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  • 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

  • Hepatitis Viruses

    Hepatitis A virus (HAV)

    Hepatitis A virus (HAV) was originally called “infectious hepatitis” or “short-incubation hepatitis,” and has an incubation period of 3-4 weeks (range, 2-6.5 weeks). HAV is highly contagious. During active infection it is excreted in the stool and is usually transmitted via fecal contamination of water or food. However, infection by fecal contamination can also spread from person to person. Although urine and saliva are less infectious than stool, they can transmit HAV infection. The greatest quantity of virus excretion in stool occurs before clinical symptoms develop, although much lower levels of excretion may occur for a few days after onset of clinical illness. Clinical illness is usually not severe, and fatality is rare. However, cases of severe HAV hepatitis with a high fatality rate have been reported. There usually is complete recovery in 1-3 weeks and no carrier state. Occasional patients may have more prolonged illness, lasting as long as a year. One report indicates that 8%-10% of cases have a fluctuating clinical and laboratory test course, sometimes for as long as 12-15 months.

    There is increased incidence of HAV infection in children, and epidemics occur within institutions for mentally retarded children, day-care centers, and orphanages. These children frequently infect institution staff, and day-care patients infect parents and other household contacts. Occasional epidemics are confined to adults, usually associated with eating contaminated food or shellfish from contaminated water. About 40%-50% (range, 30%-60%) of adults in the United States who have been tested have antibody against HAV; in some “third world” countries, this may be as high as 90%-100%. More than 50% of acute HAV infections are subclinical (“anicteric hepatitis”), including almost all infants, 75% of children less than 2 years old, and 60% of those 4-6 years old. In adults, only about 10% are asymptomatic (range, 0-60%).

    Tests for HAV infection

    At present, serologic tests are not available to detect HAV antigen. Electron microscopy (EM) can detect HAV virus in stool as early as 1-2 weeks after exposure; this period ends about 1-4 days after onset of symptoms (range, 1 week before to 2 weeks after symptoms). Virus in stool is not detectable on admission in 40%-65% of patients. Presence of HAV in blood terminates just before or at the beginning of symptoms, too late to be detected in most patients.

    Antibody testing currently is the best method for diagnosis. Both RIA and ELISA methods have been used. Tests for HAV antigen are not yet commercially available. When they do become available, the major problem will be the disappearance of antigen before or shortly after onset of clinical symptoms. Two types of antibody to HAV antigen are produced. One is a macroglobulin (IgM), which appears about 3-4 weeks after exposure (range, 15-60 days), or just before the beginning of the AST increase (range, 10 days before to 7 days after the beginning of the AST increase). Peak values are reached approximately 1 week after the rise begins, with return to normal (nondetectable) in about 2 months (range, 1-5 months or 1-2 weeks after clinical symptoms subside to about 4 months after symptoms subside). However, in a few cases detectable IgM antibody has remained as long as 12-14 months. The second type of antibody is IgG, which appears about 2 weeks after the beginning of the IgM increase (between the middle stages of clinical symptoms and early convalescence), reaches a peak about 1-2 months after it begins to rise, and then slowly falls to lower titer levels, remaining detectable for more than 10 years (Fig. 17-2).

    Serologic tests in HAV infection

    Fig. 17-2 Serologic tests in HAV infection.

    If the IgM antibody is elevated but the IgG antibody is not, this proves acute HAV infection. If the IgM antibody is nondetectable and the IgG antibody is elevated, this could mean residual elevation from old HAV infection or a recent infection in the convalescent stage. If clinical symptoms began less than 1 week before the specimen was obtained, an old HAV infection is more likely. If the test for IgM antibody is not done and the IgG antibody is elevated, this could mean either a recent infection or residual elevation from a previous infection. A rising titer is necessary to diagnose recent infection using the IgG antibody alone. If the test for IgM antibody is not done and the IgG test is nonreactive, it could mean either no infection by HAV or that the specimen was drawn before the IgG antibody titer began to rise. Whether another specimen should be drawn 2 weeks later to rule out a rising titer depends on the length of time that elapsed since clinical symptoms began or ended. Therefore, interpretation of HAV antibody test results depends on when the specimen was obtained relative to onset of clinical symptoms and which antibody or antibodies are being assayed.

