Articles on Medical Diseases and Conditions

Tag: Culture

  • Acute Rheumatic Fever (ARF)

    ARF is a disease that has a specific etiologic agent yet has some similarities to the rheumatoid-collagen-vascular group. The etiologic agent is the beta-hemolytic Lancefield group A Streptococcus. Apparent hypersensitivity or other effect of this organism causes connective tissue changes manifested by focal necrosis of collagen and the development of peculiar aggregates of histiocytes called “Aschoff bodies.” Symptoms of ARF include fever, a migratory type of polyarthritis, and frequently cardiac damage manifested by symptoms or only by electrocardiogram (ECG) changes. Diagnosis or confirmation of diagnosis often rests on appropriate laboratory tests.

    Culture. Throat culture should be attempted; the finding of beta-hemolytic streptococci, Lancefield group A, is a strong point in favor of the diagnosis if the clinical picture is highly suggestive. However, throat cultures often show negative results by the time ARF symptoms develop, and a positive throat culture is not diagnostic of ARF (since group A streptococci may be present in 15%-20% of clinically normal children). Blood culture findings are almost always negative.

    Streptolysin-O tests. Beta streptococci produce an enzyme known as streptolysin-O. About 7-10 days after infection, antibodies to this material begin to appear. The highest incidence of positive results is during the third week after onset of ARF. At this time, 80%-85% (range, 45%-95%) abnormal results are obtained; thereafter the antibody titer drops steadily. At the end of 2 months only 70%-75% of test results are positive; at 6 months, 35%; and at 12 months, 20%. Therefore, since the Streptococcus most often cannot be isolated, antistreptolysin-O (ASO) titers of more than 200 Todd units may be helpful evidence of a recent infection. However, this does not actually prove that the disease in question is ARF. Group A streptococcal infections are fairly frequent, so that occasionally a group A infection or the serologic effects of such an infection may coexist with some other arthritic disease. Another problem with ASO elevation is that the elevation persists for varying periods of time, raising the question whether the streptococcal infection that produced the antibodies was recent enough to cause the present symptoms. Commercial tests vary somewhat in reliability, and variations of 1 or 2 dilutions in titer on the same specimen tested by different laboratories are not uncommon.

    Antibodies against other streptococcal enzymes. Commercial slide latex agglutination tests that simultaneously detect ASO plus several other streptococcal antibodies, such as antideoxyribonuclease-B (AND-B) are available. The best known of these multiantibody tests is called Streptozyme. Theoretically, these tests ought to be more sensitive for detection of group A beta-hemolytic streptococcal infections than the streptolysin-O test alone, since patients who develop acute glomerulonephritis are more apt to produce antibodies against AND-B than streptolysin-O, and the antibodies against streptococcal enzymes may be stimulated unequally in individual patients. However, there is considerable debate in the literature on the merits of the combination-antibody tests versus the single-antibody tests. The American Heart Association Committee on Rheumatic Fever published their opinion in 1988 that Streptozyme gave more variable results than the ASO method and therefore was not recommended. If both the streptolysin-O test plus the AND-B test are performed, the combined results are better than either test alone and even a little better than the single combination-antibody slide test. However, relatively few laboratories perform the AND-B test, and even fewer routinely set up both the ASO test plus the AND-B test in response to the usual order for an ASO titer.

    Diagnosis. When a patient has an acute-onset sore throat and the question involves etiology (group A Streptococcus vs. some other infectious agent), throat culture is the procedure of choice, because it takes 7-10 days before ASO antibody elevation begins to occur. On the other hand, ARF and acute glomerulonephritis develop some time after the onset of the initiating streptococcal infection. The average latent period for ARF is 19 days, with a reported range of 1-35 days. Therefore, the ASO (or Streptozyme and its equivalents) is more useful than throat culture to demonstrate recent group A streptococcal infection in possible ARF or acute glomerulonephritis.

    Other tests. The ESR is usually elevated during the clinical course of ARF and is a useful indication of current activity of the disease. However, the ESR is very nonspecific and indicates only that there is an active inflammatory process somewhere in the body. In a minority of ARF patients, peculiar subcutaneous nodules develop, most often near the elbows. These consist of focal collagen necrosis surrounded by palisading of histiocytes. In some cases, therefore, biopsy of these nodules may help confirm the diagnosis of ARF. However, biopsy is not usually done if other methods make the diagnosis reasonably certain. Also, the nodules are histologically similar to those of RA. During the acute phase of the disease there usually is moderate leukocytosis, and most often there is mild to moderate anemia.

  • Cerebrospinal Fluid Examination and Neurologic Disorders

    Pressure

    Reference values for cerebrospinal fluid (CSF) pressure are 100-200 mm H2O. Elevations are due to increased intracranial pressure. The two most common causes of elevated CSF pressure are meningitis and subarachnoid hemorrhage. Brain tumor and brain abscess will cause increased intracranial pressure in most cases but only after a variable period of days or even weeks. An increase is present in many cases of lead encephalopathy. The CSF pressure varies directly with venous pressure but has no constant relationship to arterial pressure. The Queckenstedt sign makes clinical use of this information; increased venous pressure via jugular vein compression increases CSF pressure at the lumbar region, whereas a subarachnoid obstruction above the lumbar area prevents this effect.

    Appearance

    Normal CSF is clear and colorless. It may be pink or red if many red blood cells (RBCs) are present or white and cloudy if there are many white blood cells (WBCs) or an especially high protein content. Usually there must be more than 400 WBCs/mm3 (0.4 Ч 109/L) before the CSF becomes cloudy. When blood has been present in the CSF for more than 4 hours (literature range, 2-48 hours), xanthochromia (yellow color) may occur due to hemoglobin pigment from lysed RBCs. Protein levels of more than 150 mg/100 ml (1.5g/L) may produce a faint yellowish color that can simulate xanthochromia of RBC origin. Severe jaundice may also produce coloration that simulates xanthochromia.

    Glucose

    Reference values are 45 mg/100 ml (2.5 mmol/L) or higher (“true glucose” methods). Values of 40-45 mg/100 ml are equivocal, although in normal persons it is rare to find values below 45 mg/100 ml. The CSF glucose level is about 60% of the serum glucose value (literature range, 50%-80%). In newborns, the usual CSF level is about 80% of the serum glucose level. It takes between 0.5 and 2 hours for maximum change to occur in CSF values after a change in serum values.

