Laboratory diagnosis of syphilis

162 CLINICAL I M M U N O L O G Y Newsletter

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Laboratory Diagnosis of Syphilis Sheila A. Lukehart Department of Medicine, Division of Infectious Diseases, University of Washington, Seattle, Washington yphilis is one of the oldest recognized sexually transmitted infections, first clearly described as epidemic in Europe during the 15th century. The early manifestations were quite severe by modern standards, and syphilis was often fatal in the secondary stage. The disease was termed "the great pox" in contrast to the other epidemic disease, smallpox. Although the sexual mode of transmission and the early clinical manifestations were recognized early, the late complications were recognized only centuries later. The causative agent, Treponema pallidum subsp, pallidum, was first identified by Schaudinn and Hoffman in 1905 and the first serologic test (the Wassermann test) was developed in 1906.

S

Cfinical Course of Syphilis Syphilis is the prototype for spirochetal infections, which are generally characterized by discrete clinical stages and long periods of asymptomatic infection. As shown in Figure 1, the clinical course of untreated syphilis spans the lifetime of the individual, with only intermittent clinical disease. The primary lesion, or chancre, is a painless indurated ulcer, usually found in the genital region and accompanied by regional lymphadenopathy. This lesion is initially teeming with treponemes, but heals spontaneously after several weeks following clearance of the organisms. This host immune response is thought to be mediated primarily by cytokine-activated macrophages and opsonic antibody) 3 The patient may then enjoy an asymptomatic period before development of the skin rash and generalized lymphadenopathy that mark the secondary stage. The skin lesions represent loci of treponemal multiplication, with cellular infiltration and subsequent bacterial clearance and healing. The patient then enters the latent stage, which, by definition, has no clinical manifestations. Even untreated, 70% of individuals remain in the latent stage for their

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lifetimes. The other 30% develop, after many years or decades, one or more of the tertiary complications of syphilis. These include symptomatic neurosyphilis, cardiovascular disease (aortitis, aneurysm), and gummatous destruction of skin, bones, cartilage, or other tissues.

tions that viable organisms are poorly reactive with immune serum. This may also contribute to the ability of the organism to survive in the face of an active humoral and cellular host response. Although several protein and lipoprotein antigens have been identified, these are now thought to be located in the cytoplasmic membrane and or the periplasmic space and are not exposed to circulating antibody. A number of these antigens have been shown to induce secretion of inflammatory mediators, including interleukin- 1 (unpublished) and tumor necrosis factor,16by macrophages, or macrophage-like cell lines. This may contribute to the ability of the organism to disseminate and to trigger the cellular infiltration that defines the various clinical manifestations of syphilis. T. pallidum is not known to elaborate any toxins or to have cytopathic capacity; thus, the tissue destruction observed during syphilis has been ascribed to the host's immune response rather than to the organism itself. The histologic appearance of early and late

Pathogenesis of Syphilis The pathogenesis of syphilis is poorly understood. The organisms, which can achieve very high numbers at localized tissue sites, appear to be located primarily in the extracellular spaces, although the ability of this organism to invade virtually any tissue via endothelial tight junctions clearly contributes to its characteristic multiple organ system involvement. T. pallidum can also reside intracellularly, which may contribute in part to the chronic nature of the infection. Recent freeze fracture electron microscopic studies ~7'2°have revealed that the outer surface of the intact organism is largely devoid of surface exposed proteins, extending early observa-

Figure I. Clinical stages of syphilis

I PrimarYl I Secondary I I Latent

I Central Nervous System Involvement-Asymptomatic I Meningeal I I Mening°vascular I I Paresis I,

,

/

/

Weeks Months © 1994ElsevierScienceInc.

I Tabes dorsalis Cardiovascular

I

Gumm,

Years/Decades

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lesions, consisting primarily of T lymphocytes and macrophages, is consistent with this hypothesis.

Diagnostic Approaches to Syphilis The diagnosis of syphilis is based upon clinical presentation, identification of the causative agent, and serological testing. No single diagnostic method is sufficiently sensitive and specific for all stages of syphilis, and each stage has different diagnostic requirements. Clinical Presentation Syphilis has been called "the great imitator" because of its multiple organ involvement and varied clinical manifestations. Syphilis must be considered in the differential diagnosis of widely disparate signs and symptoms including skin rashes, dementia, stroke, visual or hearing loss, headaches, alopecia, neurological abnormalities, and liver or kidney dysfunction. Although syphilis is responsible for approximately 20% of genital ulcer disease in the United States and Europe, it must be considered in the differential diagnosis of all genital ulcers. While the classical primary chancre is a painless, well-circumscribed, indurated ulcer, many patients have atypical lesions that can be readily confused with genital herpes or chancroid. Similarly, the rash of secondary syphilis can be confused with eczema, scabies, tinea versicolor, and condylomata lata can be misdiagnosed as the more common genital warts. In latent syphilis, there are no clinical manifestations upon which to base a diagnosis and few, if any, clinical findings that are pathognomonic for tertiary syphilis. The accuracy of clinical diagnosis of syphilis in good STD clinics has been estimated to be only 42 to 78%; 2,3 in settings where syphilis is seen less frequently, the accuracy would be even lower. Thus, laboratory testing is essential in establishing an accurate diagnosis. Demonstration of Treponema pallidum