    HAV Antibodies

    HAV-IgM ANTIBODY
    Appearance

    About the same time as clinical symptoms (3-4 weeks after exposure, range 14-60 days), or just before beginning of AST/ALT elevation (range, 10 days before 7 days after)

    Peak

    About 3-4 weeks after onset of symptoms (1-6 weeks)

    Becomes nondetectable

    3-4 months after onset of symptoms (1–6 months). In a few cases HAV-IgM antibody can persist as long as 12-14 months.

    HAV-TOTAL ANTIBODY

    Appearance

    About 3 weeks after IgM becomes detectable (therefore, about the middle of clinical symptom period to early convalescence)

    Peak

    About 1-2 months after onset

    Becomes nondetectable

    Remains elevated for life, but can slowly fall somewhat

    Summary
    HEPATITIS A ANTIGEN AND ANTIBODIES

    HAV-Ag by EM (in stool)

    Shows presence of virus in stool early in infection

    HAV-Ab (IgM)

    Current or recent HAV infection

    HAV-Ab (total)

    Convalescent or old HAV infection

    Summary: Diagnosis of HAV Infection

    Best all-purpose test(s) to diagnose acute HAV infection = HAV-Ab (IgM)
    Best all-purpose test(s) to demonstrate past HAV infection/immunity = HAV-Ab (Total)
    (see also the box)

  • Rubella

    Rubella (German measles) is a very common infection of childhood, although primary infection can occur in adults. The major clinical importance of rubella is maternal infection during pregnancy, which may produce the congenital rubella syndrome in the fetus. The congenital rubella syndrome includes one or more of the following: congenital heart disease, cataracts, deafness, and cerebral damage. Diagnosis is made by documenting active rubella infection in the mother during early pregnancy and by proving infection of the infant shortly after birth. Rubella antibody tests are used to determine (1) if a woman is susceptible to rubella infection (and, therefore, should be immunized to prevent infection during pregnancy), (2) to prove that a woman is immune (and therefore, does not have to be immunized or be concerned about rubella infection), (3) to determine if possible or actual exposure to rubella infection during pregnancy actually produced maternal infection, (4) to determine if an infant has been infected, (5) to determine if symptoms that might be rubella (such as a rash) really are due to rubella or to something else.

    Serologic tests in rubella infection

    Fig. 17-1 Serologic tests in rubella infection.

    Rubella has an incubation period of about 14 days (range, 10-23 days), followed by development of a skin rash that lasts about 3 days (range, 1-5 days). Illness can be subclinical in up to 25% of cases. The patients are contagious for about 2 weeks (range, 12-21 days), beginning 7 days (range, 5-7 days) before and ending about 7 days (range, 5-10 days) after onset of the rash. Subclinical illness is also infective. Virus can be cultured in the nasopharynx (posterior end of the nose is best) about 7 days before the rash until about 7 days (range, 4-15 days) after onset of the rash. Serologic tests have mostly replaced culture except for epidemiologic purposes.

    Commercially available kits for antigen are not available. Those for antibody include hemagglutination inhibition (HI or HAI), indirect hemagglutination (IHA), ELISA, and LA. Most of the kits detect only IgG antibody, but some ELISA kits for IgM are also available. Some kits detect both IgM and IgG. Most current IgG kits appear to have greater than 95% sensitivity, although there is some variation between kits. There sometimes is confusion due to the large variety of kits and methods. Some kits detect both IgM and IgG, but do not differentiate between them and generally behave as though they detect IgG alone. Also, some procedures are reported as a titer and some as positive or negative. Also, HI (HAI) used to be the standard method but has been mostly replaced by ELISA and LA. Hemagglutination inhibition-reacting antibodies appear during the first week after onset of a rash; they are sometimes detectable after only 2-3 days. Peak levels are reached near the beginning of the second week after onset of the rash. Afterward the titer slowly falls, but an elevated titer persists for many years or for life. Although the standard HI test detects both IgM and IgG antibodies, the HI time sequence just described is similar to that of rubella IgG antibodies. Complement fixation-reacting or immunofluorescent-demonstrable antibodies develop in the more conventional time of 7-14 days after onset of the rash, reach a peak about 14-21 days after the rash and usually disappear in a few years.