    The most important change in the CSF glucose level is a decrease. The classic etiologies for CSF glucose decrease are meningitis from standard bacteria, tuberculosis, and fungi. Occasionally in very early infection the initial CSF glucose value may be normal, although later it begins to decrease. A frequent impression from textbooks is that one should expect a decreased CSF glucose value in nearly all patients with acute bacterial meningitis. Actually, studies have shown that only 60%-80% of children with acute bacterial meningitis have CSF glucose levels below the reference range. Studies on patients of various ages have shown decreased glucose levels in 50%-90% of cases. A major problem is the effect of blood glucose levels on CSF glucose levels. Since elevated blood glucose levels may mask a decrease in CSF values, it is helpful to determine the blood glucose level at the same time that the CSF specimen is obtained, especially in diabetics or if intravenous (IV) glucose therapy is being given. On the other hand, a low CSF glucose level may be due to peripheral blood hypoglycemia, especially if the CSF cell count is normal. Other conditions that may produce a decrease in CSF glucose level are extensive involvement of meninges by metastatic carcinoma and in some cases of subarachnoid hemorrhage, probably due to release of glycolytic enzymes from the RBCs. In leptospiral meningitis and in primary amebic meningoencephalitis, the CSF glucose level is decreased in some patients but not in others. In most other central nervous system (CNS) diseases, including viral meningitis, encephalitis, brain abscess, syphilis, and brain tumor, CSF glucose levels typically are normal when bacterial infection of the meninges is not present. However, decreased levels are sometimes found in aseptic meningitis or in meningoencephalitis due to mumps, enteroviruses, lymphocytic choriomeningitis, and in both herpes simplex virus types 1 and 2 CNS infections. For example, one investigator reports that 10% of cases of meningitis due to enterovirus had decreased CSF glucose. The box summarizes glucose level patterns.

    Protein

    The normal protein concentration of CSF is usually considered to be 15-45 mg/100 ml (0.15-0.45 g/L) (literature range, 9-90 mg/100 ml; 0.09-0.9 g/L). There is considerable discrepancy in the literature regarding the upper reference limit, which is most often listed as 40, 45, and 50 mg/100 ml (international system of units [SI] 0.4, 0.45, 0.5 g/L). Newborn values are different and even more uncertain. From birth to day 30, the range is 75-150 mg/100 ml (0.75-1.50 g/L), with the literature range being 20-200 mg/100 ml and the upper limit varying from 140-200 mg/100 ml. For days 30-90, the reference range is 20-100 mg/100 ml (0.2-1.0 g/L). From day 90 to 6 months of age, the reference range is 15-50 mg/100 ml (0.15-0.50 g/L). Values slowly decline, reaching adult levels by 6 months of age. Over age 60 years, some persons increase the upper reference limit to 60 mg/100 ml (0.6 g/L). As a general but not invariable rule, an increased protein concentration is roughly proportional to the degree of leukocytosis in the CSF. The protein concentration is also increased by the presence of blood. There are, however, certain diseases in which a mild to moderate protein concentration increase may be seen with relatively slight leukocytosis; these include cerebral trauma, brain or spinal cord tumor, brain abscess, cerebral infarct or hemorrhage (CVA), CNS sarcoidosis, systemic lupus, lead encephalopathy, uremia, myxedema, multiple sclerosis (MS), variable numbers of hereditary neuropathy cases, and chronic CNS infections. Diabetics with peripheral neuropathy frequently have elevated CSF protein levels without known cause. Blood in the CSF introduces approximately 1 mg of protein/1,000 RBCs. However, when the RBCs begin to lyse, the protein level may appear disproportionate to the number of RBCs. In acute bacterial meningitis, the CSF protein is elevated in about 94% of cases (literature range, 74%-99%).

    A marked protein elevation without a corresponding CSF cell increase is known as “albuminocytologic dissociation.” This has usually been associated with the Guillain-Barrй syndrome (acute idiopathic polyneuritis) or with temporal (giant cell) arteritis. Actually, about 20% of patients with the Guillain-Barrй syndrome have normal CSF protein levels, and less than 25% have CSF protein levels of 200 mg/100 ml (2.0 g/L) or more.

    Protein may be measured in the laboratory quantitatively by any of several methods. A popular semiquantitative bedside method is Pandy’s test, in which CSF is added to a few drops of saturated phenol agent. This agent reacts with all protein, but apparently much more with globulin. Chronic infections or similar conditions such as (tertiary) syphilis or MS tend to accentuate globulin elevation and thus may give positive Pandy test results even though the total CSF protein level may not be greatly increased. Contamination by blood will often give false positive test results.

    The technical method used can influence results. The three most common methods are sulfosalicylic acid, anazolene sodium (Coomassie Blue dye), and trichloracetic acid. Sulfosalicylic acid and Coomassie Blue are more influenced by the ratio of albumin to globulin than is trichloracetic acid.

    In some CNS diseases there is a disproportionate increase in gamma-globulin levels compared with albumin or total protein levels. Several investigators have noted that increased CSF gamma-globulin levels occur in approximately 70%-85% (literature range, 50%-88%) of patients with MS, whereas total protein levels are elevated in only about 25% of the patients (range, 13%-34%). Various other acute and chronic diseases of the brain may elevate CSF gamma-globulin levels, and this fraction may also be affected by serum hyperglobulinemia. The latter artifact may be excluded by comparing gamma-globulin quantity in serum and CSF.

    The colloidal gold test also depends on changes in CSF globulins and at one time was considered very helpful in the diagnosis of MS and syphilitic tabes dorsalis. However, the colloidal gold procedure has very poor sensitivity (only 25% of MS patients display the classic first zone pattern) and poor specificity and is considered obsolete. When measurement of CSF protein fractions is ordered, the current standard procedure is some method of immunoglobulin quantitation.

