Articles on Medical Diseases and Conditions

Month: July 2009

  • 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.

  • Dermatophytes

    The dermatophytes include a number of fungi that attack the nails and skin. A presumptive etiologic diagnosis may be made by examining scrapings of the affected area microscopically in a wet mount of 10% KOH. Calcofluor white or similar fluorescent methods enhance detection. Definitive diagnosis is by culture, usually on an all-purpose medium such as Sabouraud’s agar, although some special media are available.

  • Fungal Cultures

    When fungal culture is indicated, the laboratory must be notified, because bacterial culture media are not generally suitable for fungi. All-purpose mycotic culture media such as Sabouraud’s agar under aerobic conditions are satisfactory for most of the systemic fungi.

  • Opportunistic Fungi

    Other fungi may, under certain circumstances, produce visceral or systemic infection. The most common of these conditions are candidiasis, aspergillosis, and mucormycosis. Persons predisposed to infection include aged persons, cachectic or debilitated patients, persons with diseases such as AIDS or leukemia that affect the body’s immunologic mechanisms, and, most commonly, persons under treatment with certain drugs that impair the same immunologic mechanisms. Such drugs include many types of antileukemic or anticancer chemotherapeutic agents and sometimes adrenocortical steroids if use has been heavy or prolonged. Occasionally, overgrowth of Candida may be caused by prolonged oral antibiotic therapy that destroys normal gastrointestinal tract bacterial flora. Candida fungemia may be associated with indwelling intravenous (IV) catheters. Diagnosis in many of these patients is difficult, since the original underlying disease usually overshadows advent of the fungal infection. If the patient has a condition that predisposes to infection by fungi, culture of a fungus from an area where it is normally absent should not be disregarded as mere contamination but should be investigated further.

    Candida infections

    Candida organisms may be present as either normal nasopharyngeal area or gastrointestinal tract (GI) inhabitants (20%-40% of persons); as colonization without infection (10%-15% of vaginal cultures in asymptomatic women, with a range of 0%-50%); as superficial infection (e.g., the oral cavity or the vaginal area); or as deep or disseminated infection. On the other hand, in one study of Candida GI infection, only 75% had positive stool cultures. Some species are more often pathogenic than others. Of these, C. albicans is by far (up to 90%) the most frequent and important. Serious C. albicans infections (including fungemia) are associated with diabetes, extensive surgery, AIDS and other conditions (e.g., leukemia and lymphoma) producing decreased immunologic resistance, chemotherapy or immunosuppressive therapy, and also in some patients on hyperalimentation or following antibiotic therapy. Localized skin, oral mucous membrane (thrush), or vaginal infection by C. albicans (Monilia) is a fairly common fungus problem, associated with the same conditions as the more serious infections. Other Candida species of importance are C. tropicalis and C. parapsilosis. C. tropicalis infections are next in frequency to those of C. albicans, and are associated with bone marrow transplants, leukemia, and lymphomas, and with fungemia due to colonized venous catheters or with hyperalimentation (although C. albicans is still the most frequent organism). C. parapsilosis frequently colonizes the skin and grows especially well in glucose-containing solutions. It is most frequently associated with narcotic addiction and with hyperaimentation. C. krusei and C. guilliermondii are starting to appear in immunocompromised patients.

    Diagnosis consists of wet-mount or stained slide preparations from accessible areas (which provide a presumptive diagnosis only), culture, and serologic tests. A potassium hydroxide (KOH) wet preparation detects Candida in about 60%-70% of vaginal KOH preparations (40%-85% of all KOH cases, depending on number of organisms present, compared to culture). One report found that nonmicrobiologists could detect only about 20%. Use of fluorescent stains such as Calcofluor white could increase detection rates. A new slide LA test for Candida antigen in vaginal specimens has recently become available through at least 3 companies, with 47%-80% sensitivity. Another LA test had 53% sensitivity. Papanicolaou-stained slides are reported to detect about 45%-50% of vaginal candidiasis.

    Various serologic tests for antibody are available. The serum LA procedure is the easiest to perform. The one most often evaluated is called Cand-Tec. It has a reported sensitivity of 50%-60% (range, 30%-94%). Immunosuppressed patients tend to have lower detection rates. An IgG EIA procedure was said to have 82% sensitivity. Several tests for serum mannan, a Candida cell wall antigen, have reported sensitivity of 40%-60% (range, 29%-84%) in invasive candidiasis. At present, none of the commonly used serologic tests will reliably differentiate between superficial infection (or colonization) and deep infection, and neither will cultures from accessible specimen sites. Isolation of Candida from both blood and urine culture, however, is very suggestive of disseminated candidiasis. However, blood cultures are positive in only about 40% of cases (range, 32%-50%).

