78% of patients positive) or third (sometimes the fourth) week (85%-90% positive; range, 75%-100%), then begin to decline in titer during the fourth or fifth week, most often becoming undetectable 8-12 weeks after beginning of clinical illness. However in some cases some elevation is present as long as 1-2 years (up to 20% of patients). In children less than 2 years old, only 10%-30% develop heterophil antibodies; about 50%-75% of those 2-4 years old develop heterophil antibodies. One report states that these antibodies are rarely elevated in Japanese patients of any age. Once elevated and returned to undetectable level, heterophil antibodies usually will not reelevate in reactivated IM, although there are some reports of mild heterophil responses to other viruses.

    The original Paul-Bunnell test was based on the discovery that the heterophil antibody produced in IM would agglutinate sheep red blood cells (RBCs). In normal persons the sheep cell agglutination titer is less than 1:112 and most often is almost or completely negative. The Paul-Bunnell test is also known as the “presumptive test” because later it was found that certain antibodies different from those of IM would also attack sheep RBCs. Examples are the antibodies produced to the Forssman antigen found naturally in humans and certain other animals and the antibody produced in “serum sickness” due to certain drug reactions. To solve this problem the differential absorption test (Davidsohn differential test) was developed. Guinea pig kidney is a good source of Forssman antigen. Therefore, if serum containing Forssman antibody is allowed to come in contact with guinea pig kidney material, the Forssman antibody will react with the kidney antigen and be removed from the serum when the serum is taken off. The serum will then show either a very low or a negative titer, whereas before it was strongly positive. The IM heterophil antibody is not significantly absorbed by guinea pig kidney but is nearly completely absorbed by bovine (beef) RBCs, which do not significantly affect the Forssman antibody. The antibody produced in serum sickness will absorb both with beef RBCs and guinea pig kidney.

    The level of Paul-Bunnell titer does not correlate well with the clinical course of IM. Titer is useful only in making a diagnosis and should not be relied on to follow the clinical course of the disease or to assess results of therapy.

    In suspected IM, the presumptive test is performed first; if necessary, it can be followed by a differential absorption procedure.

    “Spot” tests were eventually devised in which the Paul-Bunnell and differential absorption tests are converted to a rapid slide agglutination procedure without titration. Most of the slide tests use either horse RBCs, which are more sensitive than sheep RBCs, or bovine RBCs, which have sensitivity intermediate between sheep and horse RBC but which are specific for IM heterophil antibody and therefore do not need differential absorption. Slide test horse cells can also be treated with formalin or other preservatives that extend the shelf life of the RBC but diminish test sensitivity by a small to moderate degree.

    Heterophil-negative infectious mononucleosis.

    This term refers to conditions that resemble IM clinically and show a similar Wright-stained peripheral blood smear picture, but without demonstrable elevation of Paul-Bunnell heterophil antibody (see the box) About 65% (range, 33%-79%) are CMV infection, about 25% (15%-63%) are heterophil-negative EBV infections, about 1%-2% are toxoplasmosis, and the remainder are other conditions or of unknown etiology.

    Diagnosis of infectious mononucleosis. When all three criteria for IM are satisfied, there is no problem in differential diagnosis. When the results of Paul-Bunnell test or differential absorption test are positive, most authors believe that the diagnosis can be made, although there are reports that viral infections occurring after IM can cause anamnestic false positive heterophil reelevations. When the clinical picture is suggestive of IM but results of the Paul-Bunnell test, differential absorption procedure, or the spot test are negative, at least one follow-up specimen should be obtained in 14 days, since about 20%-30% of IM patients have negative heterophil test results when first seen versus 10%-15% negative at 3 weeks after onset of clinical symptoms (although the usual time of antibody appearance is 7-10 days, it may take as long as 21 days and, uncommonly, up to 30 days). About 10% (range, 2%-20%) of patients over age 5 years and 25%-50% or more under age 5 years never produce detectable heterophil antibody. Another potential problem is that several evaluations of different heterophil kits found substantial variation in sensitivity between some of the kits. If the clinical picture is typical and the blood picture is very characteristic (regarding both number and type of lymphocytes), many believe that the diagnosis of IM can be considered probable but not established. This may be influenced by the expense and time lapse needed for specific EBV serologic tests or investigation of CMV and the various other possible infectious etiologies.

    Some “Heterophil-Negative” Mononucleosis Syndrome Etiologies

    Viruses

    EBV heterophil-negative infections
    Cytomegalovirus
    Hepatitis viruses
    HIV-1 seroconversion syndrome
    Other (rubella, herpes simplex, herpesvirus 6, mumps, adenovirus)

    Bacteria

    Listeria, tularemia, brucellosis, cat scratch disease, Lyme disease, syphilis, rickettsial diseases

    Parasites

    Toxoplasmosis, malaria

    Medications

    Dilantin, azulfidine, dapsone, “serum sickness” drug reactions

    Other

    Collagen diseases (especially SLE, primary or drug-induced)
    Lymphoma
    Postvaccination syndrome
    Subacute bacterial endocarditis (SBE)

    In summary, the three classic criteria for the diagnosis of IM are the following:

    1. Lymphocytes comprising more than 50% of total WBC count.
    2. Atypical lymphocytes comprising more than 10% (liberal) or 20% (conservative) of the total lymphocytes.
    3. Significantly elevated Paul-Bunnell test and/or differential absorption test result. A positive slide agglutination test result satisfies this criterion.

