Articles on Medical Diseases and Conditions

Tag: Serologic Tests

  • Giardia Lamblia

    This protozoan lives in the duodenum and proximal jejunum and is said to be the most frequent intestinal parasite in the United States. An estimated 3%-7% of U.S. adults may have the disease. The organism is usually transmitted through fecal (sewage) contamination of water. Chloridation will not kill Giardia lamblia, but iodine will. Some reports suggest a high incidence in male homosexuals. G. lamblia can also be transmitted by fecal-oral contact, especially in day-care centers, where it has been estimated that 5%-15% of young children of diaper age become infected. Acute infection typically shows foul-smelling watery diarrhea, usually accompanied by greasy floating stools, considerable intestinal gas, and epigastric pain. The symptoms may last 3-4 days or 1-3 weeks or may become chronic. In severe cases, steatorrhea and intestinal malabsorption have been reported.

    Diagnosis. Diagnosis is made through the same type of stool examinations discussed under amebiasis. Overall detection rate of stool microscopy (direct examination plus stained slide) is usually considered to be 50%-70%. Cathartics do not increase detection rates. Permanent stain techniques detect about one-third more cases than wet mounts. Cyst excretion in the stool may be irregular; Giardia cysts may be passed at 1, 2-3, or even 7-8 day intervals. Although three specimens (one specimen collected every 2 days) are usually sufficient, more may be necessary. The first specimen detects about 75% of cases diagnosed through multiple specimens. Duodenal aspiration has been found to detect more infections (about 80% of cases) than repeated stool specimens. A commercial company has a string apparatus (Entero-Test) that can be swallowed instead of resorting to duodenal aspiration.

    Serologic tests. ELISA tests for antibody have not been widely used, as current tests do not differentiate between past or currently active infection. A commercial test (Pro Spec T Giardia) for antigen detection in stool is commercially available and in several evaluations was found to have a sensitivity of 92%-100% compared to the total positive patients by ELISA plus standard O& P examinations. A commercial indirect immunofluorescent test is also available, and the manufacturer claims 97% sensitivity. To date, I have not seen any independent evaluations.

  • Entamoeba Histolytica

    E. histolytica is a unicellular single-nucleus protozoan that is said to infect 10%-12% of the world’s population, the majority of these being in the tropics. In the United States, the population at greatest risk are travelers to third-world countries, immigrants or migrants from these areas, immunocompromised persons, and about 20%-32% of male homosexuals. The organism life cycle consists of a trophozoite free-living stage that occurs in the human colon. There are two subgroups (strains or possibly species) of E. histolytica (different by enzyme analysis). One strain can invade tissue (10% of E. histolytica infections) and causes diarrhea with or without mucosal ulceration, sometimes entering the bloodstream and producing abscesses in the liver (or occasionally other organs). The much more common, relatively nonpathogenic strain can be asymptomatic, cause mild nonspecific gastrointestinal (GI) symptoms, or cause bloodless diarrhea that usually is not severe. Both types have the same basic life cycle in which the trophozoite forms a cyst, develops four nuclei, and passes outside the body in the feces. The cyst is said to be fairly resistant to the environment and can survive up to 3 months under the right conditions. The cyst is the infective stage and produces infection after being ingested in contaminated water or on food that has been in contact with either contaminated water or soil or fertilizer contaminated with human feces. Once the cyst reaches the colon the trophozoite inside emerges and divides, eventually forming eight trophozoites. About 10% of persons infected have symptoms; of these, about 10% (range 2%-20%) develop extraintestinal amebic infection and the remainder have diarrhea or the more severe colon mucosal inflammation known as amebic dysentery. Interestingly, male homosexuals with or without HIV-1 virus infection usually do not develop the invasive type of amebiasis. Because of the two subtypes of E. histolytica, there is a wide clinical spectrum of infection: a severe acute colitis (amebic dysentery) that may resemble severe ulcerative colitis (sometimes with blood and mucus in the stool) or shigellosis; chronic diarrhea similar to milder ulcerative colitis; intermittent mild diarrhea; asymptomatic carriers; and even a group with constipation. Acutely ill patients are usually afebrile and have normal white blood cell counts and hemoglobin values; although patients with severe amebic colitis or hepatic abscess frequently have low-grade fever, leukocytosis between 10,000 and 20,000/cu mm, and mild anemia. E. histolytica usually does not produce eosinophilia.