    Torulopsis glabrata is a species from the genus Torula that clinically resembles Candida in many respects. It can be found normally in the human mouth, GI tract, urinary tract, and respiratory tract. As a pathogen it is most frequently found in urine. In some hospitals it may surpass C. tropicalis in frequency and is isolated under essentially the same conditions (associated with IV catheters, antibiotic therapy, Foley catheters, and extensive surgery). Pathogenicity is considered to be less than that of Candida, so that isolation may or may not be clinically significant. In one study of fungal vaginitis, C. albicans was isolated in 81% of cases and T. glabrata was second with 16%.

    Aspergillus

    Aspergillus is a fungus with branching septated (bamboo-like) hyphae present normally in soil and different vegetation. In patients, A. fumigatus is most frequently isolated, followed by A. flavus and A. niger. The organism spreads through dust or airborne spores; therefore, human entry involves the respiratory tract. There is widespread exposure in the environment; but with the exception of some individuals with allergy, persons with normal immune systems usually do not develop Aspergillus-related disease. About 5%-10% of hospitalized patients have nasopharyngeal colonization. Aspergillus-related medical problems may occur in several forms.

    1. Reactive allergic disease. This is caused by allergy to inhaled Aspergillus organisms without Aspergillus colonization or infection.
    2. Allergic bronchopulmonary aspergillosis. This is usually associated with preexisting asthma and is a type of hypersensitivity lung disease. After inhalation, Aspergillus produces a localized small infection of the respiratory tract that is a source of antigen to an already sensitized person. Affected patients all have a history of asthma that becomes much worse. There frequently are low-grade fever and chest x-ray abnormalities of various types. However, the chest film may be normal.
    3. Aspergilloma. This is a “fungus ball” type of localized infection within cystic spaces of the lung due to preexisting chronic lung disease. About 85% are located in the upper portions of the lungs. Chronic tuberculosis is the most frequent precursor (25%-72% of aspergilloma cases). An aspergilloma may be asymptomatic or may result in hemoptysis (50%-80% of cases). Cough and sputum production is also common.
    4. “Invasive” aspergillosis. This may be localized, usually in the lung, or disseminated. Invasive aspergillosis is usually associated with patients with leukemia or lymphoma, patients with solid tumors undergoing chemotherapy, or patients who are immunosuppressed. The patients frequently have neutropenia. The same group of patients have a markedly increased incidence of Candida infection.

    The lungs and skull sinuses are frequently involved infection sites. Contaminated air conditioning systems or air ducts have often been implicated as the source of Aspergillus infection. Diagnosis requires the same procedures that were described for candidiasis. Demonstration of Aspergillus in sputum is more alarming than finding Candida. However, colonization rather than infection may be present. In one series of patients, one third of those with Aspergillus in the sputum failed to yield evidence of Aspergillus infection. A. fumigatus and A. flavus are much more likely to be respiratory tract pathogens than the other species of Aspergillus. On the other hand, in invasive pulmonary aspergillosis, only 13%-34% produced sputum that grew Aspergillus. Biopsy of lesions with culture or tissue examination are the best ways to make a definitive diagnosis. Aspergillus typically invades blood vessel walls in tissue specimens. If biopsy material is obtained, special stains are usually needed to find and identify the organism. Various serologic test procedures have been reported, but the ones most commonly used are some type of agar gel diffusion (AGD) or ELISA. In well-established Aspergillus infections, AGD detects about 65% and the ELISA about 80%-90% (range, 57%-100%). False positive results have been reported in about 3% of the general population and in up to 25% of patients with asthma. A. fumagatus specific IgE antibody detected by ELISA or radioimmunoassay methods is reported to be present in about 85% of patients with allergic broncho-pulmonary aspergillosis but less than 20% of those with aspergilloma or Aspergillus-associated asthma. A skin test for Aspergillus that gives results similar to those of the serologic tests, including the false positive reactions, is available. A nucleic acid probe with PCR amplification has recently been reported.

    In patients with aspergilloma, helpful laboratory findings include eosinophils in sputum and a peripheral blood eosinophilia. In classic cases the sputum contains golden brown plugs that contain Aspergillus hyphae. In one series, Aspergillus hyphae were seen microscopically in 40%, and sputum culture was positive in about 55%. Fiberoptic bronchoscopy is reported to increase the isolation rate (55%-100%). Results of skin tests and serologic tests are usually positive, as noted previously, but results of these tests may be positive without evidence of aspergillosis in persons with asthma.