    Serologic tests based on specific antibodies against EBV. The other type of serologic test for IM detects patient antibodies against various components of the EBV (Table 17-3). Tests are available to detect either viral capsid antigen-IgM or IgG (VCA-IgM or IgG) antibodies. VCA-IgM antibody is usually detectable less than one week after onset of clinical illness and becomes nondetectable in the late convalescent stage. Therefore, when present it suggests acute or convalescent EBV infection. Rheumatoid factor (RF) may produce false positive results, but most current kits incorporate some method to prevent RF interference. VCA-IgG is usually detectable very soon after VCA-IgM, but remains elevated for life after some initial decline from peak titer. Therefore, when present it could mean either acute, convalescent, or old infection. Tests are available for Epstein-Barr nuclear antigen (EBNA) IgM or IgG antibody, located in nuclei of infected lymphocytes. Most kits currently available test for IgG antibody (EBNA-IgG or simply EBNA). EBNA-IgM has a time sequence similar to that of VCA-IgM. The more commonly used EBNA-IgG test begins to rise in late acute stage (10%-34% positive) but most often after 2-3 weeks of the convalescent stage. It rises to a peak after the end of the convalescent stage (90% or more positive), then persists for life. Elevated EBNA/EBNA-IgG is suggestive of nonacute infection when positive at lower titers and older or remote infection at high or moderately high titer. A third type of EBV test is detection of EBV early antigen (EA), located in cytoplasm of infected cells. There are two subtypes; in one the antigen is spread throughout the cytoplasm (“diffuse”; EA-D) and in the other, the antigen is present only in one area (“restricted”; EA-R). The EA-D antibody begins to rise in the first week of clinical illness, a short time after the heterophil antibody, then peaks and disappears in the late convalescent stage about the same time as the heterophil antibody. About 85% (range, 80%-90%) of patients with IM produce detectable EA-D antibody, which usually means EBV acute or convalescent stage infection, similar to VCA-IgM or heterophil antibody. However, EA-D may rise again to some extent in reactivated EBV disease, whereas VCA-IgM does not (whether heterophil antibody ever rises is controversial, especially since it may persist for up to a year or even more in some patients). EA-D is typically elevated in EBV-associated nasopharyngeal carcinoma. EA-R is found in about 5%-15% of patients with clinical IM. It is more frequent (10%-20%) in children less than 2 years old with acute EBV infection and is typically elevated in patients with EBV-related Burkitt’s lymphoma. Expected results from the various serologic tests in different stages of EBV infection are summarized in Table 17-3 and Fig. 17-9.

    Antibody tests in EBV infection

    Table 17-3 Antibody tests in EBV infection

    Tests in EBV infection

    Fig. 17-9 Tests in EBV infection.

    Specific serologic tests for EBV are relatively expensive compared to heterophil antibody tests and are available predominantly in university centers and large reference laboratories. Such tests are not needed to diagnose IM in the great majority of cases. The EBV tests are useful in heterophil-negative patients, in problem cases, in patients with atypical clinical symptoms when serologic confirmation of heterophil results is desirable, and for epidemiologic investigations. If the initial heterophil test is nonreactive or equivocal, it is desirable to freeze the remainder of the serum in case specific EBV tests are needed later.

    Summary of Epstein-Barr Antibody Test Interpretation
    Never infected (susceptible) = VCA-IgM and IgG both negative.
    Presumptive primary infection = Clinical symptoms, heterophil positive.
    Primary infection: VCA-IgM positive (EBNA-IgG negative; heterophil positive or negative)
    Reactivated infection: VCA-IgG positive; EBNA-IgG positive; EA-D positive (heterophil negative, VCA-IgM negative)
    Old previous infection: VCA-IgG positive; EBNA-IgG positive; EA-D negative (VCA-IgM negative, heterophil negative).

  • Hepatitis D Virus (HDV)

    Hepatitis D is also called “delta hepatitis.” It is a partially defective virus that must enter the hepatitis B virus in order to penetrate and infect liver cells. Therefore, a person must have HBV present, either as a carrier or in clinical infection, in order to acquire HDV infection. When HBV infection is over, HDV infection will also disappear. HDV infection is acquired by the same routes as HBV infection (predominately parenteral, less commonly transmucosal or sexual). In the United States, infection is predominately, but not entirely, spread through blood or blood products. The highest incidence is in intravenous-use drug addicts, followed by transfusion recipients of blood or blood products. HDV infection is relatively uncommon in male homosexuals (0.2% in one study) and in the Orient, in contrast to the high incidence of HBV infection in these two populations. HDV is endemic in southern Europe, the Middle East, South America, the South Pacific, and parts of Africa, as well as in major U.S. cities.