    Serologic tests for rubella IgM antibody are available. Immunoglobulin M antibody titer begins to rise about the time of onset of the rash, peaks about 1 week after onset of the rash, and becomes nondetectable about 4-5 weeks after onset of the rash (range 21 days-3 months). Therefore, the rubella IgM and IgG antibody rise and peak are relatively close together, in contrast to serologic behavior in most other viral diseases, in which IgG usually follows IgM by at least 1 week. Some IgM procedures, but not others, may be affected by IgM produced against nonrubella antigen (e.g., rheumatoid factor). If so, this might lead to a false positive result. Besides primary infection, rubella reinfection can occur. If this happens there is often a rise in IgG antibody, but IgM antibody is not produced. Reinfection of the mother during pregnancy is not dangerous to the fetus, in marked contrast to primary infection. The ELISA method generally detects about 94%-97% of nonneonatal patients with well-established rubella compared to the HI method and can be modified to detect either IgG or IgM or both together. Most LA kits detect over 95% of cases but detect only IgG.

    Vaccination produces immune (IgM and IgG) response in about 95% of persons. Antibodies develop 10-28 days after vaccination. Some persons take up to 8 weeks to respond. Most of those who do not respond originally will do so if revaccinated. IgG elevation declines significantly in 10% of vaccinated persons by 5-8 years and becomes nondetectable in a small number of these persons (one study found about one third had no detectable IgG antibody at 10 years). IgM lasts longer than usual in vaccinated persons; in one study 72% still had detectable IgM at 6 months. Reinfection can occur, usually subclinical, more often in vaccinated persons than in those who had previous wild-type virus infection. Reinfection does not produce a detectable IgM response but may elevate the preexisting IgG level. Reinfection apparently does not harm a fetus.

    When a test is reported either as positive or negative, this is a screen for immunity to rubella infection and is performed on a 1:8 serum dilution (the 1:8 dilution is the HI titer level that has become accepted as demonstrable of an immune IgG antibody response). If multiple serum dilutions are tested, the antibody responses detected by LA are similar in time sequence to the IgG response of HI.

    Summary of Rubella Antibodies
    HAI (HI): IS A TOTAL ANTIBODY TEST (IgM + IgG)
    Appearance

    1-3 days after onset of rash

    Peak

    About 14 days (range, 10-17 days) after onset of rash

    Becomes nondetectable

    Usually decreases about 2 serial dilutions by 1 year, then stable for life
    Titer of 1:8 considered adequate immune level

    IgM ANTIBODIES

    Appearance

    About 1-2 days after onset of rash

    Peak

    About 10 days (range, 7-21 days) after onset of rash

    Becomes nondetectable

    About 5-6 weeks (range, 10 days-12 weeks) after onset of rash; in congenital rubella, remains elevated after birth for 3-6 months

    IgG ANTIBODIES

    Appearance

    About 3-4 days after onset of rash

    Peak

    About 14 days (range, 10-21 days) after onset or rash

    Becomes nondetectable

    Remains elevated for life

    Absence of HI IgG (1:8 level) or LA antibody indicates susceptibility to rubella since elevated IgG levels usually persist for many years, whereas titers of other antibodies return to normal. Presence of LA antibody means either past or recent infection. In a person who is clinically well, this means immunity to subsequent infection. In a person with clinically suspected rubella, an immediate serum specimen and a second one drawn 2 weeks later should be obtained, the standard procedure for all serologic tests. A fourfold rise in titer confirms very recent (active) infection. However, if the first serum specimen was not obtained until several days after onset of a rash, the LA antibody titer peak may already have been reached, and no further increase may occur. If tests for rubella IgM antibody are available, presence of this antibody means recent acute infection. Absence of IgM antibody in a single specimen, however, does not completely rule out acute or recent infection, since the specimen could have been obtained either before antibody rise or after antibody fall. If IgM antibody tests are not available, a significant two-tube dilution or fourfold rise in titer of CF or fluorescent antibody may be demonstrable, since these antibodies develop later than LA. However, if both the LA and CF antibodies are at their peak, it is impossible with this information alone to differentiate between recent infection and infection occurring months or even years previously. Height of titers by itself is not reliable in differentiating acute from old infection; only a sufficient change in titer can provide this information.