    Stool examination. E. histolytica is more difficult to diagnose than most of the common intestinal parasites and requires special precautions. If the stool specimen is soft or liquid, it may contain motile trophozoites and should be sent to the laboratory immediately (with the time of collection noted) or placed into a special fixative, because trophozoites are fragile and quickly degenerate. Wet mounts should be done within 30 minutes after the specimen is obtained. Well-formed stools usually contain protozoan cysts rather than trophozoites and may be either temporarily refrigerated or placed into fixative. For collection procedure, three specimens, one specimen collected every other day, are more reliable than a single specimen. If results of the stool specimens on three alternate days are all negative, and if strong clinical suspicion is still present, a saline purge should be used. After a saline purge (e.g., Fleet Phosphosoda), the patient should pass liquid stools within a few hours. Oily laxatives (e.g., mineral oil or magnesia) make the stools useless for examination. Enema specimens are not advisable because they are too dilute to be of much value, and, in addition, the trophozoites may be destroyed. Barium, if present, also makes the preparation unfit to read. If stool specimens for amebae must be sent by mail, they should be placed in a preservative (one part specimen to three parts of 10% formalin). If possible, a second portion preserved in a special polyvinyl alcohol fixative (PVA) (in the same proportions) should be included along with the formalin-fixed portion. Formalin preserves ameba cysts and also eggs and larvae of other parasites. Polyvinyl alcohol fixative is used to make permanent stained preparations of protozoan trophozoites and cysts, which is not possible after formalin fixation (there is some disagreement about preservation of cysts with PVA). Stained slides considerably increase the chances of finding protozoan trophozoites and cysts and provide better cytologic detail for identification purposes.

    Serologic tests. Serologic tests for amebiasis are available in reference laboratories. The most widely used procedures are gel diffusion, IHA, and slide LA. Various ELISA methods have been reported, based on several purified or recombinant antigens from E. histolytica. In patients with intestinal amebiasis, these tests detect about 10% of those who are asymptomatic E. histolytica carriers, less than 50% of those with mild amebic diarrhea, and about 85%-90% of those with invasive amebiasis. The IHA, LA, and ELISA tests are slightly more sensitive than gel diffusion. Thus, results of the more severe cases are more likely to be positive. The IHA and LA antibody levels persist for several years, so a positive test result does not necessarily mean active infection. Gel diffusion antibodies may become undetectable in 6 months, although some have reported persistent elevation for 1-2 years. Nucleic acid probes for antigen in stools have been reported and may be available in some university medical centers or large reference laboratories.

    Extraintestinal amebiasis

    The preceding discussion was concerned with the usual type of amebiasis—amebiasis localized to the colon. Visceral amebiasis is not common. Liver involvement with abscess formation is seen in a majority of these cases. Clinical hepatic amebiasis is always associated with chronic rather than acute ameba infestation. Only 30%-50% of patients provide a history of diarrhea. Only about 25% of patients have amebae detectable in the stool. Patients with classic hepatic amebiasis have hepatomegaly, right upper quadrant pain, elevation of the right hemidiaphragm, leukocytosis as high as 20,000/mm3 (20 x 109 L), and fever. Surprisingly, the alkaline phosphatase level is normal in more than one half of patients. Liver scan is often very helpful, both for detection and localization of a lesion. Results of the various serologic tests for amebiasis are positive in 90%-95% of cases.

    Amebic encephalitis is a rare condition produced by free-living amebae of the Acanthamoeba and Naegleria species. Naegleria infection takes place in normal individuals, usually with a recent history of swimming in rivers, fresh-water lakes, or fresh-water swimming pools. Acanthamoeba infections occur in persons with decreased immunologic defenses, frequently without any history of swimming. Acanthamoeba keratitis is also being reported due to contamination of contact lenses. Diagnosis can be made through phase contrast examination of spinal fluid or permanent slides of centrifuged spinal fluid stained by Wright’s stain or trichrome stain. Centrifugation or refrigeration decreases motility of the organisms, which would hinder phase contrast examination but not permanent stained slide examination. Gram-stained smears are not recommended. Calcofluor white stain used for detection of fungus is reported to detect Acanthamoeba cysts as well (but not trophozoites).