    There are three types of clinical infection. In the first type, HBV and HDV infection are transmitted at the same time (simultaneous acute infection or “coinfection”). Clinically, this resembles an HBV infection that is more severe than usual or has a second transaminase peak or clinical episode (“biphasic” transaminase pattern). The mortality rate of acute HDV infection due to fulminating hepatitis is about 5% (range, 2%-20%); this contrasts to a HBV mortality rate of about 1%. On the other hand, less than 5% of HDV acute coinfection patients develop chronic HDV infection compared to 5%-15% incidence of chronic HBV after acute HBV.

    In the second type of relationship between clinical HDV and HBV infection, acute HDV infection is superimposed on preexisting chronic HBV disease (HDV “superinfection”). Clinically, this resembles either an acute exacerbation of the preexisting HBV disease (if the HBV infection had previously been symptomatic), or it may appear to be onset of hepatitis without previous infection (if the initial HBV infection has been subclinical). As noted previously, there is considerably increased incidence of fulminating hepatitis. About 80% (range, 75%-91%) of patients with acute HDV superimposed on chronic HBV develop chronic HDV.

    HDV antigen and antibodies

    Fig. 17-8 HDV antigen and antibodies.

    In the third type of HDV-HBV relationship, there is chronic HDV infection in addition to chronic HBV infection. Seventy percent to 80% of patients with chronic HDV hepatitis develop cirrhosis. This contrasts to chronic HBV infection where 15%-30% develop cirrhosis. Cirrhosis is rapidly progressive (developing is less than 2 years) in 15%-20% of chronic HDV infection.

    Hepatitis D virus antigen and antibodies

    Commercially available immunoassays are available for HDV-IgM and HDV-Total antibodies. In addition, HDV antigen can be detected by immunoassay or by nucleic acid probe, available at some reference laboratories and university centers. HDV antigen is present in serum for only a few days, and most often is not detectable by the time the patient is seen by a physician. Antigen detection is more likely using nucleic acid probe since the probe technique is more sensitive than immunoassay.

    There is a very short time span for HDV antigen detection by immunoassay or by DNA probe in acute HDV infection (12.5% of cases positive on admission in one study). Sensitivity of different immunoassay systems for HDV can vary considerably (24%-100% on the same specimen in one study). Duration of elevation for both HDV-Ab (IgM) and HDV-Ab (Total) is also relatively short. Failure of HDV-Ab (Total) to persist for several years resembles IgM antibody more than IgG. HDV-Ab (Total) typically does remain elevated (usually in high titer) if acute HDV progresses to chronic HDV. HDV-Ab (IgM) may also be detectable in chronic HDV (usually in low titer), depending on sensitivity of the test system and degree of virus activity. HDV-Ab (IgM) level of elevation depends on active HDV replication and on degree of liver cell injury. It rises during active viral replication and decreases when replication ceases. HDV-Ab (Total) also tends to behave like IgM antibody but takes longer to rise and to decrease. In acute HDV infection that resolves, HDV-Ab (Total) usually decreases to a low titer and sometimes can become nondetectable. In chronic infection, due to continual presence of the virus, HDV-Ab (Total) is usually present in high titer.

    In HDV superinfection (on chronic HBV) HBsAg may temporarily decrease in titer or even transiently disappear.

    Delta Hepatitis Coinfection (Acute HDV + Acute HBV) or Superinfection (Acute HDV + Chronic HBV)

    HDV-AG

    Detected by DNA probe, less often by immunoassay
    Appearance: Prodromal stage (before symptoms); just at or after initial rise in ALT (about a week after appearance of HBs Ag and about the time HBc Ab-IgM level begins to rise)
    Peak: 2-3 days after onset
    Becomes nondetectable: 1-4 days (may persist until shortly after symptoms appear)

    HDV-AB (IGM)

    Appearance: About 10 days after symptoms begin (range, 1-28 days)
    Peak: About 2 weeks after first detection
    Becomes nondetectable: about 35 days (range, 10-80 days) after first detection (most other IgM antibodies take 3-6 months to become nondetectable)

    HDV-AB (TOTAL)

    Appearance: About 50 days after symptoms begin (range, 14-80 days); about 5 weeks after HDV-Ag (range, 3-11 weeks)
    Peak: About 2 weeks after first detection
    Becomes nondetectable: About 7 months after first detection (range, 4-14 months)

    Delta Hepatitis Chronic Infection (Chronic HDV + Chronic HBV)

    HDV-Ag

    Detectable in serum by nucleic acid probe

    HDV-Ab (IgM)

    Detectable (may need sensitive method; titer depends on degree of virus activity)

    HDV-Ab (total)

    Detectable, usually in high titer

    In chronic HDV infection, HDV-Ab (Total) is usually present in high titer. HDV-Ab (IgM) is present in low titer (detectability depends on sensitivity of the assay). HDV-Ag is usually not detectable by immunoassay in serum but is often demonstrable by immunohistologic stains in liver biopsy specimens. In these patients, HDV-Ag can often be detected in serum (and most often in liver biopsy specimens) by DNA probe.