Treponema pallidum cannot be grown in vitro, making culture diagnosis of syphilis impossible. Similarly, the organism stains poorly with normal bacteriological stains, (pallidum, meaning "pale"), and Gram stain examination is useless. The organism can be seen in lesion exudates, however,

by darkfield microscopy, and a trained microscopist can distinguish T. pallidum from the harmless commensal spirochetes by its characteristic morphology and motility. This is the simplest and least expensive method for establishing a diagnosis of early syphilis. Unfortunately, darkfield microscopes and the requisite trained microscopists are a vanishing breed, consigned largely now only to STD clinics. Alternatively, treponemes in lesion exudate can be identified in the laboratory using the Direct Fluorescent Antibody--T. pallidum (DFA-TP) test. In this test, fluoresceintagged polyvalent rabbit anti-T, pallidum (absorbed with commensal treponemes to remove crossreactivity with the endoflagellar antigens) is used to stain exudate specimens on transported slides. This test is as sensitive and specific as darkfield microscopy, but is used infrequently by clinicians. A similar monoclonal antibody-based test is available in research laboratories) New antigen detection tests are currently under development. In an unpublished study, the ELISA-based Visuwell test was evaluated on 188 swab specimens from patients with genital ulcer disease. Compared to final diagnosis based upon serologic findings and darkfield microscopy, the Visuwell was found to be 81% sensitive and 90% specific. Given that the swab specimens must be transported to a laboratory for testing, this test has no apparent advantages over the DFA-TP. PCR has been applied to the diagnosis of many infectious diseases, including syphilis. 1,4'~,7,18'21While T. pallidum DNA can be identified in clinical specimens from chancres, CSF, and amniotic fluid, a careful analysis of the sensitivity and specificity of PCR in clinical specimens has not been conducted. In laboratory mock-up specimens, the limit of detection of T. pallidum by PCR is 1 to 10 organisms. With the exception of CSF and amniotic fluid specimens, any antigen detection method for syphilis will be limited to those patients with active clinical manifestations. From a public health viewpoint, these infectious cases are important to identify, although they account for only a quarter of the total cases of syphilis reported each year.

Serologic Testing Because of unavailability of darkfield microscopy and the inconvenience of other T. pallidum detection methods, many clinicians rely heavily on serologic tests for diagnosis of syphilis. Serologic testing is based upon a two-tiered system (shown in Table I) in which nontreponemal tests are used for screening and determination of antibody titer, and the treponemal tests are used for confirmation. There are several reasons for the use of a two-tiered system: Over 40 million serological tests for syphilis are performed in the United States every year (including blood banking, prenatal, and premarital screening), and a sensitive, easy and inexpensive test is ideal for these purposes. The nontreponemal tests, which measure antibodies to a cardiolipin-cholesterol-lecithin antigen, meet these criteria. Further, some of the nontreponemal tests (e.g., RPR and TRUST) can be performed macroscopically on unheated serum, making them amenable to a STD clinic setting where the physician's goal is to establish a diagnosis and initiate therapy at the initial visit. The sensitivities of the various screening tests are comparable, but vary with stage of infection (Table 2), Antibodies generally first appear during the primary stage, and it is possible that a patient with a recently appearing chancre will be nonreactive in serologic tests; thus a nonreactive serologic test cannot rule out syphilis in a patient with suspicious genital ulcer. The tests are uniformly reactive, usually in high titer, during the secondary stage. Rarely, a very high-titer serum will give an unusual "rough" or negative reaction when tested undiluted. This is due to the prozone phenomenon and can be overcome by testing dilutions of the serum. All sera that are reactive in nontreponemal tests should be tested quantitatively to determine the patient's antibody titer. A significant increase (fourfold) in liter indicates active infection, while a fourfold decline in titer after therapy indicates efficacy of the therapeutic regimen. Either the RPR or VDRL test can be used quantitatively; however, titer comparisons should always employ the same test for both specimens. The titer will decline reproducibly after effective therapy, and cliII

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pallidum. The FTA-ABS is more labor-in-

TABLE 1. STANDARD SEROLOGICALTESTS FOR SYPHILIS O a ~ of test

Nontmponemal

Uses

Type of test

Examples

tensive and expensive than the hemagglutination tests, but has the advantage of being slightly more sensitive in primary syphilis. All treponemal tests are equally sensitive after the primary stage. In the United States, the treponemal tests are rarely used quantitatively, except in research settings. Unlike the nontreponemal tests, the treponemal tests will often remain reactive even after adequate therapy; their utility is limited, then, in patients with prior syphilis.