    Infants with congenital rubella infection can produce both IgM and IgG antibody before birth, beginning in the late second trimester. In addition, the fetus acquires passively transferred maternal IgG antibody, whether or not the mother acquired the infection during pregnancy, so that neonatal serum IgG antibodies could represent either old or current maternal infection. Therefore, neonatal serum IgG antibodies might originate from the infant, the mother, or both. By age 6-8 months, maternal antibody in the child has disappeared, and persistence of IgM or IgG antibody past this time indicates congenital or neonatal infection. For some reason, however, at least 20% of children with congenital rubella lose their HI titer by age 5 years. Congenital rubella can also be diagnosed by detection of specific rubella IgM antibody in the blood of the newborn. If the specimen is drawn before 10 days of life (the incubation period of rubella acquired during or just after birth before postnatal antibody has a chance to rise), specific rubella IgM antibody is diagnostic of intrauterine infection. If the specimen is obtained later, this antibody may be highly suggestive of congenital rubella but is not absolutely diagnostic, since there could be a small chance that infection was acquired after delivery.

    The ELISA and LA tests are, in general, more reliable than the HI test in the average laboratory. However, false positive or negative results may occur for various reasons, just as they may occur with any test in any laboratory. If the patient is pregnant and test results may lead to some action, it may be advisable to split each sample, keeping part of each frozen, if the specimens are sent to an outside laboratory, in case a recheck is desired. If the tests are performed in-house, immediate redraw of a specimen that suggests active maternal infection might be useful. Because of technical factors, most laboratories list a specific titer below which the antibody level is not considered significant. This depends to some extent on the test being used. The cutoff titer level most frequently is 1:8 or 1:10. This fact is mentioned because theoretically any antibody titer ought to be significant in terms of indicating previous infection. However, in actual practice, antibody levels below the cutoff value are considered negative since it is not certain how much artifact is involved in very low titers.

    Summary of Rubella Testing
    For immune status = Single IgG antibody test
    For primary acute infection diagnosis = IgM (if negative, repeat in 2 weeks) or IgG (using acute and convalescent specimens)
    For congenital infection diagnosis = fetal/maternal IgM
    For possible reinfection = IgG acute and convalescent (assuming IgG was known to have been elevated before the presumed reinfection occurred)

    Summary of rubella test results
    To test for immunity to rubella in a pregnant or nonpregnant woman, an LA test (or other standard rubella test) is obtained. If the result is negative, the woman is susceptible to infection. A positive test result means immunity; and in a nonpregnant woman and in many pregnant women, this is usually enough information. However, a positive test result could either be due to past infection or recent infection. If there is some reason to rule out recent infection in a pregnant or nonpregnant woman, a rubella IgM titer could be obtained. An alternative could be a titer of the original specimen plus another specimen for titer in 2 weeks. To determine whether recent infection took place, the time relationship of two critical events—date of exposure or date of rash—is extremely important regarding what test to use and when to obtain the test specimen or specimens. To determine the presence or absence of immunity only, such timing is not important.

    If a pregnant woman has been exposed to someone with rubella, and the question is whether infection has been acquired, serum should be obtained immediately for rubella antibody titer. A significant titer obtained less than 10 days after exposure usually means immunity because of previous disease (the incubation period of rubella is 10-21 days). If the result is negative or a borderline low titer, a second specimen should be obtained 3-4 weeks later to detect a rising titer (to permit sufficient time for antibody to be produced if infection did occur). If exposure was more than 10 days previously and the LA titer is borderline or elevated, a second specimen should be obtained 2-3 weeks later to detect a possible rising titer. Alternatively, a rubella IgM antibody test could be obtained about 3 weeks after exposure. Significantly elevated IgM proves recent primary infection.

    If a person develops a rash, and the question is whether it was due to rubella, two specimens for rubella antibody titer should be drawn, one immediately and the other 2 weeks later. Alternatively, a rubella IgM antibody test could be obtained 7 days after the rash onset.

  • Diagnosis of Viral Diseases

    Culture. Until the 1980s, except in a relatively few cases the only available laboratory methods were culture and serologic tests for antibodies. There have been significant advances in culture techniques in the past few years, but most virus culture still is difficult and expensive. Culture is performed using living cell preparations or in living tissues. This fact in itself rules out mass production testing. Partly for this reason, facilities for culture are limited and are available mainly at sizable medical centers, regional reference laboratories, or large public health laboratories. In addition, culture and identification of the virus takes several days. Finally, cultural isolation of certain viruses does not absolutely prove that the virus is causing actual patient disease, since many viruses are quite prevalent in the general clinically healthy population. In these instances, confirmation of recent infection is helpful, such as presence of IgM antibodies or a fourfold rising titer of antibodies.