    Although many use “viral hepatitis” as a synonym for infection by hepatitis viruses A, B, C, D, and E, a wide range of viruses may infect hepatic cells with varying frequency and severity. The most common (but not the only) examples are infectious mononucleosis (E-B virus), and the cytomegalic inclusion virus. Nonviral conditions can also affect the liver (please refer to the box on this page).

    Summary: Diagnosis of HDV Infection

    Best current all-purpose screening test = ADV-Ab (Total)
    Best test to differentiate acute from chronic infection = HDV-Ab (IgM)

    Some Causes for Liver Function Test Abnormalities that Simulate Hepatitis Virus Hepatitis

    Epstein-Barr virus (Infectious Mononucleosis)
    Cytomegalovirus
    Other viruses (herpes simplex, yellow fever, varicella, rubella, mumps)
    Toxoplasmosis
    Drug-induced (e.g., acetaminophen)
    Severe liver hypoxia or passive congestion (some patients)
    HELLP syndrome associated with preeclampsia
    Alcohol-associated acute liver disease (some patients)
    Reye’s syndrome

    Summary of Hepatitis Test Applications

    HBs
    -AG
    HBs Ag: Shows current active HBV infection
    Persistance over 6 months indicates carrier/chronic HBV infection
    HBV nucleic acid probe: Present before and longer than HBsAg
    More reliable marker for increased infectivity than HBsAg and/or HBeAg
    -Ab
    HBs Ab-Total: Shows previous healed HBV infection and evidence of immunity
    HBc
    -Ab
    HBc Ab-IgM: Shows either acute or very recent infection by HBV
    In convalescent phase of acute HBV, may be elevated when HBs Ag has disappeared (core window)
    Negative HBc Ab-IgM with positive HBsAg suggests either very early acute HBV or carrier/chronic HBV
    HBc Ab-Total: Only useful to show past HBV infection if HBs Ag and HBc Ab-IgM are both negative
    HBe
    -Ag
    HBe-AbAg: When present, especially without HBe Ab, suggests increased patient infectivity
    HBe Ab-Total: When present, suggests less patient infectivity
    HDV
    -Ag
    HDV-Ag: Shows current infection (acute or chronic) by HDV
    HDV nucleic acid probe: Detects antigen before and longer than HDV-Ag by EIA
    -Ab
    HDV-Ab (IgM): High elevation in acute HDV; does not persist
    Low or moderate elevation in convalescent HDV; does not persist
    Low to high persistent elevation in chronic HDV (depends on degree of cell injury and sensitivity of the assay)
    HDV-Ab (Total): High elevation in acute HDV; does not persist
    High persistent elevation in chronic HDV
    HCV
    -Ag
    HCV nucleic acid probe: Shows current infection by HCV (especially using PCR amplification)
    -Ab
    HCV-Ab (IgG): Current, convalescent, or old HCV infection
    HAV
    -Ag
    HAV-Ag by EM: Shows presence of virus in stool early in infection
    -Ab
    HAV-Ab (IgM): Current or recent HAV infection
    HAV-Ab (Total): Convalescent or old HAV infection

  • Hepatitis E Virus (HEV)

    This is a NANB virus with an incubation period, clinical course, and epidemiology similar to that of HAV. HEV is currently thought to be a calcivirus. In 1994 no HEV antigen or antibody tests are commercially available, although several antibody tests using homemade reagents have been reported. Sensitivity of the tests is similar to that of HAV.

  • Hepatitis C virus (HCV)

    After serologic tests for HAV and HBV were developed, apparent viral hepatitis nonreactive to tests for HAV and HBV or to other viruses that affect the liver was called non-A, non-B (NANB) hepatitis virus. Eventually, hepatitis D virus was discovered and separated from the NANB group. It was also known that NANB represented both a short-incubation and a long-incubation component, so the short-incubation component was separated from NANB and called hepatitis E. The long-incubation component retained the designation NANB. When the first serologic test for viral antibody reactive with a single antigen from NANB infectious material became commercially available in 1991, the virus identified was named hepatitis C (HCV). A second-generation test for HCV antibody became commercially available in early 1993, using 3 antigens from the HCV infectious agent. A third generation test became available in 1994. Both the first and second generation tests detect only IgG antibody. A test for HCV antigen is not commercially available in 1994, although nucleic acid RNA probe methods for HCV antigen have been developed by some investigators. HCV appears to be a group of closely related viruses, at least some of which have subgroups. In addition, it is not proven that HCV is the only hepatitis virus that produces long-incubation NANB.

    HBV e antigen and antibody

    Fig. 17-6 HBV e antigen and antibody.

