Articles on Medical Diseases and Conditions

Tag: Drawbacks

  • 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 The TPI test has been replaced by the FTA-ABS and the microhemagglutination (MHA) tests (to be discussed later), and it is now very difficult, almost impossible, to obtain a TPI study in the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.

  • Urine Culture

    Contamination of urine specimens by vaginal or labial bacteria is a serious problem. The most reliable way to obtain the specimen, especially in young children, is by suprapubic puncture and aspiration of the bladder with a needle and syringe. However, this technique has never become popular. Catheterization is another way to solve the difficulty, but approximately 2% of patients are reported to develop urinary tract infection following a single catheterization. A midstream “clean-catch” voided specimen is the third best procedure. The urinary opening is cleansed. The labia are held apart in the female and the foreskin pulled back in the male. The first portion of urine is allowed to pass uncollected, then the sterile container is quickly introduced into the urine stream to catch the specimen.

    Quantitative urine cultures have now replaced simple cultures, since it has been generally accepted that a titer of 100,000 (105) or more organisms/ml has excellent correlation with clinical infection, whereas a titer of less than 1,000 (103) organisms/ ml is usually not significant. However, there are certain important reservations and exceptions to this commonly accepted statement. The original studies that established this concept were performed on nonsymptomatic women using clean-catch urine collection. In addition, it was found that a single urine specimen collected this way that contained more than 100,000 organisms/ml had only an 80% chance of representing true infection (i.e., there was a 20% chance that the specimen was contaminated). Two consecutive urine specimens containing more than 100,000 organisms/ml has a 95% chance of indicating true infection. If the patient is receiving antibiotics or if the specimen is obtained by catheterization, many authorities accept a colony count of 1,000 or more organisms/ml as significant. If the specimen is obtained by suprapubic puncture or by cystoscopy, any growth at all is considered significant. If the patient has symptoms of urinary tract infection (fever, chills, flank pain, and pyuria), many authorities are willing to accept a clean-catch specimen colony count of 1,000 or more organisms/ml as significant even if the patient is not taking antibiotics. Not all patients with symptoms of urinary tract infection have colony counts over 1,000 organisms, because some females who have dysuria and frequency have urethritis (acute urethral syndrome, discussed earlier) rather than cystitis. About 20%-30% of these cases of urethritis are due to Chlamydia trachomatis, which does not grow on standard urine culture media. There is also the possibility that some cases could be chlamydial in origin but that the urine culture becomes contaminated with other bacteria, which leads to a false impression of cystitis. Finally, in some cases of pyelonephritis there are few or intermittent symptoms, and the urine cultures may be consistently negative, intermittently positive, or contain relatively low numbers of organisms. If there is a strong suspicion of pyelonephritis, repeat cultures may be necessary if the initial culture is negative or equivocal.

    Use of the quantitative (titer) technique with a cutoff level of 100,000 organisms/ml is supposed to compensate for small degrees of unavoidable contamination, but contamination can easily be severe enough to give a positive result. Various studies using midstream specimens have shown a surprisingly high rate of contamination in the female. In my experience, a microscopic examination of centrifuged specimen sediment in which more than 10 squamous epithelial cells per low-power field (10 x ocular, 10 x objective) are demonstrated suggests possible contamination. Three or more species of bacteria isolated from the same urine specimen also suggests contamination if the patient is not on long-term indwelling catheter drainage. All available information emphasizes that good technique in collecting the specimen is essential. Also essential is getting the specimen to the laboratory as soon as possible after collection. Urine is an excellent culture medium, and specimens that stand for more than 1 hour at room temperature permit bacterial incubation and proliferation to the point that quantitative counts are not reliable. If delivery to the laboratory must be delayed, the specimen should be refrigerated. The specimen can be preserved up to 12 hours in a refrigerator (4°C).

    Quantitative urine culture has two other drawbacks. Culture involves trained technical personnel and relatively expensive media, thus curtailing use for mass screening to detect urinary tract infection. Culture takes 24 hours to determine bacterial quantity and another 24-48 hours to identify the organism, thus delaying treatment in suspected infection.