    Cell count

    Normally, CSF contains up to five cells/mm3, almost all of which are lymphocytes. In newborns, the reference limits are 0-30 cells/cu mm, with the majority being segmented neutrophils. Also, one study reported at least one segmented neutrophil in 32 percent of patients without CNS disease when centrifuged CSF sediment was examined microscopically. There was correlation with elevated peripheral blood WBC count and presence of some RBCs in the sediment, presumably from minimal blood contamination of the lumbar puncture specimen not grossly evident. As a general rule, any conditions that affect the meninges will cause CSF leukocytosis; the degree of leukocytosis will depend on the type of irritation, its duration, and its intensity. Usually, the highest WBC counts are found in severe acute meningeal infections. The classic variety is the acute bacterial infection. It is important to remember that in a few cases if the patient is seen very early, leukocytosis may be minimal or even absent, just as the CSF glucose level may be normal. Although most investigators state or imply that 100% of patients with acute bacterial infection have elevated cell counts on initial lumbar puncture, in one study, 3% of patients had a WBC count less than 6 WBCs/mm3, and there have been several reports of other isolated cases. Usually, in a few hours a repeat lumbar puncture reveals steadily increasing WBC counts. Therefore, normal initial counts are not frequent but may occur and can be very misleading. Another general rule is that in bacterial infections, polymorphonuclear neutrophils usually are the predominating cell type; whereas in viral infections, chronic nervous system diseases, and tertiary syphilis, lymphocytes or mononuclears ordinarily predominate. However, there are important exceptions. One study reported that about one third of patients with usual bacterial pathogens but with CSF WBC counts less than 1,000/mm3 (1.0 x 109/L) had a predominance of lymphocytes on initial lumbar puncture (the only patient with a WBC count over 1,000 who had lymphocytosis had a Listeria infection). Another exception is tuberculous meningitis, which is both a bacterial and a chronic type of infection. In this case, the cells are predominantly lymphocytes (although frequently combined with some increase in neutrophils). A third exception is unusual nonviral organisms, such as Listeria monocytogenes (most often seen in neonates or in elderly persons or those who are immunocompromised), fungus, and spirochetes (leptospirosis and syphilis). Listeria meningitis may have lymphocytic predominance in some cases and neutrophilic predominance in others. Similarly, in active CNS syphilis the few studies available indicate that if the WBC count is elevated, lymphocytes predominate in 60%-80% of cases. On the other hand, coxsackievirus and echovirus infections may have a predominance of neutrophils in the early stages; in most of these patients, the CSF subsequently shifts to lymphocytosis. Uremia is said to produce a mild lymphocytosis in about 25% of patients. Partial treatment of bacterial meningitis may cause a shift in cell type toward lymphocytosis.

    After therapy is started, WBC values usually decrease. However, in some cases more than 50 WBCs/mm3 may persist at least 48 hours and sometimes longer than 2 weeks following adequate therapy. This tends to be more common with Haemophilus influenzae infection but may also occur with pneumococcal infection. Fungal infections are more commonly associated with persistently elevated neutrophils than bacterial infections, even though fungi typically have CSF lymphocytosis rather than neutrophilia. Nocardia meningitis or brain abscess, however, is one bacterial infection that does tend to show persistent neutrophilia more often than other bacteria.

    In cases of subarachnoid hemorrhage or traumatic spinal fluid taps, approximately 1 WBC is added to every 700 RBCs (literature range, 1 WBC/500-1,000 RBCs). This disagreement in values makes formulas unreliable that attempt to differentiate traumatic tap artifact from true WBC increase. Also, the presence of subarachnoid blood itself may sometimes cause meningeal irritation, producing a mild to moderate increase in polymorphonuclear leukocytes after several hours that occasionally may be greater than 500 WBCs/ mm3. Occasionally a similar phenomenon occurs in patients with intracerebral hematoma. Another exception is the so-called aseptic meningeal reaction that is secondary either to a nearby infection or sometimes to acute localized brain destruction. In these cases, which are not common, there may be a wide range of WBC values, with neutrophils often predominating. When this occurs, however, the CSF glucose level should be normal, since the meninges are not directly infected. Aseptic meningitis is not the same as aseptic meningeal reaction. Aseptic meningitis is due to direct involvement of the meninges by nonbacterial organisms. Viruses cause most cases, but organisms such as amebae sometimes may cause meningitis. Bacterial CSF cultures are negative. The CSF glucose level is usually normal. Protein concentration is usually but not always increased. The WBC count is elevated to a varying degree; the predominant type of cell depends on the etiology. True aseptic meningitis also has to be differentiated from bacterial organisms that do not grow on ordinary culture media (anaerobes, leptospira, mycobacteria, bacteria inhibited by previous or current antibiotic therapy, etc.).

    Neonatal CSF reference range differences from childhood and adult values

    Neonates have higher CSF reference ranges for protein, glucose, and cell count than adults have. Protein was discussed earlier. Cell counts 1-7 days after birth average about 5-20/mm3 (range, 0-32 mm3), with about 60% being segmented neutrophils. Glucose is about 75%-80% of the blood glucose level.

    Culture

    The diagnosis of acute bacterial meningitis often depends on the isolation of the organisms in the spinal fluid. In children, there is some regularity of the types of infection most commonly found. In infants under the age of 1-2 months, group B streptococci are most frequent, closely followed by Escherichia coli. Listeria monocytogenes is often listed as third, followed by other enteric gram-negative bacteria. In children from age 3 months to 5 or 6 years, H. influenzae is the most common organism; Meningococcus is second, and Pneumococcus is third. In older children and adolescents, Meningococcus is first and Pneumococcus is second. In adults, Meningococcus and Pneumococcus are still dominant, but Pneumococcus is more prevalent in some reports. Staphylococci are the etiology in about 4%-7% of cases, most often associated with CNS operations (e.g., shunt procedures), septicemia, or endocarditis. In old age, pneumococci generally are more common pathogens than meningococci, with gram-negative bacilli in third place. However, there can be infection by nearly any type of organism, including Listeria and fungi. Also, in patients who are debilitated or have underlying serious diseases such as leukemia or carcinoma, Listeria and fungi are not uncommon. In many cases, a centrifuged spinal fluid sediment can be smeared and Gram stained so that the organisms can be seen. A (bacterial) culture should be done in all cases where bacterial meningitis is suspected or is even remotely possible. Special provision should be made for spinal fluid to reach the laboratory as quickly as possible; and if any particular organism is suspected, the laboratory should be informed so that special media can be used if necessary. For example, meningococci grow best in a high carbon dioxide atmosphere, and H. influenzae should be planted on media provided with a Staphylococcus streak. Culture is said to detect about 85% (range, 54%-100%) of cases. In one series there was thought to be about 5% false positive culture results due to contamination. Previous or concurrent antibiotic therapy is reported to decrease culture detection rates by about 30% and Gram stain detection rates by about 20%.