    HCV antigen and antibody

    Fig. 17-7 HCV antigen and antibody.

    HCV is a small RNA virus that is currently being classified as a Flavivirus (although some have proposed reclassifying it as a Pestivirus). It has been shown to exist in at least 4 genotypes or strains; the frequency of each strain differs in various geographic locations. Average HCV incubation is 6–8 weeks. However, incubation of 2 weeks to 1 year has been reported. Most cases (80%, range 70%–90%) develop IgG antibody by 6 weeks (range, 5–30 weeks) after onset of symptoms (similar to HBV). Like HBV, HCV has been detected in serum, semen, and saliva. Transmission is thought to be similar to that of HBV (major risk groups are IV drug abusers and transfusions of blood and blood products), but some differences exist. Male homosexuals currently are much less likely to become infected by HCV (less than 5% of cases) than with HBV (40%-80% of cases). Also, HCV is less apt to be transmitted through heterosexual intercourse than HBV. Although sexual transmission can occur, it appears to be infrequent. There is some disagreement using current tests regarding frequency of HCV transmission from mother to infant. Most investigators report that fetal or neonatal infection is very uncommon. However, if the mother is coinfected with HIV, newborn HCV infection as well as HIV infection were frequent. Passive transfer of maternal anti-HCV antibody to the fetus is very common.

    HCV hepatitis now accounts for about 80%-90% of posttranfusion hepatitis cases when blood comes from volunteer donors whose serologic test results for HBV are negative. HCV is also reported to cause 12%-25% of cases of sporadic hepatitis (hepatitis not related to parenteral inoculation or sexual transmission). Only about 25% of acute HCV cases develop jaundice. The clinical illness produced is similar to HBV but tends to be a little less severe. However, progression to chronic hepatitis is significantly more frequent than in HBV; occurring in about 60% (range, 20%-75%) of posttransfusion cases by 10 years. About 30% (range, 20%–50%) of HCV patients develop cirrhosis by 10 years. Apparently, HCV acquired by transfusion is more likely to become chronic and lead to cirrhosis than community-acquired HCV (in one study, liver biopsy within 5 years of HCV onset showed 40% of transfusion-related cases had chronic active hepatitis and 10%-20% had cirrhosis, whereas in community-acquired cases less than 20% had chronic active hepatitis and 3% had cirrhosis).

    HCV has been proposed as a major etiology for hepatocellular carcinoma (hepatoma), similar to HBV. Antibodies to HCV have been reported in about 40%-60% of patients (range, 5%-94%) with hepatoma. The incidence varies considerably in different geographical areas, different regions within the same geographical area, and different population groups. In some areas HBV predominates; in others, HCV is more common and may even exceed HBV in frequency. HCV (or HBV) proteins can be detected in hepatoma cancer cells in varying number of cases.

    HCV-Ag
    Nucleic acid probe (without PCR)

    Appearance: About 3-4 weeks after infection (about 1-2 weeks later than PCR-enhanced probe)
    Becomes nondetectable: Near the end of active infection, beginning of convalescence

    Nucleic acid probe with PCR

    Appearance: As early as the second week after infection
    Becomes nondetectable: End of active infection, beginning of convalescence

    HCV-Ab (IgG)
    2nd generation (gen) ELISA

    Appearance: About 3-4 months after infection (about 2-4 weeks before first gen tests); 80% by 5–6 weeks after symptoms
    Becomes nondetectable: 7% lose detectable antibody by 1.5 years; 7%-66% negative by 4 years (by 1st gen tests; more remain elevated and for longer time by 2nd gen tests)

    Summary: Hepatitis C Antigen and Antibody
    HCV-Ag by nucleic acid probe

    Shows current infection by HCV (especially using PCR amplification)

    HCV-Ab (IgG)

    Current, convalescent, or old HCV infection (behaves more like IgM or “total” Ab than usual IgG Ab)

  • Hepatitis B Virus (HBV)

    HBV was originally called “serum hepatitis,” or “long-incubation hepatitis,” and has an incubation period of 60-90 days (range, 29-180 days). HBV is found in blood and body secretions. Infection was originally thought to be limited to parenteral inoculation (blood transfusion or injection with a contaminated needle). Although this is still the major source of infection, the virus may be contracted by inoculation of infected blood, saliva, or semen through a small break in the skin or a mucous membrane (e.g., the rectum) or by sexual intercourse. The virus seems less infectious through nonparenteral transmission than is HAV. At least 30% of persons with serologic evidence of HBV infection (past or present) do not have a currently identified risk factor.