    Antigen detection. In the 1980s, several other diagnostic techniques that can detect viral antigen have appeared. These include electron microscopy, fluorescent antibody (FA or IFA) methods, enzymelinked immunoassay (ELISA), latex agglutination (LA) methods, and, even more recently, nucleic acid (DNA) probes (Chapter 14). These methods can provide same-day results. However, many of them are relatively expensive, especially the DNA probes, particularly when only one patient specimen is tested at a time. Except in large-volume reference laboratories, most institutions do not receive a large number of orders for virus tests in general; and with the possible exception of rubella, hepatitis B virus (HBV), human immunodeficiency virus type 1 (HIV-1), Epstein-Barr virus (EBV), and possibly rotavirus, laboratories usually receive very few requests for diagnosis of any one particular virus. This makes it difficult for the average laboratory to keep reagents for testing many different viruses; and without having the advantage of testing many specimens at the same time, costs (and therefore, prices) are much higher.

    Antibody detection. In addition to culture and tests for viral antigen, serologic tests for antibody are available for most viruses. There are many techniques, including complement fixation (CF), hemagglutination (HA or HAI), radioimmunoassay (RIA), ELISA, FA, and LA. Some of these methods can be adapted to detect either antigen or antibody and either IgM or IgG antibody. Although they are considerably less exacting than culture, most techniques other than LA and ELISA monoclonal spot test modifications are still somewhat tedious and time-consuming. Therefore, these tests are not immediately available except at reference laboratories. Serologic tests have the additional disadvantage that antibodies usually take 1-2 weeks to develop after onset of illness, and unless a significantly (fourfold or two-tube) rising titer is demonstrated, they do not differentiate past from recent infection by the viral agent in question. One serum specimen is obtained as early in the disease as possible (“acute” stage) and a second sample is obtained 2-3 weeks later (“convalescent” stage). Blood should be collected in sterile tubes or Vacutainer tubes and serum processed aseptically to avoid bacterial contamination. Hemolyzed serum is not acceptable. To help prevent hemolysis, serum should be separated from blood clot as soon as possible. The serum should be frozen as soon as possible after collection to minimize bacterial growth and sent still frozen (packed in dry ice) to the virus laboratory. Here a variety of serologic tests can be done to demonstrate specific antibodies to the various organisms. A fourfold rise in titer from acute to convalescent stage of the disease is considered diagnostic. If only a single specimen is taken, an elevated titer could be due to previous infection rather than to currently active disease. A single negative test result is likewise difficult to interpret, since the specimen might have been obtained too early (before antibody rise occurred) or in the case of short-lived antibodies such as IgM, a previously elevated antibody value may have decreased to nondetectable levels.

    There is one notable exception to the rule of acute and convalescent serologic specimens. In some circumstances, it is desirable to learn whether a person has an antibody titer to a particular virus that is sufficient to prevent onset of the disease. This is especially true for a woman in early pregnancy who might be exposed to rubella. A single significant antibody titer to rubella suggests immunity to the virus.

    Two types of antibodies are produced in most, but not all, bacterial or viral infections. A macroglobulin (IgM) type appears first, usually shortly before or just after onset of clinical illness; reaches a peak titer about 1-2 weeks after clinical symptoms begin; and then falls to normal levels within a few weeks (usually in less than 6 months). A gamma-globulin (IgG) type appears 1 or more weeks after detection of IgM antibody. The IgG antibody reaches a peak 1-3 weeks (sometimes longer) after the peak of the IgM antibody. The IgG antibody typically persists much longer than the IgM antibody (several years or even for life). Therefore, presence of the IgM antibody usually indicates recent acute infection. Presence of the IgG antibody usually requires that a rising titer be obtained to diagnose acute infection (although in some diseases there are circumstances that alter this requirement), since without a rising titer one does not know whether the IgG antibody elevation is due to recent or to old infection.