    Mucormycosis

    Mucormycosis (zygomycosis) is usually caused by saprophytic bread mold fungi with nonseptate hyphae of the genus Mucor or Rhizopus. Infection is characteristically associated with diabetic ketoacidosis and is also more common in patients with leukemia or lymphoma and immunocompromised persons. Infection is most common in the nasopharynx region and paranasal sinuses. There may be spread to the brain or blood-borne dissemination. Diagnosis is through culture and biopsy.

  • 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.

  • Other Rickettsial Diseases

    Bartonellosis (Oroya fever or Carrion’s disease) is caused by the rickettsial organism Bartonella bacilliformis and occurs only in Andean mountain regions of South America. The vector is the sandfly Phlebotomus (species). The organism is related to Rochalimaea quintana, the rickettsial etiology of trench fever and therefore is also related to Rochalimaea henselae, one of the etiologies of cat scratch fever.

    Q fever is a rickettsial disease whose etiology is Coxiella burnetii. Goats and cows are the major vectors; infection occurs through exposure to contaminated milk or animal placentas, or may occur by inhalation of infected material. The organism is very resistant to drying, so that dust inhalation can spread the disease in dry areas. The incubation period is 2-4 weeks. Clinical disease is similar to moderately severe influenza. Infection usually (not always) does not produce a rash, which is unusual among the rickettsiae. Liver function tests are abnormal in 80% of patients, but only to a small degree. Diagnosis is usually by immunofluorescent serologic tests, generally performed in large reference laboratories or public health laboratories. The Weil-Felix test panel is nonreactive.

    Ehrlichiosis is a rickettsial disease caused by Ehrlichia chaffeensis. The disease is spread by a tick vector and is clinically similar to Rocky Mountain Spotted Fever, except that only about 20% of patients develop a rash. Over half of Ehrlichiosis patients develop some degree of leukopenia (74%-85%) or thrombocytopenia (72%-84%), and mild aminotransferase enzyme level elevations (78%-100%) are common. Usually the disease is not severe. About one third of patients have symptoms that raise the question of CNS involvement. CSF shows elevated WBC count in 67%-71% of cases, with lymphocytes predominating in one third of cases. Total protein is elevated in 33%-62% of cases. There is also a canine Ehrlichiosis caused by a related but not identical tick-borne Rickettsia. In fact, for several years the usual diagnostic test for human Ehrlichiosis was an immunofluorescent test based on cross-reaction with canine Ehrlichiosis organisms. Now indirect immunofluorescent assays (IFA) are available that are specific for E. chaffeensis and that are being done in some large reference laboratories or public health laboratories. Acute and convalescent serum specimens may be required to document active or recent infection (vs. old infection).

  • Rickettsial Diseases

    The rickettsiae to some extent resemble small bacteria but are not stained with Gram stain and cannot be cultured on artificial media. These organisms are spread only by insect vectors that have fed on blood from a patient with the disease, not from personal contact with a patient. Blood cultures can sometimes isolate and identify rickettsiae, especially in Rocky Mountain spotted fever. The blood specimens should be frozen and sent to the laboratory packed in dry ice. Since artificial culture media are not available, chick embryo or live animal inoculation must be done. Very few laboratories will perform rickettsial culture. Therefore, serologic tests or other procedures by far overshadow culture as diagnostic aids. The most commonly used procedure is the Weil-Felix reaction. This test takes advantage of the fact that certain rickettsial diseases produce antibodies that also react (or cross-react) with antigen contained in certain strains of Proteus bacteria. These Proteus groups are called OX-19 and OX-K. Titers of 1:80 are suspicious, and titers of 1:160 are definitely significant. Antibodies appear 7-10 days after onset of illness. Rickettsial diseases that may be diagnosed by means of the Weil-Felix reaction are the following:

    Rickettsial Diseases

    Unfortunately, there are serious limitations to the Weil-Felix test. First, there are a fairly large number of borderline false positive results as well as occasional outright false positives. Since the Weil-Felix reaction depends on Proteus antigen, urinary tract infection by Proteus should be ruled out if the Weil-Felix test result is positive. Second, about two thirds of patients with Rocky Mountain spotted fever or Brill’s disease (recrudescent typhus) have false negative reactions. In these two diseases, other serologic tests (e.g., the microimmunofluorescence procedure) are preferred to the Weil-Felix test.