  • Varicella-Zoster Virus (VZV)

    Varicella-zoster virus (VZV) is a member of the herpesvirus group. Infection is spread through direct contact with skin lesions or through droplet inhalation. The incubation period is about 14 days (range, 9-21 days). Primary infection is usually varicella (chickenpox). The period of skin rash lasts about 4-6 days. This may be preceded by a short prodromal period. The period of contagion is said to be from 2 days before the rash until no new skin lesions appear and all old ones become crusted. Usually there is lifelong immunity to new infection (although not always). Complications are not common but are not rare. They include pneumonia, encephalitis, and Reye’s syndrome (20%-30% of Reye’s syndrome follows varicella infection). Incidence and severity of complications are increased in immunocompromised persons. Twenty-three percent to 40% of bone marrow transplant patients develop primary VZV infection or reinfection. Varicella infection in pregnancy may affect the fetus in 5%-10% of cases.

    After the varicella syndrome is over, the virus begins a latent period in sensory nerve ganglion cells. Later on, it may reactivate in the form of zoster. Reactivation is more common in persons with malignancy or in those who are immunocompromised. It becomes more frequent with increasing age. About 10%-20% of the population is affected. Neuralgia is the most frequent symptom. A rash is also relatively frequent, often in the distribution of a dermatome. Encephalitis, sensory and motor neurologic abnormality, and ocular abnormality may occur.

    Laboratory tests include Tzanck test smears of varicella-zoster lesions. Sensitivity is said to be 50% or less in varicella and 80% or less in zoster. This procedure is described in the section on simplex and the microscopic appearance is the same. Culture of lesions can be done, but results in varicella are reported to be 34%-78% positive and in zoster to be 26%-64%. Serologic tests can be done using fluorescent antibody (FA), ELISA, and slide LA. EIA is said to be 50% sensitive (range, 36%-94%); FA, about 75% (range, 69%-93%); and LA, about 60% (52%-76%). It appears that antibody production (and, therefore, sensitivity) is greater in otherwise healthy children than in adults. IgM antibody rises in varicella about 5-6 days after the rash begins and peaks at about 14 days; it rises in zoster about 8-10 days after onset of the rash and peaks at about 18-19 days. Some patients with VZV infection who later are infected by herpesvirus type 1 experience an anamnestic rise in VZV antibody. Nucleic acid (DNA) probe methods have also been reported for skin lesions and for CSF 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 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

  • 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.

  • Systemic Mycoses

    Certain fungi, known as the deep or systemic fungi, are characterized by involvement of visceral organs or penetrating types of infection. Besides true fungi, actinomycetes and nocardiae are bacteria that produce disease resembling deep fungal infection in many ways. These are discussed in the chapter on bacterial infections. The systemic fungi include Blastomyces dermatitidis (blastomycosis), Coccidioides immitis (coccidioidomycosis), Cryptococcus neoformans (cryptococcosis), Histoplasma capsulatum (histoplasmosis), and Sporothrix schenckii (sporotrichosis). Certain Candida species (especially Candida albicans and Candida tropicalis), Aspergillus species (Aspergillus fumigatus and Aspergillus flavus), and certain zygomycetes (Rhizopus species and Mucor species) may, on occasion, produce infection that would qualify as a systemic mycosis. Blastomyces, Coccidioides, Histoplasma, and Sporothrix organisms are considered diphasic (dimorphic) fungi, since they grow as a mycelial phase in culture but in a yeast (budding) phase within tissue infections.

    Diagnosis of Fungal Infections

    The diagnosis of mycotic infection can be assisted in several ways:

    1. Wet mount of scraping, exudate, fresh swab smear, or other specimen such as sputum; usually done with 10% potassium hydroxide (KOH). India ink or nigrosin preparations are used for cryptococcosis. The advantages of wet mounting are same-day results and, in some instances, reasonably accurate diagnosis. Disadvantages are that few laboratory personnel are expert in this technique, and consequently there are frequent false positive and negative results. A recent aid to wet-mount examination are naturally fluorescing compounds, such as Calcofluor white, that bind nonspecifically to fungus cell walls and can outline the organism when it is viewed with the proper filters under a fluorescent microscope. Unfortunately, false negative results are frequent regardless of technique because the specimen obtained may not contain organisms. Also, in most cases (except possibly cryptococcosis) the most the technique can offer is recognition that a mycotic infection may be present without reliable identification of what the organism is, or its species. Speciation may be important because some species are more likely to be true pathogens in certain body areas than other species. When material such as sputum is examined, there is often a problem in deciding whether an organism is causing infection, is colonizing the area without actual infection, or is a contaminant.
    2. Stained smear of a clinical specimen. Gram stain or Papanicolaou stain can be used. Wright’s stain or Giemsa stain is used for histoplasmosis. The advantages of the stained smear are same-day results and a permanent preparation that may be a little easier to interpret than a wet-mount preparation. However, others find the wet preparation easier to examine. The disadvantages are the same as those of the wet-mount preparation.
    3. Tissue biopsy with demonstration of the organism by special stains, such as periodic acid-Schiff or methenamine silver. The yield from this procedure depends on whether the biopsy specimen contains organisms, the number of organisms present, and whether the organism is suspected so that the special stains are actually used.
    4. Culture of a lesion. This permits definite isolation of an organism with speciation. The yield depends on whether the proper specimen is obtained, whether the organism is still alive by the time it reaches the laboratory, whether proper culture media are used, and the experience of the technologists. Because of the locations involved in deep mycotic infections, it may be difficult to secure a specimen or obtain material from the correct area. The organisms usually take several days to grow.
    5. Serologic tests. The major advantage is that specimens for other types of tests may not be available or the results may be negative. The disadvantages are the usual need for two specimens (“acute” and “convalescent”) and the long time period involved, the second specimen being obtained 1-2 weeks after the first to see if there is a rising titer. This means 2-3 weeks’ delay, possibly even more, since the specimens usually must be sent to a reference laboratory. There are usually a significant percentage of false negative results (sometimes a large percentage), and there may be a certain number of false positive and nondiagnostic results as well.
    6. Skin tests. The advantage is a result in 24-48 hours. The disadvantages include the time period needed to develop antibodies, false positive or negative test results, and the problem of differentiating a positive result due to old infection from one due to recent or active infection. In addition, the skin test in some cases may induce abnormality in the serologic tests, so a rising titer does not have its usual significance.

    Blastomycosis

    Blastomycosis may involve primarily the skin or the visceral organs. Granulomatous lesions are produced that are somewhat similar histologically to the early lesions of tuberculosis. Skin test results are unreliable, reportedly being positive in only about 50% of cases. Complement fixation (CF) tests also detect fewer than 50% of cases and produce false positive results in Histoplasma infections. Immunodiffusion tests are more specific for blastomycosis and are reported to detect about 80% of active cases. These procedures usually must be sent to a reference laboratory, since the number of requests for these tests in most institutions is quite small.

    Coccidioidomycosis

    Coccidioidomycosis is most often contracted in the San Joaquin Valley of California but occasionally appears elsewhere in the Southwest. It has a predilection for the lungs and hilar lymph nodes but occasionally may become systemic to varying degrees. Clinical symptoms are most often pulmonary, manifested usually by mild or moderate respiratory symptoms and sometimes by fever of unknown origin. Rarely, overwhelming infection much like miliary tuberculosis develops. Diagnosis is usually made through either biopsy or serologic tests. The most sensitive tests are tube precipitin (results of which become positive 1-3 weeks after onset of infection, with about 80% of cases positive by 2 weeks), latex agglutination (LA; slightly more sensitive than tube precipitin, but with 6%-10% false positive results), and immunodiffusion. The tube precipitin test usually reverts to negative by 6 months after onset of infection. A CF test is also widely used, especially for spinal fluid specimens. In cerebrospinal fluid (CSF) specimens it detects more than 90% of active coccidioidomycosis infections, whereas the tube precipitin test is not reliable on spinal fluid. These tests usually must be sent to a reference laboratory unless the laboratory receiving the request is located in an area where coccidioidomycosis is endemic. The coccidioidomycosis skin test is very useful, being equally as sensitive as the serologic tests. It does not produce an antibody response that would interfere with the other tests.

