Articles on Medical Diseases and Conditions

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  • Effect of Therapy on Test Results

    Studies before 1990 indicated that if patients with their first syphilitic infection in the primary or secondary stage were given adequate treatment, an elevated VDRL titer would decline (on the average) 2 dilutions (fourfold) by 3 months and 3 dilutions by 6 months. The rate of decline was said to be faster when the pretreatment VDRL titer was higher and more slowly when the VDRL titer was lower or when the infection was long-standing or was a reinfection. A large Canadian study published in 1991 confirmed some of these observations and disagreed with others. The Canadian study found that the first infection and the earlier stages of syphilis were more likely to respond serologically to treatment and responded faster, in agreement with previous studies. However, the Canadian study, using the RPR and current treatment recommendations, noted a slower rate of RPR decline, with a 2-tube dilution decrease (on the average) not attained until 6 months and a 3-tube decrease not achieved until 12 months. Although the rate of decrease was similar at all dilutions, the time needed to decrease to nonreactive was (as expected) much earlier with lower pretherapy titers than with high titers. In first-infection primary syphilis, if the initial titer was 1:8, the RPR (on the average) became nonreactive in 26% of patients by 3 months, in 54% by 6 months, in 70% at 1 year, and in 81% by 3 years. If the initial titer was 1:256, no patients were nonreactive at 3 months, only 6% at 1 year, and 31% at 3 years. In the early latent stage with a pretherapy titer of 1:8, 7% were nonreactive at 3 months, 31% at 1 year, and 66% at 3 years.

    In the Canadian study the FTA-ABS became nonreactive after therapy in 11% of first-infection primary stage patients at 1 year and 24% at 3 years. The FTA-ABS or the MHA-TP seldom became nonreactive when therapy began in the secondary stage and in no patients after the secondary stage.

    One study reported that HIV infection in its late stage may result in loss of FTA-ABS and MHATP reactivity in 17% of cases. In one study, BFP results were obtained using the RPR method in 4% of HIV-positive patients (all in males) versus 0.8% incidence in non-HIV patients. Another publication noted that whereas HIV infection may delay VDRL seropositivity, eventually the test does become reactive. Another noted that the term “dils” (dilutions) in a VDRL or RPR report refers to the titer by using the total volume of the diluted specimen (i.e., a titer of 1:8 may be reported as 8 dils).

  • Selection of Tests

    The selection of tests for syphilis is dictated by the clinical situation. If the patient has possible primary syphilis, a dark-field examination would be helpful, although it is becoming difficult to find laboratories with the necessary equipment and laboratorians with sufficient expertise. If the dark-field test result is negative or cannot be performed, an FTA-ABS test should be done. If the FTA-ABS result is nonreactive and clinical suspicion is strong, the physician, for practical purposes, has the option of treating the patient without a conclusive diagnosis or of repeating the FTA-ABS test in 2-3 weeks.

    If the patient has confirmed early syphilis, an RPR result should nevertheless be obtained. If the RPR result is reactive, the degree of reactivity should be titered, since a falling titer after treatment is evidence that treatment was effective.

    If the patient has possible, equivocal, or late syphilis, an FTA-ABS (or MHA) and STS test should be done. If the FTA-ABS (or MHA) test result is reactive (2+ or more), the diagnosis is probable syphilis. If the FTA-ABS (or MHA) test result is weakly reactive (borderline or 1+ reactive), the test should be repeated in 1 month. If it is still weakly reactive, the diagnosis is possible or probable syphilis, depending on the clinical picture. The RPR results are useful mainly as additional evidence in equivocal cases.

    If a routing screening RPR result is found to be positive in a person with no history or clinical evidence of syphilis, a confirmatory test should be done. If the confirmatory test result is negative, the patient should be screened for diseases known to cause a high incidence of BFP reactions. In this respect, a weakly positive RPR result may be due only to an acute BFP etiology, and the RPR result should be negative in 2-3 months. If the confirmatory test result is positive, past or present syphilis is a strong probability. Nevertheless, since even the confirmatory tests may occasionally produce a false positive (or false negative) reaction, in certain patients it may be necessary to repeat the confirmatory test.

  • Sensitivity and Specificity of Syphilis Tests

    Studies have been done in which duplicate samples from known syphilitic patients in various stages of their disease and also from normal persons were sent to various laboratories. Besides this, many reports have appeared from laboratories all over the world comparing one test with another in various clinical stages of syphilis, in nonsyphilitic diseases, and in apparently normal persons. These results are summarized in Table 15-3.