    Interpretation of Hepatitis B Serologic Tests

    I     HBSAg positive, HBCAb negative*
    About 5% (range, 0%-17%) of patients with early stage HBV acute infection (HBCAb rises later)

    II     HBSAg positive, HBCAb positive, HBSAb negative
    a. Most of the clinical symptom stage
    b. Chronic HBV carriers without evidence of liver disease (“asymptomatic carriers”)
    c. Chronic HBV hepatitis (chronic persistent type or chronic active type)

    III     HbSAg negative, HBCAb positive,* HBSAb negative
    a. Late clinical symptom stage or early convalescence stage (core window)
    b. Chronic HBV infection with HBSAg below detection levels with current tests
    c. Old previous HBV infection
    IV     HBSAg negative, HBCAb positive, HBSAb positive
    a. Late convalescence to complete recovery
    b. Old infection
    *HBCAb=combined IgM+IgG. In some cases (e.g., category III), selective HbCAb-IgM assay is useful to differentiate recent and old infection.

    There is a considerably increased incidence of HBV infection in male homosexuals (about 10% of yearly reported cases and 40%-80% with serologic evidence of infection), in intravenous drug abusers (about 25%-30% of yearly reported cases and 60%-90% with serologic evidence) of infection; and in renal dialysis patients or dialysis unit personnel. Although serologic evidence of infection in heterosexual males is low (about 5%-6%; range, 4%-18%), heterosexual HBV transmission is now about 20%-25% of yearly reported cases. Thirty percent or more of regular sex partners of actively infected persons become infected. There is an increased risk in renal transplant patients, and in persons with leukemia or lymphoma. Hospital personnel are also at risk for HBV infection, comprising about 5% (range, 2%-6%) of yearly reported cases, most often due to accidental needle stick after drawing blood from an infected patient. Thirteen percent to 24% of dentists and dental workers have serologic evidence of infection. It is reported that the risk of contracting HBV infection from a contaminated needle stick is 6%-30%. There is disagreement regarding risk of HBV spread in day-care centers. It has also been reported that 26%-77% of institutionalized mentally handicapped patients have antibodies against HBV and about 20% (range, 3%-53%) had detectable HBV antigen.

    HBV infection is especially prevalent in Taiwan, various other areas of Southeast Asia, and parts of Africa. About 10%-15% of these populations are said to be HBV carriers. For comparison, U.S. male homosexuals have a carrier rate of about 4%-8% and intravenous (IV) drug abusers have a rate of about 7%.

    Hepatitis B virus infection has a wide range of severity and is fatal in about 1% (range, 1%-3%) of patients. In general, only about 30%-40% (range, 10%-50%) of patients with acute HBV develop clinically apparent acute hepatitis. Neonates almost always are asymptomatic and most children do not develop jaundice.

    Some 5%-15% of HBV infections become chronic, either as the carrier state or as chronic hepatitis. Although various definitions of these terms can be found in the literature, the carrier state is usually defined as persistence of HBV surface antigen for more than 6 months but with normal liver function tests and normal microscopic findings on liver biopsy. Chronic hepatitis can be divided into chronic persistent hepatitis (abnormal liver function tests plus relatively normal liver biopsy findings) and chronic active hepatitis (abnormal liver function tests plus abnormal findings on liver biopsy). The abnormalities on liver biopsy may exist in a spectrum of severity and may progress to cirrhosis. About 2% of HBV infections (15%-20% of chronic HBV) exist in the asymptomatic carrier state, about 6% are chronic persistent hepatitis, and about 3% are chronic active hepatitis. About 15%-30% of patients with chronic HBV infection (roughly 3% of all HBV patients; range, 0.75%-4.5%) develop cirrhosis. There is also a considerably increased risk for hepatocellular carcinoma (hepatoma); the relative risk for HBV carriers is quoted as 30-106 times noncarriers, while the relative risk for a carrier who has cirrhosis rises to nearly 500.

    Mothers who acquire HBV infection during the third trimester or early postpartum, or who are HBV carriers, frequently transmit HBV infection to their infants during or after birth. Incidence varies from 12.5%-40% and may be as high as 70%-90% of cases when the mother is positive for HBV antigen by nucleic acid probe as well as positive by both HBV surface antigen by immunoassay plus the HBV e antigen. A lesser number (5%-10% in one study) become infected if the mother is negative by nucleic acid probe even though HBV surface antigen by immunoassay and HBV e antigen are both positive.

    Without therapy, 80%-90% of infected infants become chronic carriers of HBV surface antigen. These infants are said to have a 25% risk of fatal cirrhosis or hepatoma. A combination of HBV vaccine and HBV immune globulin administered to the newborn can reduce risk of the chronic carrier stateby 85%-95%.

    Tests for Hepatitis B virus infection

    Studies have shown that the intact HBV molecule (Dane particle) has a double shell structure that contains several different antigens or antigenic material. There is an outer envelope that incorporates the hepatitis B surface antigen (HBsAg, formerly known as the Australia antigen, or HAA antigen). There is an inner core that contains an HBV core antigen HBcAg). Also within the core is a structure consisting of double-stranded viral deoxyribonucleic acid (DNA), as well as the material called HBV e antigen (ABeAg) and an enzyme known as DNA polymerase.

    Currently, there are three separate HBV antigen-antibody systems: surface antigen, core antigen, and e antigen.