Specimens

Screening and Rapid Plasma Reagin quantitation (RPR)

Macroscopic fiocculation

Unheated plasma or serum

Screening and Venereal Diseases quantitation Research Laboratory

Macroscopic flocculation

Heat-inaaivated serum Cerebrospinal fluid

(VDRL)

TreponemM

Screening

Toluidine Red Unheated Serum Test (TRUST)

Macroscopic flocculation

Unheated serum or plasma

Screening

Unheated Serum Reagin CUSR)

Macroscopic flooeulation

Unheated serum

Screening

Reagin Screen Test (RST)

Macroscopic flocculation

Unheated senma or plasma

Confirmatory

Fluorescent Treponemal Antibody-Absorbed

Immuno~uoresccnce

Senma (CSF rarely)

Serologic Testing in HIV-Infected Patients

(FTA-ABS) Confirmatory

MicrohemagglutinationT. pallidum (MHA-TP)

Hemagglutination Senun (CSF rarely)

Confirmatory

7".pallidum

Hemagglutination Serum (CSF rarely)

Hemagglutination Assay (TPHA)

nicians often rely on follow-up titers to detect treatment failure or relapse. Many patients treated for fast-episode early (primary or secondary) syphilis will revert to nonreactive in the nontreponemal tests, usually within 1 year of therapy. The disadvantage of the nontreponemal tests is that "biological false positive (BFP)" reactions can occur due to autoantibody formation. Some individuals have short-term (acute) BFP reactions following viral infections or immunizations; others have BFP reactions for months or years (chronic), often due to underlying collagen vascular disease, systemic lupus erythematosus, other autoimmune disorders, aging, or injection drug use. The BFP rate in young healthy individuals is <1%; however, in geriatric populations, the rate can be as high as 10%. When false positive tests occur, they are usually, but not always, of low titer (<8 dils). In order to rule out BFP reactions, all sera that are reactive in nontreponemal tests are routinely examined using a confirmatory treponemal test. The treponemal tests measure antibodies to whole, fixed (FTA-ABS) or sonicated (MHA-TP, TPHA) T. pallidum. Because most people have antibodies that cross-react with T. pallidum (produced in response to antigens shared with oral or mucosal 0197-1859/94/$0.00 + 7.00

commensal treponemes), the specificity of the treponemal tests is increased by dilution of the test serum in "sorbent," a culture filtrate of Treponemaphagedenis, biotype Reiter. Even these steps are not sufficient, however, to remove all cross-reactive antibodies: patients with Lyme disease are often reactive in the FTA-ABS test, though not in the nontreponemal tests. In the FTA-ABS test, antibodies are detected by fluorescein-tagged anti-human IgG following reaction of the patient's serum with whole T. pallidum fixed to a microscope slide. The presence of brightly fluorescing treponemes on the slide indicates the presence of anti-treponemal antibodies in the test serum. In the MHA-TP and TPHA tests, anti-treponemai antibodies are detected by their ability to agglutinate erythrocytes coated with sonicated T.

Several case reportss':9 have suggested that persons with biopsy-proven syphilis and coexisting HIV infection may be falsely nonreactive in both treponemal and nontreponemal tests, suggesting that serologic diagnosis of syphilis may be unreliable in HIV-infected persons. Although large controlled studies have not addressed this issue, such reports appear to be isolated cases. Most experts agree that existing serologic tests are accurate for diagnosis of syphilis in the vast majority of HIV-infected individ~tal~, In seeming contrast to these reports, several studies have demonstrated higher RPR titers in HIV-infected patients with early syphilis than in HIV-uninfected patients) °':s There is controversy in the literature about the rate of titer decline following therapy in HIV-infected patients: some studies show no difference in rate of decline between HIV-infected and uninfected patients,TM while others suggest a slower rate of decline in HIVinfected individuals. It should be noted that treatment failure appears to he more common in HIV-infected patients, suggesting that those patients with slow declines in titers should be evaluated carefully to determine if re-treatment is warranted.