    Comparison of serologic tests for syphilis

    Table 15-3 Comparison of serologic tests for syphilis (approximate percentage reported reactive)*

    Table 15-3 demonstrates considerable variation in results. Several factors must be involved besides the inherent sensitivity and specificity of the individual tests themselves:

    1. Antibiotic treatment may cause some previously reactive syphilitic patients to become nonreactive.

    2. Some clinically normal persons may have unsuspected subclinical syphilis.

    3. True BFP reactions, either acute or chronic, must be taken into account.

    4. There is obvious variation in technique and ability between laboratories. Some laboratories introduce their own modifications into standard techniques.

    5. The time of appearance differs for the various antibodies. In general, the FTA-ABS test result becomes positive in significant numbers of patients in the middle or end of the primary stage, followed by the MHA and RPCF, and then the VDRL. All of these procedures usually give positive results in the secondary stage, and also probably in the early latent stage.

  • Specific Treponema Pallidum Tests

    Treponema pallidum immobilization (TPI) test. Syphilitic spirochetes can grow in rabbits. Nelson devised a Treponema pallidum immobilization (TPI) test in which syphilitic spirochetes are incubated with the patient’s serum. If specific antisyphilitic antibody is present, it will attack the spirochetes and immobilize them, causing them to stop moving when viewed under the microscope. This involves an antibody that is different from reagin and is specific against pathogenic Treponema spirochetes. Besides syphilis, other Treponema spirochetal diseases such as yaws may give positive reactions. The main disadvantages of this test are that it requires working with live spirochetes and use of an animal colony, is difficult to perform accurately, and is expensive. The TPI test has been replaced by the FTA-ABS and the microhemagglutination (MHA) tests (to be discussed later), and it is now very difficult, almost impossible, to obtain a TPI study in the United States.

    Reiter protein complement fixation (RPCF) test. The TPI test is done using the Nichol strain of pathogenic spirochetes. It was discovered that a certain nonpathogenic Treponema spirochete called the “Reiter strain” could be cultured more easily and cheaply on artificial media. Antigen prepared from this organism was adapted to a CF technique, and the result was the Reiter protein complement fixation (RPCF) test.

    The Reiter antibody is different from the Treponema-immobilizing antibody of the TPI test. Apparently, the nonpathologic Reiter and the pathologic Nichol spirochete share a common protein antigen, and it is this protein that is used in the RPCF test. In addition, the Nichol organism has a specific antigen that results in the immobilizing antibody response of the TPI assay. Several investigators have found the RPCF test to be almost as sensitive and specific as the TPI test, although others are less enthusiastic about its sensitivity in late syphilis. The Reiter antibody also appears at a different time than does the TPI antibody. The disadvantages of the RPCF are those inherent in all CF tests. The RPCF test is practically never used in the United States.

    Fluorescent treponemal antibody (FTA) test. In the FTA procedure, dead Nichol strain spirochetes are fixed to slides; these spirochete-containing slides can be prepared in the laboratory or obtained ready for use from commercial sources. The patient’s serum is placed on the slide with the spirochetes and allowed to incubate with the organisms. Antispirochete antibody in the patient serum coats the surface of the spirochetes. The serum is then washed off and replaced by antibodies against human gamma globulin that are tagged with a fluorescent dye. Since human antibodies against syphilis produced after the first month of infection are gamma globulins, the antibodies against human gamma globulin, with the fluorescein dye attached, will attack and adhere to any patient antitreponemal antibody that coats the spirochetes on the slide. The spirochetes will then appear fluorescent when viewed with an ultraviolet microscope.

    Unfortunately, fluorescent work is not as simple as this description or the recent literature would imply. Many technical problems remain. These tests at present are not suitable for mass screening, although they are less time consuming than the RPCF test and easier than the TPI test. Many substances give varying degrees of natural fluorescence, and it is sometimes difficult to decide whether a preparation is actually positive or not. There may be nonspecific antigen-antibody binding of the cross-reacting type, as well as specific reaction. When the animal anti-human globulin antibody is conjugated with fluorescein, not all of the fluorescein binds to it. Any remaining free fluorescein may nonspecifically stain various proteins, including the spirochetes, when the tagged mixture is added to the patient’s serum.

    Because of the problem of nonspecific fluorescence, the FTA underwent modification to become the FTA test with absorption (FTA-ABS). Reiter Treponema antigen is used to absorb nonspecific cross-reacting antibodies out of the patient’s serum. Antibody to T. pallidum is not absorbed out by this technique. The absorbed patient serum replaces nonabsorbed patient serum in the standard FTA procedure.