    HBV surface antigen

    HBV surface antigen (HBsAg) can be detected by nucleic acid probe or by immunoassay.

    About 20%-60% of chronic persistent HBV hepatitis and 9%-60% of HBV chronic active hepatitis have detectable HBsAg by immunoassay. It has been reported that the new recombinant hepatitis B vaccines produce a transient (detectable) passive transfer antigenemia in infants (but not adults), lasting about a week but occasionally as long as 2-3 weeks.

    Antigenic subgroups of HBsAg exist; the most important to date are adw, ayw, adr, and ayr, but others have been discovered. These are thought to indicate possible subgroups (strains) of HBV.

    HBs Ag by Immunoassay

    Appearance

    2-6 weeks after exposure (range, 6 days-6 months). 5%-15% of patients are negative at onset of jaundice

    Peak

    1-2 weeks before to 1-2 weeks after onset of symptoms

    Becomes nondetectable

    1-3 months after peak (range, 1 week-5 months)

    HBsAg by nucleic acid probe (DNA probe)

    HBsAg-DNA is somewhat more sensitive than HBsAg by immunoassay in the very early stage of acute HBV infection. In one study, HBV-DNA was positive in 53% of patients seen before the peak of ALT elevation. It is also somewhat more sensitive than HBsAg by immunoassay in chronic HBV infection, both in serum and when applied to liver biopsy specimens. HBsAg-DNA using the polymerase chain reaction (PCR) amplification method is said to increase HBsAg detection rates by up to 66% over nonamplified HBsAg-DNA probe.

    HBsAg-DNA is most often used as an index of HBV activity or infectivity. Detection of HBV-DNA in serum more than 4 weeks after the alanine aminotransferase (ALT) peak (over 8 weeks after onset of symptoms) is said to be a reliable predictor of progression to chronic HBV infection. Loss of serum HBV-DNA with HBeAg still positive in acute HBV infection commonly precedes loss of HBeAg and seroconversion to HBeAb (total).

    HBsAb-Total behaves like a typical IgG antibody, rising (most often) after HBsAg is no longer detectable and remaining elevated for years. Presence of HBsAb-Total therefore usually means the end of acute HBV infection and predicts immunity to reinfection. However, there are reports that HBsAg and HBsAb-Total may coexist at some point in time in about 5% of patients (range, 2%-25% of cases); this most often happens in association with decreased immunologic mechanisms; such as occurs with acquired immunodeficiency syndrome (AIDS). However, it possibly could also result from subsequent infection by a different subgroup (strain) of HBV. Also, about 15% of patients have been reported to lose HBsAb-Total in less than 6 years.
    Hepatitis B virus core antigen and antibodies HBV Core Antigen (HBc Ag)

    Currently, there is no commercially available test to detect HBcAg.

    HBV core antibodies (HBc Ab)

    Tests are commercially available for IgM and for total antibody (IgM + IgG)

    In chronic HBV infection, there is disagreement in the literature whether HBcAb-IgM is detectable, with some investigators stating it is usually absent and others finding it elevated in varying numbers of patients. This disagreement partially is due to a tendency of the HBcAb-IgM antibody to increase titer in relation to the degree of HBV activity. The ongoing quantity of liver cells being injured is less in most cases of chronic HBV than in acute HBV. In addition, sensitivity of the HBcAb-IgM test is not the same for all manufacturer’s kits. For example, one manufacturer (Abbott) dilutes the patient’s serum specimen to a degree that only a considerably elevated HBcAb-IgM titer will be detected. This is done so that HBcAb-IgM will only be detected in patients with active acute or recent acute HBV infection. Other manufacturer’s kits who use lesser patient serum dilution may detect lower HBcAb-IgM titers, such as may be present in some cases of chronic HBV infection.

    surface antigen and antibody (HbsAg and HBsAb-Total)

    Fig. 17-3 surface antigen and antibody (HbsAg and HBsAb-Total).

    HBV Surface Antibody (HBsAb-Total; Both IgM + IgG)

    Appearance

    2-6 weeks after disappearance of HBsAg (range, HBsAg still present to over a year after HBsAg is gone); about 10% of patients do not produce HBsAb

    Peak

    2-8 weeks after initial appearance

    Becomes nondetectable

    About 85% of patients have persistent HBsAb-Total for many years or life, although there is often a slow decline to lower titers. About 15% (range, 2%-33%) of patients lose HBsAb-Total in less than 6 years

    Summary: HBV Surface Antigen and Antibody

    HBsAg by Immunoassay

    1. Means current active HBV infection.
    2. Persistance over 6 months indicates carrier/chronic HBV infection.

    HBsAg by Nucleic Acid Probe

    1. Same significance as detection by immunoassay.
    2. Present before and longer than HBsAg by immunoassay.
    3. More reliable marker for increased infectivity than HBsAg by immunoassay and/or HBeAg.