    Rocky Mountain spotted fever

    Rocky Mountain spotted fever occurs in spring and summer, predominantly in the eastern two thirds of the United States (the area east of the Rocky Mountains). The disease is transmitted through the bite of an infected tick, with an incubation period of 3-12 days. Fever, severe frontal headache, severe myalgias, and a macular or maculopapular skin rash that develops on the third to fifth day and eventually develops a petechial component are the most characteristic clinical findings, with the rash typically involving the palms and soles. Nausea, vomiting, and diarrhea are reported in about 60% of patients and abdominal pain is reported in about 35%. The WBC count is usually normal, but thrombocytopenia is said to be common. In severe cases disseminated intravascular coagulation may develop. Serologic tests include the Weil-Felix test, CF test, microimmunofluorescence (MIF) test, latex agglutination (LA) test, and tissue biopsy with immunofluorescent staining. The Weil-Felix test has very poor sensitivity and is not specific, and the antibody does not develop until some time between 7-21 days after infective contact (compared with incubation period of 3-12 days). Complement fixation is more specific but detects 50% or fewer cases and is very laborious to perform, the antibody does not appear until 14-21 days after infection, and antibody elevations persist for years. Antibodies detected by the MIF and LA tests begin to appear 7-10 days after onset of illness. These tests are reported to detect considerably more than one half of Rocky Mountain spotted fever cases but are available only in reference laboratories or public health laboratories. The MIF test is currently considered the gold standard. LA is reported to detect about 80%-85% of patients detected by MIF. Skin biopsy of a lesion with immunofluorescent antirickettsial antibody studies on the tissue can also be done but the degree of sensitivity is controversial (50%-70%) and the test is not widely available.

    Cat scratch disease

    This condition has two possible etiologies. One is a small bacillus that stains faintly gram-negative, and the other is a member of the Rickettsial family. About 80% of patients affected are children (about 90% are less than 18 years old and about 70% are less than 10 years old). About 85% (range, 83%-90%) have a history of contact with cats, most of which (about 80%) are kittens or less than 1 year old. Cat inoculation via scratch (57%-83% of patients), bite, or lick is followed in 4-14 days (range, 3-50 days) by development of a primary inoculation site pustule or papule in 54%-93% of patients. The primary lesion persists 1-3 weeks but can persist up to 1-3 months. Regional lymphadenopathy develops in 1-3 weeks after appearance of the primary site lesion; the nodes are unilateral in about 90% (range, 85%-95%) of patients and are tender in about 80%. Cat scratch disease is possibly the most common cause of chronic lymphadenopathy in children. The node or nodes are located in the head and neck area in about 25% of cases; the axilla (occasionally the epitrochlear area) in about 45%, and the groin in about 20%. They generally regress in size in 2-4 months but can persist longer. Adenopathy is accompanied by fever and malaise in about 35% of patients (range, 30%-59%), but the fever is usually (90%) less than 102°F (38.9°C). The lymph nodes suppurate in 10%-30% of cases and may drain in 10%-20% of cases. Parinaud’s oculoglandular syndrome (unilateral conjunctivitis with regional lymphadenitis) is present in 4%-6% of patients. The lymph nodes often raise the clinical question of mycobacterial infection and sometimes of malignant lymphoma. Biopsy specimens display a characteristic lesion consisting of lymphocytes and also histiocytes with scanty cytoplasm surrounding a central area containing segmented neutrophils that usually becomes necrotic. The lesions may be single or can be stellate (branching) due to coalescence of the original lesions. The same histologic pattern can be found in tularemia and in lymphogranuloma venereum. It is extremely difficult to find the organisms by Gram stain, and more success is obtained with the Warthin-Starry silver stain. Interestingly, the more commonly used silver stain methods such as methenamine silver are not useful. Even with Warthin-Starry, the organisms may be difficult to find or may be absent. The best areas are the center of suppurative granulomas. A skin test has been described using antigen derived from lymph node pus, but the antigen is not readily available.

    In 1988, scientists at the Armed Forces Institute of Pathology (AFIP), using special media, cultured a gram-negative rod from several patients with cat scratch disease which was subsequently given the name Afipia felis. However, only a few years later it was found that considerably more patients with cat scratch disease had antibodies to a rickettsial organism named Rochalimaea henselae than evidence of Afipia felis infection. Later, R. henselae was isolated by culture from several patients with cat scratch disease and now appears to be the most frequent cause. Both organisms can be isolated using chocolate agar in 5% CO2 atmosphere; but growth may take up to 6 weeks. Both organisms also grow in Haemophilus test medium.