    Cryptococcosis

    Cryptococcosis (torulosis) is a fungal disease with a marked predilection for lung and brain. Pigeon feces seems to be the major known source of human exposure. Persons with illnesses that are associated with decreased immunologic resistance, such as acquired immunodeficiency syndrome (AIDS), Hodgkin’s disease, and acute leukemia, or those undergoing therapy with steroids or immunosuppressive agents are particularly susceptible. Pulmonary infection is apparently more common than central nervous system (CNS) infection but is often subclinical. Pulmonary infection radiologically can present in much the same way as tuberculosis and histoplasmosis, such as pneumonia or focal nodules, or occasionally as military lesions. CNS system disease typically occurs without respiratory disease and typically is very slowly progressive but may occasionally be either asymptomatic or severe and acute. In CNS disease, headache is found in about 75% of cases and fever in about 35%. Peripheral blood complete blood cell count and erythrocyte sedimentation rate are most often normal in respiratory and CNS cryptococcosis. Laboratory findings in CNS disease are described in Chapter 19. Diagnosis can be made through culture of sputum or CSF, by histologic examination of biopsy specimens (special stains for fungi are required), by microscopic examination of CSF using an india ink or nigrosin preparation to show the characteristic organism thick capsule (Chapter 19), and by serologic tests. Serologic tests that detect either antigen or antibody are available. Antibody is usually not present in the CSF; and in serum, antibody appearance is inconsistent in the early acute phase of the illness. In addition, some of the antibody-detection systems cross-react with histoplasmosis antibody. Cryptococcus antigen detection systems do not react when the patient is infected by other fungi.

    The most widely used serologic test is the slide latex agglutination procedure, which detects antigen. In localized pulmonary cryptococcosis, the LA test can be used on serum, but it detects fewer than 30% of cases. In patients with cryptococcal meningitis, the latex test can be used on either serum or CSF specimens. When used on CSF specimens, it detects about 85%-90% of culture-positive cases of cryptococcal meningitis (range, 75%-100%) versus 40%-50% for india ink. Testing both serum and CSF increases the number of LA-detectable cases. Rheumatoid factor may produce a false positive reaction; so patient serum must be heat inactivated, and a control for rheumatoid factor and nonspecific agglutinins must be used when either serum or CSF is tested. Some kits now incorporate pretreatment of the specimen with pronase, a proteolytic enzyme, to inactivate interfering substances. Occasional false positive results have been reported in patients with malignancy or collagen-vascular disease. False positive results also were reported due to culture medium contamination of the CSF specimen when a portion was removed by wire loop, plated on culture media, then the loop reintroduced into the CSF specimen to obtain fluid for LA testing. It should be mentioned that a few investigators have not obtained as good results on CSF specimens as most others have. Some of the test evaluations on CSF reported in the literature are difficult to interpret, however, since kits by different manufacturers apparently produce different results, and some investigators used their own reagents. As noted previously, the latex test may be nonreactive in low-grade chronic infections. The half-life of cryptococcal polysaccharide antigen is about 48 hours, so that once positive, the results of a test detecting antigen may remain positive for several days even if therapy is adequate. Besides LA, an enzyme-linked immunosorbent assay (ELISA) test that is a little more sensitive than the latex tests is commercially available.

    Histoplasmosis

    Histoplasmosis is the most common of the systemic fungal infections. It is most often encountered in the Mississippi Valley and Ohio Valley areas but may appear elsewhere. Certain birds, especially chickens and starlings, are the most frequent vectors in the United States. In endemic areas, 60% or more of infected persons are asymptomatic. The remainder have a variety of illness patterns, ranging from mild or severe, acute or chronic pulmonary forms, to disseminated infection.