    Patterns of Cerebrospinal Fluid Abnormality: Cell Type and Glucose Level

    POLYMORPHONUCLEAR: LOW GLUCOSE
    Acute bacterial meningitis

    POLYMORPHONUCLEAR: LOW OR NORMAL GLUCOSE
    Some cases of early phase acute bacterial meningitis
    Primary amebic (Naegleria species) meningoencephalitis
    Early phase Leptospira meningitis

    POLYMORPHONUCLEAR: NORMAL GLUCOSE
    Brain abscess
    Early phase coxsackievirus and echovirus meningitis
    CNS syphilis (some patients)
    Acute bacterial meningitis with IV glucose therapy
    Listeria (about 20% of cases)

    LYMPHOCYTIC: LOW GLUCOSE
    Tuberculosis meningitis
    Cryptococcal (Torula) meningitis
    Mumps meningoencephalitis (some cases)
    Meningeal carcinomatosis (some cases)
    Meningeal sarcoidosis (some cases)
    Listeria (about 15% of cases)

    LYMPHOCYTIC: NORMAL GLUCOSE
    Viral meningitis
    Viral encephalitis
    Postinfectious encephalitis
    Lead encephalopathy
    CNS syphilis (majority of patients)
    Brain tumor (occasionally)
    Leptospira meningitis (after the early phase)
    Listeria (about 15% of cases)

    Gram stain

    Gram stain of the sediment from a centrifuged CSF specimen should be performed in all cases of suspected meningitis. Gram stain yields about 70% positive results (literature range, 50%-90%) in culture-proved acute bacterial meningitis cases. There is some controversy over whether Gram staining need be done if the CSF cell count is normal. In the great majority of such cases bacteriologic findings are normal. However, occasional cases have been reported of bacterial meningitis without an elevated cell count, most commonly in debilitated or immunosuppressed patients. It may also occur very early in the initial stages of the disease. Gram stain yields unquestioned benefits in terms of early diagnosis and assistance in choice of therapy. However, a negative Gram stain result does not rule out acute bacterial meningitis, and some false positive results occur. The majority of false positive results are due to misinterpretation of precipitated stain or debris on the slide. In some cases the presence of bacteria is correctly reported, but the type of organism may be incorrectly identified due to overdecolorization of the organisms or insufficient familiarity with morphologic variations of bacteria.

    Latex agglutination tests for bacterial antigens

    Recently, rapid slide latex agglutination (LA) tests have become available for detection of pneumococcal, meningococcal, H. influenzae type B, and streptococcal group B bacterial antigen in CSF or in (concentrated) urine. Until the 1980s, CSF bacterial antigen detection was done by counterimmunoelectrophoresis (CIE). This method had an overall sensitivity of about 60%-70% (range, 32%-94%). The LA kits are considerably faster, easier, and have increased detection rates (about 85%-90%; range, 60%-100%). There is some variation in overall sensitivity between different manufacturers’ kits. This is especially true for antibodies against meningococci. The major reason is that several different strains of meningococci may produce infection, and it is necessary to have an antibody against each one that it is desired to detect. The most common strains are type B (about 47% of cases; range 28%-50%), type C (about 30%; range, 23%-63%), type Y (about 10%), type W135 (about 10%) and type A (about 3%). Some kits do not include antibodies against all of these strains. Besides not reaching 100% sensitivity, the LA kits are relatively expensive per patient (in part due to the multiple tests included in each kit to detect the different organisms). Besides LA, there is a commercially available kit based on a coagglutination method. This kit has an overall sensitivity about intermediate between CIE and LA, with similar results to LA for H. influenzae, and less sensitivity for pneumococci and meningococci.

    Cerebrospinal fluid lactate

    A number of published reports have evaluated CSF lactic acid assay in various diseases. In general, patients with acute bacterial, tuberculous, and fungal meningitis have elevated CSF lactate values, whereas normal persons and those with viral (“aseptic”) meningitis do not. Sensitivity of the test for acute bacterial meningitis is about 93%-95% (range, 66%-100%). Most reports indicate clear-cut separation between bacterial and viral infection values (in the sense that the values in viral meningitis are not elevated), but several investigators found that some patients with viral meningitis have elevated CSF lactate levels (about 20% of cases in these reports). However, in all instances of viral meningitis the elevation was less than twice the reference range upper limits. One advantage of CSF lactate is that it may remain elevated 2-3 days after the start of antibiotic therapy. However, values in some cases of treated or partially treated bacterial infection do return to normal relatively early.

    Xanthochromia has been reported to nonspecifically increase CSF lactate levels, although one report states that blood itself does not. CNS tissue destruction from various causes (including brain tumor, head trauma, CVAs, and intracerebral hemorrhage, cerebral hypoxia, and seizures), may also produce elevated CSF lactate levels.

    Because CSF lactate is not specific for bacterial infection, because it is not elevated in all cases of bacterial meningitis, and because there is some uncertainty as to whether lactate elevation excludes the possibility of viral meningitis, the true usefulness of the test is not clear. The availability of LA slide tests further complicates the picture. However, CSF lactate assay could be useful in patients with symptoms of meningitis if CSF Gram stain results are negative and LA test results (if available) are also negative. Such patients could have tuberculous or fungal meningitis, partially treated bacterial meningitis, or meningitis due to other organisms. Increased CSF lactic acid levels, especially if more than twice the upper reference limit, could suggest that further investigation is essential. A normal lactate level is not reliable in excluding bacterial meningitis.

    Reference range for CSF lactate is usually based on specimens from children or adults. One investigator found that neonates 0-2 days old had mean values nearly 60% higher than those obtained after 10 days of life, whereas neonates aged 2-10 days had about 25% higher levels than after age 10 days.