    The FTA-ABS is a well-established test. It has relatively good sensitivity in primary syphilis (except in very early disease) and is reported to be even more sensitive than the TPI in tertiary syphilis. If the patient is treated adequately in the primary or secondary stage, the FTA-ABS response will usually return to nonreactive state, but after the secondary stage it usually will not become nonreactive in spite of therapy. It is said to be at least as specific as the TPI, possibly even more so.

    Drawbacks. Weak reactions may cause interpretation problems. Official recommendations are that equivocal or 1+ reactive specimens should be repeated and the 1+ reclassified as borderline if the repeat test result is nonreactive and called 1+ reactive if reactivity remains 1+. This is important because some false positive and false negative results may occur in the FTA-ABS due to laboratory technical factors. Some studies have revealed a 5%-10% variance between laboratories. This is much more likely to happen in weakly (1+) reactive specimens than with specimens having reactivity graded 2+ to 4+.

    No laboratory test is free from the possibility of nonhuman error, and the FTA-ABS is no exception. Occasional false positive FTA-ABS results have been reported in persons with hyperglobulinemia due to macroglobulins and in patients with antinuclear antibodies. In addition, atypical fluorescent patterns (“beaded”) that could be misinterpreted as reactive have occurred in some patients with systemic lupus erythematosis. Cross-reaction may occur in other spirochetal diseases such as Borrelia (Lyme disease), leptospirosis, and relapsing fever. Problems of nonspecific fluorescence mentioned earlier have been reduced but not entirely eliminated. Occasional false negatives may occur even with the FTA-ABS test. Several reports suggest that concurrent infection by the human immunodeficiency virus type 1 (HIV-1) can sometimes delay the development of a positive VDRL or FTA-ABS reaction.

    Microhemagglutination (MHA-TP) test. An MHA test is available using formalin-treated RBCs coated with Nichol strain T. pallidum material. Patient serum is preabsorbed with Reiter Treponema reagent in the same manner as the FTA-ABS technique. Antibody to T. pallidum prevents normal RBC agglutination when the test is performed. About 1%-2% of sera contain nonspecific Forssman-type antibodies, so that reactive sera must be retested with nonsensitized control RBCs.

    The MHA test is not as sensitive in primary syphilis as the FTA-ABS test, although it is reactive in more than 50% of patients. It seems equally as sensitive as the FTA-ABS test in secondary and possibly in late syphilis. Compared to FTA-ABS results, various studies have shown 90%-98% overall correlation. Our laboratory has performed a comparison of this type and found that nearly 85% of the disagreements represented either nonreactive or 1+ reactive MHA results and nonreactive or 1+ FTA-ABS results. Therefore, most disagreements seem to occur at low reactivity levels in which the accuracy of either test result is open to some question. This being the case, there is reason to conclude that the MHA could be substituted for the FTA-ABS, except possibly in primary syphilis (in which case an FTA-ABS test could be done if the MHA test results were nonreactive). The MHA test is much easier to perform than the FTA-ABS test and is less expensive.

    More recently, several enzyme-linked immunosorbent assays (ELISA) have been reported, with preliminary results similar to those of the FTA-ABS. However, more independent evaluations are needed. Several research centers have also developed Western blot methods. Several investigators have published nucleic acid probe tests amplified by polymerase chain reaction (PCR). Again, initial very good results must be verified by others.

  • Cardiolipin Tests

    The first practical serologic test for syphilis (STS) was the complement fixation (CF) technique invented by Wassermann. He used extract from a syphilitic liver as the antigen that demonstrated the presence of antitreponemal antibodies. Subsequently it was found that the main ingredient of the substance he used actually had nothing to do with syphilitic infection and was present in other tissues besides liver. It is a phospholipid that is now commercially prepared from beef heart and therefore called cardiolipin. The reagent used in current screening tests for syphilis (as a group, sometimes called STS) is a mixture of purified lipoproteins that includes cardiolipin, cholesterol, and lecithin. Apparently, an antibody called “reagin” is produced in syphilis that will react with this cardiolipin-lipoprotein complex. Why reagin is produced is not entirely understood; it is not a specific antibody to T. pallidum. There is a lipoidal substance in spirochetes, and it is possible that this substance is similar enough to the cardiolipin-lipoprotein complex that antibodies produced against spirochetal lipoidal antigen may also fortuitously react with cardiolipin.

    The original Wasserman CF test was replaced by a modification called the Kolmer test; but this in turn was replaced by the much faster, easier, and cheaper flocculation tests. In earlier versions of the flocculation reaction the patient’s serum was heated; for unknown reasons, heating seemed to enhance the reaction. Then a suspension of cardiolipin antigen particles is added to the serum and mixed. In a positive (reactive) test result, the reagin antibody in the patient serum will combine with the cardiolopin antigen particles, producing a microscopic clumping or flocculation of the antigen particles. The reaction is graded according to degree of clumping. The current standard procedure for this type of test is the Venereal Disease Research Laboratory (VDRL) test. It was found that the preliminary heating step could be eliminated if certain chemicals were added to the antigen; this modification is called the rapid plasma reagin (RPR) test and gives results very similar to those of the VDRL.