    Special stains. The Tzanck test is sometimes requested in certain skin diseases associated with vesicles or bullae. One of the vesicles is carefully unroofed, and the base and undersurface of the vesicle membrane is scraped; the scrapings are gently smeared on glass slides. The smear can be stained with Wright’s stain or Giemsa stain; if so, the slide can either be methanol-fixed or air-dried. Papanicolaou (Pap) stain can also be used, in which case the slide must be immediately fixed in a cytology fixative. The slide is then examined microscopically for multinucleated giant cells or characteristic large abnormal rounded epithelial cells. If found, these are suggestive of herpes simplex or varicella-zoster infection.

    Viral test specimens

    The type of specimen needed for viral culture depends on the type of illness. In aseptic meningitis, a CSF specimen should be obtained. In addition, stool culture for virus should be done, since enteroviruses are frequent causes of meningitis. In enterovirus meningitis, stool culture is 2-3 times more effective than CSF culture.

    In any kind of meningitis with negative spinal fluid cultures or severe respiratory tract infection of unknown etiology it is a good idea to freeze a specimen of serum as early in the disease as possible. Later on, if desired, another specimen can be drawn and the two sent for virus studies. As noted, serum specimens are generally drawn 2 weeks apart.

    In suspected cases of (nonbacterial) encephalitis, whole blood should be collected for virus culture during the first 2 days of illness. During this short time there is a chance of demonstrating arbovirus viremia. This procedure is not useful in aseptic meningitis. Spinal fluid should also be sent for virus culture. Although the yield is relatively small in arbovirus infections, the specimen results sometimes are positive, and culture also helps to rule out other organisms, such as enterovirus. In upper respiratory tract illness, throat or nasopharyngeal swabs are preferred. These should be placed in trypticase broth (standard bacterial medium). Swabs not preserved in some type of medium such as trypticase or Hank’s solution are usually not satisfactory, since they dry out quickly, and most viruses are killed by drying. Throat washings or gargle material can be used but are difficult to obtain properly. In viral pneumonia, sputum or throat swabs are needed. If throat swabs are used, they should be placed in acceptable transport solutions. Whether throat swab or sputum is used, the specimen must be frozen immediately and sent to the virus laboratory packed in dry ice. In addition, a sputum specimen (or throat swab) should be obtained for Mycoplasma culture (Chapter 14).

    In possible viral gastroenteritis, the most logical specimen is stool. At present, rotavirus and Norwalk viruses cannot be cultured from stool, but stool can be examined for Norwalk virus by immune electron microscopy and for rotavirus antigen by RIA, ELISA, or slide LA. Serologic tests on serum can be used for diagnosis of rotavirus infection, but only a few laboratories are able to do this. Whenever a stool culture for virus is needed, actual stool specimens are preferred to rectal swabs, since there is a better chance of isolating an organism from the larger sample. Stool samples should be collected as soon as possible—according to the U.S. Centers for Disease Control (CDC), no later than 48 hours after onset of symptoms (to ensure the best chance of success). The stool specimen should be refrigerated, not frozen; and if sent to an outside laboratory, the specimen should be shipped the day of collection (if possible), and kept cool with dry ice. However, it is better to mail any virus specimens early in the week to avoid arrival on weekends. An insulated container helps prolong effects of the dry ice.

    An adequate clinical history with pertinent physical and laboratory findings should accompany any virus specimen, whether for culture or serologic studies. As a minimum, the date of clinical illness onset, collection date of each specimen, and clinical diagnosis must be included. The most likely organism should be indicated. This information helps the virus laboratory to decide what initial procedures to use. For example, some tissue culture cell types are better adapted than others for certain viruses. Considerable time and effort can be saved and a meaningful interpretation of results can be provided.

    Certain viruses deserve individual discussion. The method of diagnosis or type of specimen required for some of these organisms is different from the usual procedure, whereas in other cases it is desirable to emphasize certain aspects of the clinical illness that suggest the diagnosis.

  • Viral Diseases

    Viral upper respiratory tract diseases

    Respiratory disease may take several forms, and the predominant etiologies are different in different age groups. Incidence statistics also vary depending on the geographic area and the population selected. Of the known viruses, rhinoviruses are predominantly associated with acute upper respiratory tract disease (including the common cold) in adults, whereas in children, rhinovirus, adenovirus, parainfluenza virus, and the enteroviruses are important. Acute bronchitis in children is most often due to respiratory syncytial virus and parainfluenza virus. In croup, parainfluenza is said to be the most important virus.