    Histoplasmosis begins with a small primary focus of lung infection much like the early lesion of pulmonary tuberculosis. Thereafter, the lesion may heal or progress or reinfection may occur. Mild acute pulmonary infection with influenza-like symptoms may develop. The illness lasts only a few days, and skin test results, cultures, and chest x-ray films are usually normal. More severe acute pulmonary involvement produces a syndrome resembling primary atypical pneumonia. Chest x-ray films may show hilar adenopathy and single or multiple pulmonary infiltrates. Results of histoplasmin skin tests and CF or latex agglutination tests are negative during the first 2-3 weeks of illness but then become positive. Sputum culture is sometimes positive but not often. Chronic pulmonary histoplasmosis resembles chronic pulmonary tuberculosis clinically. Cavitation sometimes develops. Results of skin tests and CF tests usually are positive by the time the disease is chronic. Sputum is the most accessible material for culture, although results are reportedly negative in 50%-60% of cases. In clinically inactive pulmonary disease, such as coin lesions, sputum culture is usually negative. Even if the organism is present in the specimen, it takes 3-4 weeks for growth and identification. Histoplasmosis is a localized pulmonary disease in the great majority of patients, so there is usually little help from cultures obtained outside the pulmonary area. In the small group that does have disseminated histoplasmosis, either acute or chronic, there is a range of symptoms from a febrile disease with lymphadenopathy and hepatosplenomegaly to a rapidly fatal illness closely resembling miliary tuberculosis. In disseminated (miliary) histoplasmosis, standard blood cultures are positive in 40%-70% of patients (probably 60%-80% with newer culture methods). Bone marrow aspiration is the diagnostic method of choice; it is useful both for cultures and for histologic diagnosis of the organisms within macrohages on Wright-stained smear or fungus stain on a marrow clot section. Occasionally lymph node biopsy may be helpful. If it is performed, a culture should also be taken from the node before it is placed in fixative. Bone marrow aspiration and liver or lymph node biopsy are not helpful in the usual forms of histoplasmosis, which are localized to the lungs.

    The most commonly used diagnostic test in histoplasmosis is the CF test. Titers of 1:16 are considered suspicious, and 1:32 or more are strongly suggestive of histoplasmosis. Two types of CF test are available, based on mycelial antigen and yeast phase antigen. The test based on yeast phase antigen is considerably more sensitive than that based on mycelial antigen. Neither test result is likely to be positive in the early phase of acute infection. Later on (about 3-4 weeks after infection), results of the yeast antigen CF test become positive in 70%-85% of cases. Some additional cases may be detected with the mycelial CF antigen. About 3.5%-12% of clinically normal persons demonstrate positive results, usually (but not always) in titers less than 1:16. Thirty-five percent to 50% of patients with positive CF test results in the literature could not be confirmed as having true histoplasmosis infections. How many of these positive results were due to previous old infection or localized active infection without proof is not known. Because of the false positive and negative results, a fourfold (two-dilution level) rise in titer is much more significant than a single result, whether the single result is positive or negative. The CF test result may be negative in 30%-50% of patients with acute disseminated (miliary) histoplasmosis and when the patient has depressed immunologic defenses or is being treated with steroids.

    LA tests are also available and are reported to be a little more sensitive than the CF tests. However, there are conflicting reports on their reliability, with one investigator unable to confirm 90% of the cases with positive results and other studies being more favorable. Differences in reagents may be a factor. ELISA serologic methods have been reported with sensitivity equal to or better than CF. DNA probe methods have also been reported.

    Besides serologic tests, a skin test is available. Results of the skin test become positive about 2-3 weeks after infection and remain positive for life in 90% of persons. The skin test result is falsely negative in about 50% of patients with disseminated (miliary) histoplasmosis and is said to be negative in about 10% of patients with cavitary histoplasmosis. A (single) skin test result is difficult to interpret, whether it is positive (because of past exposure) or negative (because it may be too early for reaction to develop, or reaction may be suppressed by miliary disease, depressed immunologic status, or steroid therapy). Also, about 15% of patients develop a positive CF test result because of the skin test. The histoplasmin skin test reacts in about 30% of patients who actually have blastomycosis and in about 40% of those with coccidioidomycosis. For these reasons, routine use of the histoplasmin skin test is not recommended.

    In serious localized infection or widespread dissemination of the deep fungi, there is often a normocytic-normochromic or slightly hypochromic anemia. The anemia is usually mild or moderate in localized infection. In acute disseminated histoplasmosis, various cytopenias (or pancytopenia) are present in 60%-80% of cases, especially in infants.

    Sporotrichosis

    Sporotrichosis is caused by S. schenckii, which lives in soil and decaying plant material. Most of those who contract the disease are gardeners, florists, or farmers. The fungus is acquired through a scratch or puncture wound. The lymphocutaneous form constitutes two thirds to three fourths of all cases. A small ulcerated papule develops at the site of inoculation and similar lesions appear along lymphoid channels draining the original lesion area. Lymph nodes are not involved. The classic case is a person who does gardening and has contact with roses who develops a small ulceration on one arm followed by others in a linear ascending distribution. In children it is found as frequently on the body or face as on the extremities. Other than the lesions the patient usually has few symptoms.