    Drawbacks of the cardiolipin serologic tests for syphilis

    Variation in test modifications. Not all sera from known syphilitics gave positive reactions in these tests. It was discovered that the number of positives could be increased by altering the ratio of antigen ingredients. However, usually when the percentage of positive results increases significantly, more false positives are reported. One report indicates that about 2% of VDRL or RPR tests in primary and secondary syphilis are falsely negative due to antigen excess (prozone phenomenon).

    Effect of antibiotic therapy. A peculiarity of the STS exists when antibiotic treatment is given. If the patient is treated early in the disease, the STS will revert to nonreactive state. In patients with primary syphilis, one study found that the VDRL or RPR returned to nonreactive state in 60% of patients by 4 months and 100% of patients by 12 months. In secondary syphilis, the VDRL became nonreactive in 12-24 months. Another study of patients with primary and secondary syphilis reported that successful treatment usually produced a fourfold decrease in VDRL titer by 3 months and an eightfold decrease by 6 months. The VDRL decline may be slower if the patient had other episodes of syphilis in the past. However, the longer the disease has been present before treatment, the longer the VDRL takes to become nonreactive. In many cases after the secondary stage it will never become nonreactive, even with adequate treatment (this is called “Wassermann fastness”).

    Spontaneous loss of test reactivity. In tertiary syphilis, there is a well-documented tendency for a previously reactive VDRL/RPR test result to revert spontaneously to nonreactive, even if the patient is untreated. This is reported to occur in about 20%-30% (literature range, 5%-45%) of patients.

    Biologic false positive reactions. Some patients have definitely positive results on RPR but just as definitely do not have syphilis or any exposure to it. These are called biologic false positive (BFP) reactions. The major known causes of BFP reactions can be classified under three headings:

    1. Acute BFP reactions, due to many viral or bacterial infections and to many febrile reactions such as hypersensitivity or vaccination. These usually give low-grade or moderate (1 to 2 +) STS reactions and return to normal within a few weeks.
    2. Chronic BFP reactions, due to chronic systemic illness such as antiphospholipid antibodies (Chapter 8), rheumatoid-collagen diseases, malaria, or chronic tuberculosis. There is also an increased incidence of BFP reactions in old age. Whereas there is less than 2% incidence of BFP reactions in men before the age of 60 years, some studies report an incidence as high as 9%-10% after age 70.
    3. Nonsyphilitic treponemal diseases such as yaws, pinta, Borrelia (Lyme disease), or relapsing fever.

    Summary. The cardiolipin STS is cheap, easy to do, and suitable for mass testing. Its sensitivity and specificity are adequate, and positivity develops reasonably early in the disease. Reagents are well standardized and reproducibility is good. Disadvantages are relatively poor sensitivity in primary syphilis, the tendency in late syphilis for spontaneous reversion of a reactive test result to a nonreactive result, and the problem of BPF reactions. To add further to the confusion, some patients with BFP reactions may have syphilis in addition to one of the diseases known to give BFP reactions. Because of this, everyone hoped for a way to use T. pallidum organisms themselves as antigen rather than depend on the nonspecific reagin system.

  • Immunologic Tests

    Immunologic tests for syphilis depend on the fact that the diseases caused by infectious organisms are characterized by development of antibodies toward that organism. These antibodies may be specific or nonspecific (Table 15-1). Nonspecific antibodies may be either of the cross-reacting type (sharing a common antigen with another organism) or of the fortuitous type (provoked by some nonspecific portion of the organism).