    Computerized tomography, magnetic resonance imaging, and radionuclide brain scanning

    Computerized tomography (CT) and magnetic resonance imaging (MRI) are now important aids in screening for abnormality in the CNS. Both can visualize the ventricular system as well as detect mass lesions both within CNS tissue and outside. In addition, the nature of the lesion can frequently be deduced from density characteristics. Both CT and MRI technology have been changing rapidly, and assessment of their capabilities relates to data currently available. Detection of brain tumors is to some extent influenced by location and type of tumor as well as by technical factors such as use of IV contrast media. Reports indicate CT abnormality in approximately 90%-95% of patients with mass lesions or areas of tissue destruction. Statistics from CT and MRI are not always comparable to radionuclide brain scans, since data from the scans vary according to the number of head positions employed, the isotope preparation used, the time between administration of isotope and scanning, and whether a blood flow study was included. In general, CT is about 10%-15% more sensitive than brain scan in cerebral tumor or about 5%-10% more sensitive when brain scanning is performed with optimal technique. It is somewhat more reliable than brain scanning in detecting posterior fossa lesions. MRI is about 5% more sensitive than CT. In chronic subdural hematoma, detection with CT is about equal to that achieved when brain scanning is combined with cerebral blood flow study. In acute subdural or epidural hematoma, CT is significantly better than radionuclide brain scanning. An important advantage over radionuclide techniques in either acute or chronic subdural hematoma is that CT can frequently permit a more exact diagnosis, whereas abnormalities found by radionuclide techniques are often not specific. MRI is reported to be equal to or slightly better than CT. In cerebral infarct, all three techniques are affected by the time interval after onset. During the first week, CT is somewhat more sensitive than MRI or radionuclide scanning without blood flow study but still detects only 50%-60% of infarcts. The sensitivity of all techniques increases to the 80% range by 3-4 weeks. Intracerebral hematoma is much better seen on CT than brain scan regardless of the time interval and better than MRI in the early stages. None of the three techniques is perfect. In most of the various focal lesion categories, a certain percentage are detected by one technique but not the other, although CT and MRI are generally superior to radionuclide scans. When Hakim’s syndrome (normal pressure hydrocephalus) is a possibility, CT or MRI can rule out the disorder by displaying normal ventricular size. If ventricular dilation is seen, cerebral atrophy may be inferred in some cases, but in many the differentiation between atrophy and normal pressure hydrocephalus cannot be made with adequate certainty.

    Overall advantages of CT over standard radionuclide procedures are ability to visualize the ventricular system, a relatively small but definite increase in detection rate for brain tumors, more specificity in the appearance of many lesions, and better delineation of CNS anatomy. Advantages over MRI are lower cost, better results in early CVA or early hemorrhage, and ability to detect calcifications and show details of bone (which is seen poorly on MRI). Advantages of radionuclide procedures are elimination of the need for x-ray contrast media (which many CT patients must receive), lower cost for equipment and lower charge to the patient, and ability to inspect major blood vessels via blood flow studies. Advantages of MRI are absence of any radiation, slightly and sometimes significantly better detection rate of many lesions compared to CT, better tissue detail, much better visualization of the spinal cord, and in some cases ability to suggest a more exact diagnosis. The major disadvantages are considerably higher cost, slower imaging time, and in some cases, problems with patients who have cardiac pacemakers or internal metal objects.

  • 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.

  • Human Immunodeficiency Virus 1 (HIV-1)

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

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

    Clinical findings

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

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

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

    Laboratory findings

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

    Diagnosis of HIV-1 infection

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

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

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

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

    Tests in HIV-1 infection

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

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

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

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

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

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

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

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

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

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

  • 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.

  • 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.

  • Obtaining a Specimen for Culture

    After material has been taken for culture, three steps should be followed. First, the specimen must be taken to the laboratory as soon as possible, since many organisms die on prolonged exposure to air or drying. This is especially true for swab preparations. Swab kits are available that contain a carrier medium into which the specimen is placed. This is a great help in preserving most bacteria, but the medium is not ideal for all organisms. For example, gonococci or anaerobes must have special transport systems. Anaerobic specimens require special precautions when the specimen is obtained and during transport to the laboratory. Second, the source of the culture should be written on the request sheet. This tells the laboratory what normal flora organisms are to be expected and provides some information on the pathogens that should be looked for and thus what media should be used. Finally, if a specific organism is suspected, this information should also be written on the request, so that if special culture methods are required, the requisite techniques will be anticipated and used.

  • Tuberculosis and Mycobacterial Disease

    Tuberculosis is caused by Mycobacterium tuberculosis (MTB), a rod-shaped bacterium that requires special media for culture and that has the peculiarity of “acid-fastness” (resistance to decolorization by strong acidic decolorizing chemicals such as acid alcohol after being stained by certain stains such as carbol fuchsin). Tuberculosis is still very important and common despite advances in drug therapy. It has been reported that about 25% of persons exposed to MTB will become infected; and of those infected, about 10% will develop clinical disease (range, 5%-40%). The disease usually begins in the chest due to inhalation of airborne infectious material. This material is carried to some localized area of the lung alveoli and provokes a host response of granulomatous inflammation around the material (the “Ghon complex”). It is thought that in many cases there is also a silent hematogenous spread of the organisms. In most cases the host is able to contain and eventually destroy the organisms in the chest and those reaching other locations. Those in the lungs seem better able to survive than those deposited elsewhere. In some cases the organisms remain dormant, and the infection can be reactivated at a later date; in some cases the initial infection spreads; and in some cases reinfection takes place. If infection in the lungs progresses to clinical disease, the most important symptoms are cough, fever, and hemoptysis. (The most important diseases to rule out are lung carcinoma and bronchiectasis.) The kidney is involved in a small percentage of advanced cases, with the main symptom being hematuria (Chapter 12). A small number of patients develop widespread extrapulmonary disease, known as miliary tuberculosis. The laboratory findings in tuberculosis depend to some extent on the stage and severity of the disease.

    Chest x-ray films

    Chest x-ray films often provide the first suggestion of tuberculosis and are a valuable parameter of severity, activity, and response to therapy. Depending on the situation, there are a variety of possible roentgenographic findings. These may include one or more of the following:

    1. Enlargement of hilar lymph nodes.
    2. Localized pulmonary infiltrates. These occur characteristically in an upper apical location or, less commonly, in the superior segment of the lower lobes. Cavitation of lesions may occur.
    3. Miliary spread (small punctate lesions widely distributed). This pattern is not common and may be missed on routine chest x-ray films.
    4. Unilateral pleural effusion. The most common causes are tuberculosis, carcinoma, and congestive heart failure. Tuberculosis has been reported to cause 60%-80% of so-called idiopathic pleural effusions, although this percentage varies greatly depending on the patient’s geographic location and other factors.