    The major diagnostic tests are culture of the lesions, biopsy, and serologic tests. The LA test, tube agglutination, and immunofluorescent test on serum are the most sensitive (±90% detection rate). CF tests detect approximately 65% of cases.

  • Lyme Disease

    Lyme disease is caused by the spirochete Borrelia burgdorferi by means of several tick vectors, the principal one in the Northeast and North Central United States being the deer tick Ixodes dammini and in the Pacific Coast states, Ixodes pacificus, the Western black-legged tick (both morphologically “hard” ticks). The three major affected areas in the United States are the northeastern states (New Jersey to Connecticut), the far western states, and the upper midwestern states. However, cases have been reported elsewhere and also in Canada, Europe, and Australia.

    Ixodes dammini has a 2-year, three-form life cycle. The very young ticks (called larval stage, although the organism has a tick shape) feed on a vector organism, usually the white-foot mouse, and then are dormant until the following spring. The larval ticks are very small and have only three pairs of legs, like insects. The following year in the spring the larval tick changes to the nymph stage, which has four pairs of legs like the adult stage.

    In 50%-80% of patients, about 1 week (range, 3-68 days) after the tick bite, a reddish macular expanding lesion with central clearing (“erythema chronicum migrans”) develops on the skin at the inoculation site often followed by similar skin lesions in some other areas. This usually fades within 2-3 weeks (range, 1 day-4 weeks) and is usually accompanied by low-grade fever, weakness, fatigue, and regional lymphadenopathy. Although this characteristic skin lesion should strongly suggest Lyme disease, only 20%-30% of patients recall such a lesion. Migratory arthralgias and myalgia are frequently present. About 10% of patients develop anicteric hepatitis. In the second stage of illness, CNS (most often aseptic meningitis) or peripheral nervous system abnormalities (Bell’s palsy or Bannwarth’s polyneuritis syndrome) occur about 4 weeks (range, 2-8 weeks) after the tick bite in about 15%-20% of patients (range, 11%-35%). About 7% (range, 4%-10%) of patients develop transitory ECG abnormalities or myocardial inflammation, usually about 5 weeks after the tick bite (range, 4 days-7 months). In the third stage of illness, about 40% (range, 26%-60%) of patients develop recurrent arthritis. This is the most famous manifestation of Lyme disease and involves one or more joints, most commonly the knee, beginning about 6 weeks-6 months after the tick bite (range, 4 days-2 years).

    Laboratory test abnormalities include elevated erythrocyte sedimentation rate in about 50% of cases. Peripheral blood WBCs are elevated in only about 10%; fluid aspirated from arthritic joints is similar to that from patients with rheumatoid arthritis. CSF in patients with meningeal or peripheral nerve symptoms usually show increased numbers of WBCs with lymphocytes predominating, normal glucose and mildly increased protein levels, oligoclonal bands similar to those of multiple sclerosis, and CSF-IgM antibody present.

    Culture can be done from biopsy of the erythema migrans (ECM) skin lesion; best results are obtained from the advancing edge of the lesion. Transport of the specimen and specimen culture in the same special BSK culture media plus incubation for several weeks if necessary has produced best results; but even so the spirochetes were isolated in less than 45% of cases (range, 5%-71%). Warthin-Starry silver stains on ECM lesion biopsy demonstrates spirochetes in less than 40% of cases. Blood cultures may be positive in the second stage of illness but only in 2%-7% of cases and therefore is not cost-effective. Culture of CSF in second-stage symptomatic patients may be positive in about 10% of patients. DNA probes with PCR amplification have been reported to have a sensitivity of 80% when performed on a biopsy of the ECM skin lesion, the same or better than the best culture results. However, thus far, DNA probe for Borrelia antigen in blood has not substantially improved serologic test results.