    Screening tests for bacteriuria

    Because of the delay inherent in culture methods, several screening tests for rapid detection of significant degrees of bacteriuria have been introduced. If organisms are seen on Gram stain of uncentrifuged urine, reports indicate about 93% (range, 88%-95%) probability of a positive (100,000 organisms/ml) quantitative culture. The same is said to be true of direct examination of unstained centrifuged urine sediment. Gram-stained centrifuged sediment apparently gives too many false positive results. Gram-stained uncentrifuged urine and unstained urine sediment examination are said to give relatively few false positive results. Microscopic examination of centrifuged urine sediment to detect pyuria (increased number of WBCs) is widely used to screen for urinary tract infection. However, pyuria is found in only about 70%-80% (range, 13%-93%) of patients with positive urine cultures, and about 30% (range, 2%-40%) of patients with pyuria do not have a positive urine culture. For example, in one group of infants with culture-proven urinary tract infection, 42% had normal urine sediment and no bacteria seen on Gram-stained smears of noncentrifuged urine. Leukocyte esterase dipsticks have been used as a substitute for microscopic examination. They are positive in about 80%-90% (range, 72%-98%) of patients with a positive culture. Some urine test dipsticks have both the leukocyte esterase and a biochemical nitrite test; if results of both are positive, this detects 85%-90% (range, 78%-100%) of positive results from culture. Several chemical methods have been advocated. The most successful include the triphenyl tetrazolium chloride (TTC) test, the Greiss nitrate test (previously mentioned), and the catalase test. Best results have been reported with the TTC test. Nevertheless, reports vary as to its accuracy, with a range of 70%-90% correlation with quantitative culture, including both false positive and false negative results. The Greiss nitrate test detects about 55% (range, 35%-69%) of cases with a positive culture. The catalase test is even less reliable.

    Finally, there are several other bacteriuria screening systems now available, and more keep appearing. The Marion Laboratories Bac-T-Screen filters the urine, trapping any bacteria on the filter, which is then stained to reveal presence of bacteria. Sensitivity (compared to culture) is reported to be about 93% (range, 88%-99%). However, false positive results are about 29% (range, 16%-55%). Two companies market tests using firefly luciferase (bioluminescence) that reacts with bacterial ATP. Three companies market automated screening instruments, two detecting turbidity produced by bacterial growth (Autobac and MS-2 systems) and one (AMS systems) scanning a multiwell card containing various test media. Results from all of these systems are similar to those of the Bac-T-Screen. The advantages of these systems are that negative test results suggest that culture would not be necessary. Results are available the same day. Drawbacks are that positive test results by any of these methods must be followed by quantitative culture (except possibly the AMS system). False positive test results are relatively frequent, and negative test results do not completely rule out urinary tract infection (especially since the sensitivity figures quoted earlier are for 100,000 organisms/ml, and detection rates become less if bacterial numbers are less).

    Drawbacks of urine quantitative culture

    In summary, although quantitative culture with a cutoff titer value of 100,000 organisms/ml is widely considered the most reliable index of urinary tract infection, there are several major limitations:

    1. Using a titer of 100,000 organisms/ml as the cutoff point for a positive culture decision eliminates specimens with low-grade contamination but does not identify those with greater degrees of contamination.
    2. The cutoff point of 100,000 organisms/ml does not apply to catheterized or suprapubic aspiration specimens and may be misleading if used to interpret them.
    3. There are circumstances in which fewer than 100,000 organisms/ml may be significant in clean-catch specimens.
    4. A positive culture does not reveal which area of the urinary tract is involved.
    5. Tuberculosis of the urinary tract, anaerobic infections, and chlamydial infections of the urethra give negative culture results on ordinary culture media.
    6. Bacteremia from many causes, even if transient, involves organisms that are filtered by the kidney and may produce a temporarily positive quantitative culture result.
    7. Some cases of urinary tract infection may give negative cultures at various times. In several studies, a sizable minority of patients required repeated cultures before one became positive. This may be due to the location of the infection in the kidney and to its degree of activity.

    Therefore, two cautions are required when one is interpreting a urine culture report. If the results suggest that urinary tract infection is present, the physician must be certain that the specimen was properly collected, especially in the female. If the result is negative, this does not rule out chronic pyelonephritis. The problem of pyelonephritis and bacteriuria is also discussed in Chapter 12.

    Kits with agar-coated slides that not only yield quantitative urine culture results but also act as a culture medium for the organisms are now available. These are reported to have 90%-98% accuracy in various university hospital laboratories when compared with standard quantitative culture methods. The average is about 95% reliability. There is some question whether office laboratories can approach this figure and what provision would be made to identify the organisms.

    It should be mentioned that a few studies have reported little or no difference in contamination rates between clean-catch cultures and no-precaution cultures.