    Sputum smear

    Sputum smears provide a rapid presumptive diagnosis in pulmonary tuberculosis. The smear is usually stained by one of the acid-fast (acid-fast bacillus, or AFB) procedures (usually the Ziehleelsen or Kinyoun methods). Fluorescent Auramine-o staining methods are available, faster, and somewhat more sensitive. Smears require about 5 Ч 103 organisms/ml of specimen for microscopic detection. The more advanced the infection, the more likely it is to yield a positive smear. Therefore, the rate of positive findings is low in early, minimal, or healing tuberculosis. Also, the smear may be normal in a substantial minority of advanced cases. Culture is more reliable for detection of tuberculosis and also is necessary for confirmation of the diagnosis, for differentiation of MTB from the “atypical” mycobacteria, and for sensitivity studies of antituberculous drugs. According to the literature, false negative smears (smear negative but culture positive) have been reported in an average of 50% of cases (literature range, 16%-70%). Some of these false negative results may be due to laboratory technique problems and differences in smear staining methods. A high centrifugation speed when concentrating the specimen is said to increase the yield of positive smears. False positive smears (positive smear but negative culture) have been reported, averaging about 1%-5% of positive smears (literature range, 0.5%-55%). Some of these were apparently due to contamination of water used in the smear-staining procedure by saprophytic mycobacteria. Control slides are necessary to prevent this. Some authorities believe that only 1-2 acid-fast organisms/300 oil immersion fields should be considered negative (although indicative of need for further specimens). Smears may sometimes be positive for up to 2 months when cultures are negative if the patient is on antituberculous drug therapy (this would not be considered a genuine false positive, since the drugs inhibit mycobacterial growth or the organisms may be nonviable). After 2 months, persistence of positive smears raises the question of treatment failure. Temporary persistence of positive smears with negative cultures is more likely to occur in severe cavitary disease (in one series, this occurred in 20% of cases). Sputum specimens should be collected (for culture and smear of the concentrated specimen) once daily for at least 3 days. If the smear is definitively positive, further smears are not necessary. Also, a definitively positive smear means high probability that culture of the specimens already collected will obtain positive results, and it is not necessary to collect more than three specimens or to proceed to more complicated diagnostic procedures. If smears are negative, one must consider the possibility that the culture may also be negative, and conventional cultures on standard solid media average 20 days to produce growth from MTB smear-positive specimens and about 27 days from MTB smear-negative specimens (overall range, 2–8 weeks).

    Culture

    Sputum culture is preferred for pulmonary tuberculosis (gastric aspiration may be done if adequate sputum specimens cannot be obtained); urine culture is preferred for renal involvement; and bone marrow culture is preferred in miliary tuberculosis. Reports indicate that an early morning specimen, either of sputum or urine, produces almost as many positive results as a 24-hour specimen and has much less problem with contamination. Special mycobacteria culture media are needed. The necessity for adequate sputum culture specimens, regardless of the concentrated smear findings, should be reemphasized. Several reports indicate that aerosol techniques produce a significantly greater yield of positive cultures than ordinary sputum collection. The aerosol mixture irritates the bronchial tree and stimulates sputum production. At any rate, it is necessary to get a “deep cough” specimen; saliva alone, although not completely useless, is much less likely to reveal infection and is much more likely to be contaminated. If sputum cultures are negative or if the patient is unable to produce an adequate sputum sample, gastric aspiration may be used. Gastric contents are suitable only for culture; nontuberculous acid-fast organisms may be found normally in the stomach and cannot be distinguished from M. tuberculosis on AFB smear. If renal tuberculosis is suspected, urine culture should be done (Chapter 12). However, renal tuberculosis is uncommon; and even with urine specimens obtained on 3 consecutive days, only about 30% of cases are positive.

    Cultures should be grown in high carbon dioxide atmosphere, since this is reported to increase the number of positive cultures by at least 10%. Inoculation on several varieties of media increases the number of positive results by 5%-10%. The 4% sodium hydroxide traditionally used to digest and decontaminate sputum before concentration also kills some mycobacteria. Use of weaker digestion agents increases culture yield, but troublesome overgrowth by other bacteria may also increase.

    Culture should be done on all tissue specimens when tuberculosis is suspected. Acid-fast stains on tissue slides reveal tuberculosis organisms in only 30%-40% of cases that are positive by culture. Several newer methods such as BACTEC (which uses liquid media and a machine that detects metabolic products of bacterial growth) have been able to decrease detection time for MTB smear-positive specimens to 8 days and time for MTB smearegative specimens to 14 days (overall range, 1-3 weeks). The system is about 93% sensitive compared to conventional multimedia culture. Once culture growth occurs, the organism must be identified. Conventional methods require biochemical and growth tests to be performed that may take 3-6 weeks to complete. The BACTEC system has a nucleic acid phosphate method that can identify MTB (only) in 3-5 days. Commercial DNA probes are available that can identify MTB and certain non-MTB mycobacteria in 1 day. Gas-liquid chromatography and high-performance liquid chromatography have also been used. Antibiotic sensitivity studies are recommended when a mycobacterial organism is isolated, since multiresistant MTB is increasing in various areas and non-MTB mycobacteria have often been multiresistant. Conventional culture methods take 21 days; BACTEC takes about 5 days.

    Data on sputum culture sensitivity using conventional AFB media is difficult to find since culture is usually considered the gold standard of AFB detection. Sputum culture appear to average about 75% sensitivity (range, 69%-82%). Sensitivity using BACTEC averages about 85% (range, 72%-95%).