    Viral pneumonia

    Respiratory syncytial virus is the predominant cause of pneumonia in infants and young children, beginning at age 1 month with a peak incidence at about age 6 months, followed by adenovirus or parainfluenza virus. In older children or adults, bacterial pneumonia (most often due to Pneumococcus or Mycoplasma pneumoniae) is more common than viral pneumonia. Among viral agents known to cause pneumonia in adults, the most common is probably influenza. In any study, a large minority of cases do not yield a specific etiologic agent.

    Viral meningitis

    Viruses are an important cause of meningitis, especially in children. They typically produce the laboratory picture of aseptic meningitis: the classic cerebrospinal fluid (CSF) findings are variable, but often include mildly increased protein levels, increased cell counts with mononuclear cells predominating, normal glucose levels, and no organisms found on culture. It should be remembered, however, that tuberculous meningitis gives similar findings, except for a decreased CSF glucose level, and likewise shows a sterile culture on ordinary bacterial culture media. Some patients with mumps meningoencephalitis may have decreased CSF glucose levels in addition to CSF lymphocytosis. Enteroviruses are the largest etiologic group causing aseptic meningitis. Among the enteric viruses, poliomyelitis used to be the most common organism, but with widespread polio vaccination programs, echovirus and coxsackievirus have replaced polio in terms of frequency.

    After the enteroviruses, mumps is the most important. A small but significant number of patients with mumps develop clinical signs of meningitis, and a large number show CSF changes without demonstrating enough clinical symptoms to warrant a diagnosis and workup for meningitis. Changes in CSF or the clinical picture of meningitis may occur in patients without parotid swelling or other evidence of mumps. Lymphocytic choriomeningitis and leptospirosis are uncommon etiologies for aseptic meningitis.

    Encephalitis is a syndrome that frequently has CSF alterations similar to those of meningitis. The two cannot always be separated, but the main difference is clinical; encephalitis features depression of consciousness (lethargy, coma) over a prolonged period, whereas meningitis usually is a more acute illness with manifestations including fever, headache, vomiting, lethargy, stiff neck, and possibly convulsions. In severe bacterial infection, encephalitis may follow meningitis. Encephalitis is most often caused by viruses, of which the more common are mumps, herpes simplex type 1 (HSV-1), measles, and the arboviruses. Sometimes encephalitis is a complication of vaccination.

    Viral gastroenteritis

    Viruses are likely to be blamed for diarrhea that cannot be explained otherwise. In most cases, definitive evidence is lacking because enteric virus is present in a significant number of apparently healthy children. Bacterial infection should always be carefully ruled out. Two clinical types of viral gastroenteritis have been described. One type usually occurs in epidemics, more often in older children and in adults, with clinical signs of an acute self-limited gastroenteritis of 1-2 days’ duration. The most commonly associated etiology is the Norwalk-type group of viruses. The other type of illness is sporadic and affects mostly infants and younger children. There is severe diarrhea, usually accompanied by fever and vomiting, which lasts for 5-8 days. Rotavirus is the most frequently isolated virus in these patients. About 5%-10% of gastroenteritis in infants less than 2 years old is said to be caused by adenovirus types 40 and 41

    Viral infections in pregnancy

    By far the most dangerous viral disease during pregnancy is rubella. Statistics are variable, but they suggest about a 15%-25% risk of fetal malformation when rubella infection occurs in the first trimester (literature range, 10%-90%). The earlier in pregnancy that maternal infection occurs, the greater the risk that the fetus will be infected. However, not all infected fetuses develop congenital malformation. When the fetus is infected early in the first trimester, besides risk of congenital malformation, as many as 5%-15% of fetuses may die in utero. Risk of fetal malformation in second trimester infections is about 5%. After the fourth month of pregnancy, there is no longer any danger to the fetus. Cytomegalovirus (CMV) infection is more frequent than rubella, but CMV has a lower malformation rate. Cytomegalovirus damage is more severe in the first two trimesters. Other viruses may cause congenital malformations, but evidence is somewhat inconclusive as to exact incidence and effects. Herpes simplex and the hepatitis viruses are in this group.