    HBcAb-IgM

    Appearance

    About 2 weeks (range, 0-6 weeks) after HBsAg appears

    Peak

    About 1 week after onset of symptoms

    Becomes nondetectable

    3-6 months after appearance (range, 2 weeks-2 years)

    HBcAb-Total

    Appearance

    3-4 weeks (range, 2-10 weeks) after HBsAg appears

    Peak

    3-4 weeks after first detection

    Becomes nondetectable

    Elevated throughout life; may have slow decline to lower titers over many years

    Therefore, the HBcAb-IgM level rises during active HBV infection, remains elevated during convalescence (during the time between loss of HBsAg and rise of HBsAb-Total, known as the “core window”), and becomes nondetectable in the early weeks or months of the recovery phase.

    In the majority of patients, HBcAb-Total becomes detectable relatively early, before HBsAg has disappeared, and maintains elevation throughout the gap between disappearance of HBsAg and appearance of HBsAb-Total (the core window). It is elevated for many years. Thus, the HBcAb-Total level begins rising somewhat similar to an IgM antibody level and remains elevated like an IgG antibody. If it is the sole test used, HBcAb-Total could give positive results during late-stage active acute infection, convalescence, chronic infection, or recovery since, in its early stage, HBcAb-Total may coexist with HBsAg.

    HBV core antibodies (HBcAb = HBcAb-IgM + HBcAb-IgG combined)

    Fig. 17-4 HBV core antibodies (HBcAb = HBcAb-IgM + HBcAb-IgG combined).

    HBV surface antigen-antibody and core antibodies (note “core window”) *HBcAb = HBcAb-IgM + HBcAb-IgG (combined)

    Fig. 17-5 HBV surface antigen-antibody and core antibodies (note “core window”) *HBcAb = HBcAb-IgM + HBcAb-IgG (combined).

    In many persons with HBV there is a time lag or gap in time of variable length between disappearance of the HBV surface antigen and appearance of the surface antibody. This has been called the “core window,” because the core total antibody is elevated during this time and represents the only HBV marker elevated in acute infection that is consistently detectable (the core IgM antibody is also present during part or all of the acute infection and also during part or all of the core window, but may become nondetectable during the window period, depending on when the patient specimen was obtained and the time span of the core window). The core window typically is 2-8 weeks in length but varies from 1 week (or less) to more than a year. Elevation of HBcAb-Total in itself does not mean that one has discovered the core window; a test for HBsAg (and, if nondetectable, a test for HBsAb-Total) must be performed because both HBsAg and HBsAb-Total must be absent. The core antibody nearly always is present in chronic hepatitis when surface antigen is detectable unless the patient is severely immunosuppressed.

    HBcAb-Total (1) may be elevated in later stages of acute infection, in convalescence (core window), or in old infection; (2) is only useful to show old HBV infection if HBsAg and HBcAb-IgM are both negative.

    Summary: Diagnosis of HBV Infection
    Best all-purpose test(s) to diagnose acute or chronic HBV infection
    —HBs Ag *(active infection, acute or chronic)
    —HBc Ab-IgM (late acute and recent or convalescent stage)
    *HBV-DNA probe may be necessary in some cases.

    Hepatitis B virus e antigen and antibodies

    The e antigen is usually not employed for diagnostic purposes. Since the e antigen is considered a marker for continued replication of the HBV, the e antigen is often used as an index of HBV infectivity. It is generally accepted that the presence of the e antigen (without e antibody) means several times greater potential to infect others compared to infectivity when the e antigen is not detectable. The presence of HBsAg by DNA probe is an even stronger marker for infectivity than the e antigen (as mentioned previously).

    HBeAb appears either at the time e antigen disappears or within 1-2 weeks later. Since the disappearance of the e antigen occurs shortly before disappearance of the surface antigen, detection of e antibody usually means that the acute stage of HBV infection is over or nearly over and that infectivity for others is much less. In a few cases there is a short period of e-antigen and e-antibody coexistence. Immunologic tests for the e antigen and the e antibody (total) are commercially available.

    HBe Ag

    Appearance

    About 3-5 days after appearance of HBs Ag

    Peak

    About the same time as HBs Ag peak

    Becomes nondetectable

    About 2-4 weeks before HBs Ag disappears in about 70% of cases
    About 1-7 days after HBs Ag disappears in about 20% of cases
    Accompanies persistant HBs Ag in 30%-50% or more patients who become chronic HBV carriers or have chronic HBV infection; however, may eventually convert to antibody in up to 40% of these patients

    HBe Ab-Total

    Appearance

    At the same time as or within 1-2 weeks (range, 0-4 weeks) after e antigen disappears (2-4 weeks before HBs Ag loss to 2 weeks after HBs Ag loss)

    Peak

    During HBV core window

    Becomes nondetectable

    Persists for several years (4-6 years)

    Summary: HBV e Antigen and Antibody

    HBeAg

    When present, especially without HBe Ab, suggests increased patient infectivity

    HBeAb-Total

    When present, suggests less patient infectivity