    Nucleic acid probe

    Nucleic acid (DNA) probe methods are now becoming available that permit direct nonsmear detection of mycobacteria in clinical specimens. The first such test available (Gen-Probe, Inc.) is reported in two studies to be 83%-85% sensitive compared with culture using sputum specimens. However, it has been reported that antituberculous drugs can interfere with the probe; this study found sensitivity in nontreated patients to be over 90% (when comparing probe to culture, culture only detects about 75%-80% of cases). Ten percent of specimens were positive by probe but negative by culture, which may represent additional true positives that could not be confirmed. A DNA probe is also available specifically for M. tuberculosis. Same-day results can be obtained. Disadvantages of this first-generation method are need for considerable technologist time and certain special equipment. Compared with culture, the general Mycobacterium screen probe will not differentiate M. tuberculosis from other mycobacteria, whereas the specific M. tuberculosis probe will not detect the other mycobacteria. Neither probe would provide therapeutic drug sensitivity information. The major drawback regarding its use as replacement for the acid-fast smear is the relatively high cost of the probe method, very high if only one specimen at a time is processed. DNA probes with PCR amplification have been reported (e.g., Roche Diagnostics) that are said to have a sensitivity of 3-30 organisms/ml (compared to at least 5 Ч 103 organisms/ml required for a positive acid-fast smear). Nevertheless, one study involving 7 prestigious worldwide reference laboratories who were sent sputum or saliva specimens to which various quantities of BCG (M. Bovis) mycobacteria were added, showed false positive PCR rates of 3%-77%. In specimens containing 102 organisms, sensitivity ranged from 0%-55%; in specimens containing 103 organisms, 2%-90%; and in specimens containing 101 organisms, 20%-98%.

    Skin test (Mantoux test)

    This test is performed with an intradermal injection of purified protein derivative (PPD) or old tuberculin (Table 14-1). A positive result is represented by an area of induration having a specified diameter by 48 hours. The diameter used to be 10 mm but was redefined in 1990 to require different diameters depending on the person’s risk group (see box). In addition, a distinction was made between “reaction” (diameter or width of induration without record of previous test result) and “conversion” (increase in reaction width within 2 years from last previous reaction width). For all persons younger than 35 years of age whose previous reaction was negative, an increase in PPD induration of 10 mm or more in diameter within a period of 2 years would be considered a conversion and presumptive suspicion for occult tuberculosis (TB), whereas the change would have to be at least 15 mm for persons 35 years of age or more (that is, for nonrisk persons above or below age 35 who have had a PPD within 2 years, conversion criteria would replace reaction size criteria).

    Table 14-1 Comparison of tuberculosis skin tests*
    Comparison of tuberculosis skin tests


    A positive skin test is a manifestation of hypersensitivity to the tubercle bacillus. This reaction usually develops about 6 weeks after infection, although it may take several months. A positive reaction means previous contact and infection with TB; the positive reaction does not itself indicate whether the disease is currently active or inactive. However, in children under 3 years of age it usually means active TB infection. Apparently, once positive, the reaction persists for many years or for life, although there is evidence that a significant number of persons revert to negative reactions if the infection is completely cured early enough. In a few cases of infection the test never becomes positive. The Mantoux test may revert to negative or fail to become positive in the following circumstances:

    1. In about 20% of seriously ill patients, due to malnutrition (severe protein deficiency).
    2. In newborns and occasionally in old age.
    3. In some persons with viral infections, or within 1 month after receiving live virus vaccination.
    4. In 50% or more of patients with miliary tuberculosis.
    5. In a high percentage of patients with overwhelming pulmonary tuberculosis.
    6. In a considerable number of patients who are on steroid therapy or immunosuppressive therapy.
    7. In many persons who also have sarcoidosis or Hodgkin’s disease.
    8. In some persons with chronic lymphocytic leukemia or malignant lymphoma.
    9. In some patients with chronic renal failure or severe illness of various types.
    10. In some persons with old infection (“waning” of reactivity).
    11. When there is artifact due to improper skin test technique (e.g, subcutaneous rather than intradermal injection).

    In cachectic patients and those with protein malnutrition, treatment with an adequate protein diet can restore Mantoux test reactivity to most patients after about 2 weeks. In patients after age 50 with M. tuberculosis infection, especially those with old previous infection, the PPD skin test sometimes may slowly decrease in reactivity and eventually become negative. (How often this occurs is controversial; the best estimate seems to be 8%-10%, but studies range from 0.1%-21%, possibly influenced by the elapsed time period since infection and time intervals between testing.) If another skin test is performed, the new skin test itself stimulates body reaction and may restore reactivity (“booster phenomenon”). This phenomenon could simulate a new infection if previous infection were not known, since the next time a skin test is performed the physician would see only conversion of a negative to a positive reaction. Restoration of skin reactivity can take place in only 1 week, so that retesting 1 week after the first negative reaction can usually show whether or not there is potential for the booster reaction. (The 1-week interval would in most cases not be long enough for true conversion in persons with their first infection.) Repeated skin tests will not cause a nonexposed person to develop a positive reaction. Some investigators recommend skin testing with antigens used to demonstrate the presence of skin test anergy (e.g., Candida or Trichophyton antigen) if the Mantoux test is repeatedly negative in a person with substantial suspicion of mycobacterial infection.

    The standard procedure for skin testing is to begin with an intermediate strength PPD (or the equivalent). If the person has serious infection, some clinics recommend starting with a first-strength dose to avoid necrosis at the injection site. A significant minority of patients with tuberculosis (9%-17%) fail to react to intermediate strength PPD; a second-strength dose is then indicated.

    Miliary tuberculosis

    Miliary TB is clinically active TB that is widely disseminated in the body by hematogenous spread. Clinical symptoms are often nonspecific, such as fever, weakness, and malaise. There frequently is an associated condition, such as alcoholism, intravenous (IV) drug abuse, or malignancy, that decreases immunologic defenses. About 20% have a negative tuberculin skin test reaction. About 35% do not show a miliary pattern on chest x-ray film. If routine clinical and culture methods fail, biopsy of bone marrow or liver may be useful. Liver biopsy has a fairly good positive yield (up to 80%), considering that a needle biopsy specimen is such a tiny random sample of a huge organ. However, it is usually difficult to demonstrate acid-fast organisms on liver biopsy even when tubercles are found, and without organisms the diagnosis is not absolutely certain. Bone marrow aspiration is probably the best procedure in such cases. Bone marrow yields much better results for mycobacterial culture than for demonstration of tubercles. Routine marrow (Wright-stained) smears are worthless for histologic diagnosis in TB. Aspirated material may be allowed to clot in the syringe, then formalin-fixed and processed as a regular biopsy specimen for histologic study. Before clotting, some of the aspirate is inoculated into a suitable TB culture medium. It should be emphasized that bone marrow aspiration or liver biopsy is not indicated in pulmonary tuberculosis (since this disease is relatively localized), only in miliary TB.