  • Dark-Field Examination

    Dark-field is a wet-preparation method for direct visualization of living T. pallidum spirochetes in material from syphilitic lesions. A microscope with a special dark-field condensor is required. This condensor prevents light from passing directly through the slide being viewed into the microscope viewing lenses but instead forces the light to pass to the periphery of the slide. Objects on the slide reflect some light up into the viewing lenses, so the objects appear white on a dark background. Dark-field was the only way to make the laboratory diagnosis of syphilis before serologic tests were available. The usual indications for this test are suspected primary stage syphilitic lesions and any suitable secondary stage lesion. Dark-field examination may be the only way to make a diagnosis early in the primary stage, since immunologic test antibodies often do not appear until late in the primary stage. Obtaining the specimen without contamination by blood or surface bacteria is very important. The lesion should be cleansed thoroughly with water or 0.85% saline and a sterile gauze pad. No soap or antiseptics should be used. Care must be taken not to produce bleeding, since red blood cells (RBCs) will obscure the organisms. After the lesion is blotted dry, a clear serous exudate should accumulate in a few minutes. If it does not, the lesion may be abraded gently with gauze, but not enough to cause bleeding. The serous fluid exudate is drawn off in a pipette or capillary tube for examination with a dark-field microscope. The causative organism, T. pallidum, has a characteristic morphology and motility on dark-field examination, but experience is necessary for interpretation, since nonpathogenic varieties of spirochetes may be found normally in the genital areas. Sensitivity with reasonable experience is reported to be about 75%. Dark-field examination by experienced personnel is no longer widely available in many areas because it is rarely ordered. If it is unavailable, a serologic test using the fluorescent treponemal antibody-absorbed (FTA-ABS) method could be substituted. However, the FTA-ABS test will miss 10%-20% of cases, with the possibility of error being greatest in the first week of the primary stage.

  • Syphilis

    Syphilis is caused by the spirochete Treponema pallidum. Clinical syphilis usually is subdivided into primary, secondary, latent, and tertiary (late) stages. The primary stage begins after an average incubation period of 3-6 weeks (range, 1.5-13 weeks) and is manifested by the development of a primary stage shallow ulcer, or chancre, near the site of infection. The time of appearance is variable, and the lesion often is inconspicuous and overlooked, especially in the female. The primary stage lasts about 4-6 weeks (range, 2-8 weeks) during which the chancre usually heals, followed by the secondary stage, which lasts about 4-12 weeks. About 80% of patients develop a rash, which in addition to other areas typically involves the palms and soles. Mucous membranes are involved in about 60% of cases. There is generalized lymphadenopathy in 75%. Asymptomatic central nervous system (CNS) involvement has been documented in up to 30% of patients. During the last part of the secondary stage the visible lesions disappear and the patient enters the latent stage. This lasts, on the average, 3-5 years. About 25% of patients relapse temporarily into active disease during the first 1-2 years. By the end of the latent period about one half of untreated patients apparently achieve spontaneous cure or at least do not develop further evidence of the infection. About 25% remain in a latent (“late latent”) status, and the remaining 25% develop tertiary stage sequelae such as neurologic, cardiovascular, or ocular syphilis.

    Diagnostic procedures in syphilis include dark-field examination, immunologic tests, and cerebrospinal fluid (CSF) examination, depending on the clinical situation.

  • Antibiotic Removal

    One of the major frustrations in microbiology is the interference to bacterial growth caused by previous administration of antibiotics. Culture growth inhibition may occur even if the organism is not sensitive to the antibiotic. Several antibiotic removal devices are now available, based on membrane filtration or resin adsorption. These are useful for liquid specimens such as spinal fluid, body cavity fluid or blood. Although some evaluations have found these devices to be helpful, others have not. This technique was (and is) controversial among infectious disease specialists.

  • Serum Bacteriostatic or Bacteriocidal Concentration (Schlichter Test)

    In patients with bacterial endocarditis whose symptoms persist despite treatment, it is important to know whether antibiotic therapy really is effective in vivo. Also, some antibiotics, such as gentamicin, have a therapeutic range close to the toxic range. Blood levels of some antibiotics can be measured using various methods. This will provide some assurance that antibiotic blood levels have been reached that ordinarily should be effective. However, this does not guarantee that the organisms are in fact being sufficiently inhibited or killed. To estimate in vivo effectiveness, a Schlichter test may be performed. A patient blood specimen is obtained 15 minutes after IV infusion of the antibiotic (1 hour after an intramuscular dose). This represents the peak antibiotic level. If a trough level is desired, the specimen is drawn just before the next antibiotic dose is to be given. A standard suspension of the organisms previously cultured from the patient is placed in serial dilutions of patient serum and incubated overnight. If the lowest serum dilutions (i.e., those tubes with the least dilution of the antibiotic) do not inhibit growth of the organism, therapy is probably not effective. There is some dispute on which dilution level to use as the peak value cutoff for therapeutic effectiveness; Schlichter accepted 1:2, but currently the majority of investigators require 1:8. In fact, for a test that is widely used and relied on, there is a surprising amount of disagreement about many critical technical aspects, including whether to obtain peak or trough level or both and what their optimal values are; whether to make the patient serum dilutions with saline, Mueller-Hinton broth, nonantibiotic-containing serum, or a mixture of these; what criteria should define the bacteriocidal endpoint; and so forth. For this reason some investigators doubt the value of the test.