    The Rochalimaea genus includes two species, Rochalimaea quintana (etiology of trench fever) and R. henselae. R. henselae is now considered the etiologic agent of several conditions. One is bacillary angiomatosis, which is focal proliferation of small blood vessels, endothelial cells, and connective tissue cells (somewhat similar in appearance to Kaposi’s sarcoma), and which is found predominantly in the skin but sometimes in the liver or spleen, most often in patients with HIV-1 infection. R. quintana can also cause these lesions. Another condition is peliosis hepatis (multicystic angiomatoid structures with endothelium not seen by ordinary microscopy; usually in the liver). It also causes occasional cases of septicemia, most often in HIV-infected patients, and is one cause of cat scratch fever. The organism is a small gram-negative curved bacillus that requires special culture conditions for isolation. Diagnosis can be made by Warthin-Starry silver stains from tissue biopsies and culture on special media noted previously. There have been two published reports of a fluorescent antibody test for R. henselae and for A. felis. The sensitivity of these tests is not yet established. Nucleic acid probe methods have been used experimentally and will probably also become commercially available in the future, although most likely in many patients the disease will be treated empirically.

  • Vincent’s Angina

    Vincent’s angina (Vincent’s infection) is an infection of the mouth caused by an interesting synergistic group of organisms, including anaerobic streptococci, a fusiform gram-negative bacillus, and a spirochete. Gram-stained smears demonstrating all three organisms are usually sufficient for diagnosis.

  • Leptospirosis

    Leptospirosis is caused by several species of Leptospira organisms found most often in rats but sometimes present in some farm animals and in some cats and dogs (presumably from rat-transmitted infection). Transmission is most often through accidental contact with water contaminated by infected rat urine. Those most at risk are sewer workers and slaughterhouse employees, but farmers and campers sometimes come into contact with contaminated water. There is an incubation period of 4-20 days, then abrupt onset of fever, often accompanied by chills, headache, malaise, and conjunctivitis. Muscle pain is present in 50% of cases. The fever typically lasts 4-9 days. WBC count can be normal or elevated. Urinalysis often contains protein and some WBCs. Serum bilirubin is usually normal, but about 10% of cases have mild elevation. Alanine aminotransferase is elevated in about 50% of cases, usually to less than five times normal. About 50% of patients experience a recurrence of fever about 1 week (range, 2-10 days) after the end of the first febrile period. Patients are more likely to demonstrate signs of hepatitis in this phase and may develop symptoms of meningitis. The most severe form of leptospirosis is called Weil’s disease and occurs in about 5% of infections. The most striking findings are a combination of hepatitis and glomerulonephritis, clinically manifested by jaundice with hematuria. Therefore, the disease is sometimes considered in the differential diagnosis of jaundice of unknown etiology. Symptoms of meningitis occasionally predominate. Laboratory findings include leukocytosis with a shift to the left. A mild normocytic-normochromic anemia usually develops by the second week. Platelet counts are normal. After jaundice develops, liver function test results are similar to those in viral hepatitis. After onset of kidney involvement, the blood urea nitrogen (BUN) level is often elevated, and hematuria is present with proteinuria. CSF examination shows normal glucose levels but increased cell count, which varies according to the severity of the case; initially, these are mainly neutrophils, but later, lymphocytes predominate. Cultures on ordinary bacterial media are negative.

    Diagnosis often requires isolating the organisms or demonstrating specific antibodies in the serum. During the first week (days 1-8), spirochetes may be found in the blood by dark-field examination in about 8% of cases and can be cultured from the blood in many more. Instead of ordinary blood cultures, one to three drops of blood are inoculated into a special culture medium (Fletcher’s), since larger quantities of blood inhibit the growth of leptospires. The CSF may be cultured toward the end of the first week. During the second week the blood results quickly become negative. During the third week (days 14-21) the spirochetes may often be recovered from the urine of patients with nephritis. Animal inoculation is the most successful method. Antibodies start to appear at about day 7 and are present in most cases by day 12. Antibodies persist for months and years after cure. A titer of 1:300 is considered diagnostic, although without a rising titer, past infection cannot be ruled out completely. If a significant titer has not developed by day 21, it is very rare for it to do so later. In summary, blood cultures during the first week and serologic tests during the second and third weeks are the diagnostic methods of choice.