    PPD Reaction Size Considered “Positive” (Intracutaneous 5 TU* Mantoux Test at 48 Hours)
    5 MM OR MORE
    Human immunodeficiency virus
    (HIV) infection or risk factors for HIV
    Close recent contact with active TB case
    Persons with chest x-ray consistent with healed TB
    10 MM OR MORE
    Foreign-born persons from countries with high TB prevalence in Asia, Africa, and Latin America
    Intravenous (IV) drug users
    Medically underserved low-income population groups (including Native Americans, Hispanics, and African Americans)
    Residents of long-term care facilities (nursing homes, mental institutions)
    Medical conditions that increase risk for TB (silicosis, gastrectomy, undernourished, diabetes mellitus, high-dose corticosteroids or immunosuppression RX, leukemia or lymphoma, other malignancies
    Employees of long-term care facilities, schools, child-care facilities, health care facilities
    15 MM OR MORE
    All others not listed above
    * TU, tuberculin units.

    Renal tuberculosis

    Renal TB is almost always bilateral and presumably results from nonhealing infection produced during the transient bacteremia of the lung primary stage. There is usually a latent period of many years before clinical infection becomes evident. The estimated incidence of eventual active renal infection is about 2%-5%, but this probably represents incidence in high-risk groups. About 25% (range, 20%-75%) of patients are said to have a normal chest x-ray film. About 14% (range, 12%-15%) of patients are reported to have a negative PPD skin test result. Even the intravenous pyelogram results (IVP) are normal in about 25% (range, 14%-39%) of cases. Most patients do not have systemic symptoms, such as fever. The erythrocyte sedimentation rate was elevated in 23% of patients in one report. Only 20%-56% have urinary tract symptoms. Gross hematuria is the classic finding in renal TB but is present in only about 20% of patients. Pyuria (with negative urine culture) and microscopic hematuria are more frequent, occurring in about 65%-85% of cases. Some patients have a positive urine culture with some ordinary pathogen in addition to renal TB. Urine culture for TB was mentioned previously; 30%-80% of patients have positive cultures when three 24-hour or early morning specimens are collected (true culture sensitivity is probably less than 80%, since the diagnosis could easily be missed with negative cultures).

  • Direct Methods of Bacterial Detection

    Culture. This is the classic definitive method for detection and identification and will be discussed later in more detail. The major drawback is time; it usually takes 1 full day to grow the organism and then part or all of 1 day to identify it. It may take an additional day to isolate it before identification if there is a mixture of organisms. Some organisms take longer than 1 day to grow. There is always a certain percentage of false negative results (sometimes a large percentage) due to various factors, both clinical and technical. Several major difficulties are suppressive effects of antibiotic therapy on bacterial growth (even though clinical cure is not achieved); specimen not obtained from its best area (sampling error), inadequate or inappropriate specimens obtained, or faulty specimen transport to the laboratory; and differences in the way any individual laboratory processes the specimen compared to a research laboratory.

    Immunologic methods. Immunologic methods (immunoassay) depend on antigen-antibody reaction, either test antibody binding to patient antigen or test antigen attachment to patient antibody. There also must be a readout or indicator system to show that the reaction has taken place and to quantify the amount of patient antigen or antibody. The indicator can be a radioactive molecule (radioimmunoassay [RIA]), a fluorescent molecule (fluorescent immunoassay [FIA]), a molecule with an attached enzyme that can participate in a biochemical color reaction (enzyme-linked immunoassay [ELISA or EIA]), or some other method, such as an inert particle coated with antigen or antibody that produces particle agglutination as the endpoint of the reaction (e.g., latex particle agglutination [LA]). There can be a single-reagent antibody or antigen that captures the antigen and a second antibody that contains the readout molecule and that attaches to the captured patient antigen (“sandwich” immunoassay). The antibody used may be produced in animals and is not completely specific for the selected antigen (polyclonal antibody); or an antibody may be produced that is specific for an antigen or a particular receptor (epotope) on the antigen (monoclonal antibody). Considerably simplified, monoclonal antibodies currently are most often produced by injecting the antigen into a mouse, waiting until the mouse produces antibody against the antigen, obtaining samples of the mouse spleen and culturing different lymphocytes until one is found that produces a specific antibody, then incubating the mouse lymphocyte with a myeloma cell and providing an environment (e.g., polyethylene glycol) that causes the two cells to stick together and then fuse into one hybrid cell. The myeloma inheritance causes the cell (and its offspring) to rapidly reproduce for long periods of time, while the mouse spleen inheritance results in continued specific (monoclonal) antibody production. Some of the immunologic methods are capable of accuracy that is equivalent to culture (e.g., fluorescent anti- body method for Corynebacterium diphtheriae); others are less reliable, depending on the technique and the particular kit manufacturer. All antibodies do not behave the same, even under the same conditions.
    Nucleic acid probe (DNA probe). Greatly simplified, this technique attempts to construct a nucleic acid sequence (the “probe”) that matches a sequence in the deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of the organism to be detected. This sequence or probe is incorporated (or grafted) into a larger nucleic acid molecule, usually a single strand of DNA (although a strand of RNA could be used) that can be tagged with an indicator system (radioisotope or biochemical reaction). Then the specimen to be tested is prepared for analysis. If the target molecule is DNA, since DNA usually exists as a double-stranded molecule, the target molecule DNA double strands are first separated into single strands by various means, and then the test DNA single strands containing the probe sequence are introduced. If the probe sequence matches a sequence in the target, the probe hybridizes with the target DNA (combines with the target single strand) to again form a double strand. If the target molecule is RNA, RNA exists in living cells in single-strand state, so that the DNA single-strand test molecule containing the RNA probe area can bypass the strand separation step and hybridize directly with an RNA single strand (instead of another DNA single strand) if the probe area matches a nucleic acid sequence in the target RNA strand. After incubation, nonhybridized (nonattached) test probe-carrying single strands are washed away and the indicator system is used to demonstrate whether any probe remains combined to target molecules. In some ways this technique is similar to direct immunologic detection methods. Advantages of nucleic acid probe systems are much greater sensitivity than current antibody systems; specificity that can be varied to the genus, species, or even strain level; theoretical possibility of use for any organism in any clinical specimen; and same-day results. The major disadvantage thus far is relatively high cost of the test when performed on a single specimen basis.