    Currently, the most helpful procedures are serologic tests. IgM antibody levels rise about 2-4 weeks after onset of ECM, peak about 6-8 weeks after ECM onset, and usually become nondetectable by 4-6 months after onset. However, some patients have persistent IgM levels, presumably due to continued infection or reinfection. IgG antibody levels rise about 6-8 weeks after onset of erythema migrans and peak at about 4-6 months after onset of erythema migrans, but may not peak until later or even more than a year. The highest IgG levels tend to occur when patients develop arthritis. IgG levels typically remain elevated for life. The most commonly used tests are immunofluorescent and ELISA methods. False positive results can be obtained in patients with other spirochetal diseases, such as syphilis, relapsing fever, and leptospirosis, and according to one report also in subacute bacterial endecarditis (SBE). Some of the ELISA tests attempt to adsorb out some of these antigens if they are present. Both test methods can give about the same results, although investigators generally seem to have a more favorable opinion of ELISA. In the earliest stage of the disease (ECM present 1-7 days), serologic tests are rarely positive. Later in the first stage, 3-4 weeks after onset of ECM, the tests are positive in about 40% of patients. In the second stage of illness (coincident with systemic symptoms) about 65% are positive, and in the third (arthritic) stage, about 90%-95% (range, 80%-97%) are positive. This suggests that negative serologic tests in clinical stages one and two may have to be repeated 3-4 weeks later. ELISA tests using recombinant flagellar proteins as antigen somewhat improve IgM test specificity and may increase sensitivity a little in early disease compared to ELISA tests using whole organism alone. Sensitivity of IgG antibody is significantly greater than IgM in the second and third stages of Lyme disease because disseminated (second stage) infection raises IgG more than IgM (which has already peaked or has already started to decline).

    Evaluation of different kits has shown considerable variation in sensitivity and specificity between different kits, between laboratories, and even between evaluations in the same laboratories when the same specimen was repeated later. Western blot testing is commercially available or performed with homemade reagents. This has the advantage of visually identifying which proteins are reacting to patient antibodies. Unfortunately, there still is little agreement how to interpret the Lyme Western blot test. Some of the proteins that are rather frequently detected are shared with other organisms. Some of the more specific proteins (outer coat proteins A and B) may not appear until relatively late in some patients. Nucleic acid probe testing has recently been reported, with or without PCR amplification, mostly using homemade reagents. Although results have been more sensitive than some standard ELISA or fluorescent antibody kits, DNA probes so far have not increased usable sensitivity as much as has been achieved in some other diseases. Finally, some studies have reported that some patients with Lyme disease have a reactive antinuclear body (ANA) test, usually the speckled type. One report found that the VDRL or RPR test for syphilis is usually nonreactive.

    In one report from a Lyme disease referral center, of 788 patients with positive serologic test results for Lyme disease, 23% had active Lyme disease, 20% had previous Lyme disease, and 57% were judged not to have evidence of Lyme disease.

  • Serologic Tests

    In many cases, direct detection methods are not possible, are difficult and expensive, are unreliable, or are attempted with negative results. Serologic tests attempt to detect antibodies formed against antigens of an organism being searched for. The majority of organisms have a reasonably predictable antibody response. IgM-type antibodies appear first, most often in 7-14 days (sometimes later), generally reaching peak titer about 2 weeks later, then falling, and becoming nondetectable about 6 months later (usual range, 4-8 months, although sometimes longer). IgG-type antibodies typically appear about 2 weeks after initial appearance of IgM antibodies, peak about 2 weeks later, and typically persist for years (although some eventually disappear and others may slowly decrease in titer and persist at low titers). If antibodies to a particular organism are rare in a community, a single significantly elevated result can be at least presumptively diagnostic. However, most often it requires two serum specimens, one obtained immediately and the other 2-3 weeks later, hoping to obtain a fourfold (2-tube serial dilution) rise in titer in order to prove current or recent infection. Potential problems include circumstances in which serologic tests for certain organisms are not available, tests ordered to detect one organism when another is the culprit, patients who fail to produce detectable antibody, patient antibody formation that varies in time from the usual pattern, serum specimens obtained before antibody rise or after antibody becomes nondetectable, and specimens obtained after antibody has peaked so that past infection cannot be differentiated from current infection. In the last of these circumstances, presence of IgM antibody in high titer would suggest current or relatively recent infection. Finally, there is substantial variation in homemade or commercially available test kits, both in ability to detect the desired antibodies (sensitivity) and the number of false positive results obtained (specificity).