TABLE 2. SENSITIVITY OF SEROLOGICTESTS FOR SYPHILIS* Stage of disease

Primary

Secondary

Latent

Tertiary

VDRL, RPR

59-87

100

75-100

75-100

FTA-ABS

86-100

100

96-100

96-100

MHA-TP, TPHA

65-87

I00

96-100

94-100

aPercentreactive

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New Serologic Tests for Syphilis There has been disappointingly little advance in serologic testing for syphilis in the past 20 years. Most new tests are simply variations on old themes---for example, reworking the nontreponemal tests in an ELISA format--with higher-level technology and higher cost but no increase in sensitivity or specificity. Serologic tests based upon cloned T. pallidum antigens are currently under development, but no large trials have been conducted to date. These tests have the potential for increased specificity, but may not be as cost-effective for large-scale screening (i.e., blood bank) as the current two-tiered system. Special Considerations in Syphilis

Diagnosis

Examination of Cerebrospinal Fluid

Treponema pallidum invades the central nervous system in at least 40% of syphilis patients within the first weeks or months of infection) 2 Infection of the CNS is demonstrated by mononuclear pleocytosis, elevated protein concentration, and development of VDRL and treponemal antibodies. Many untreated patients will control their CNS infection and resolve CSF abnormalities by the end of 2 years of infection, although approximately 15 to 25% will have persistent CNS involvement and CSF abnormalities during the latent stage. CNS involvement may be completely asymptomatic, or may be manifested as chronic headaches, meningeal signs, hearing or visual deficits, uveitis, stroke, or classic paresis and tabes dorsalis. CSF examination is currently recommended for all syphilis patients with 1) coexisting HIV infection; 2) syphilis of 1 year's duration; and 3) any signs or symptoms of CNS involvement. In neurosyphilis, the degree of pleocytosis and protein elevation may be low in asymptomatic cases, and increasing in active neurosyphilis. The VDRL test is the only serologic test currently recommended for use on CSF. When reactive, it provides unequivocal proof of neurosyphilis; however, it is insensitive, and a nonreactive CSF-VDRL can never be used to rule out neurosyphilis. A recent study in HIV-infected patients, ~4in whom CSF pleocytosis and elevated protein concentrations can be due solely to HIV infection,

suggests that a nonreactive CSF MHA-TP can be used successfully to rule out neurosyphilis; on the other hand, reactive treponemal test is CSF may be present in individuals with no other evidence of CNS syphilis. Evaluation of Infants for Congenital Syphilis Infants born to women with untreated syphilis are at risk for congenital infection. Laboratory evaluation of these infants can be problematic. Maternal IgG is transferred across the placenta and can result in reactive serologic tests in the neonate regardless of infection status. In order to determine whether antibodies detected in the neonatal serum are of maternal or infant origin, one must test for anti-T, pallidum IgM. IgM is not transferred from mother to fetus, thus any specific IgM is of neonatal origin and indicates active infection. Two tests are currently available for detecting anti.T, pallidum IgM: the 19s-FTA-ABS and the Captia-M test. In the 19s-FrAABS test, the IgM fraction of the serum is separated by column chromatography prior to testing in the FTA-ABS test. This serum separation step avoids problems encounter with earlier tests, such as interference by rheumatoid factor and competition by higher avidity IgG. In the Captia-M test, IgM is captured by plasticbound anti-IgM, and then tested for reactivity with T. pallidum antigens in a modified sandwich ELISA format. Both tests are highly specific, but are not sensitive in infants with very early infection, as IgM is not yet being produced. Summary

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

Syphilis is a fascinating disease with a varied and perplexing array of clinical manifestations. Diagnosis is based upon clinical manifestations when present, identification of the etiologic agent, and serologic testing. A few new diagnostic tests have been developed in recent years but, with the exception of the IgM tests, most have no apparent advantage over existing standard approaches.

13.

14.

15. References I.

Burstain JM, Grimprel E, Lukehart SA, et al.: Sensitive detection of Treponema pallidtmz by using the polymerase chain reaction. J Clin Micro© 1994 Elsevier Science Inc.

16.

biol 29:62--69, 1991. Chapel TA, Brown WJ, Jeffries C, Stewati JA: How reliable is the moqahological diagnosis of penile ulcerations? Sex Trans Dis 4:150-152, 1977. Fast MV, D'Costa LJ, Nsanze H, et al.: The clinical diagnosis of genital ulcer disease in men in the tropics. Sex Trans Dis 11:72-76, 1984. Gordon SM, Eaton ME, George R, et al.: Response of symptomatic neumsyphilis to high dose intravenous penicillin G in persons infected with human immunodeficiency virus. In press, N Engl J Meal 331:1469, 1994. Gourevitch MV, Selwyn PA, Daveony K, et al.: Effects of HIV infection on the serologic manifestations and response to treatment of syphilis in intravenous drug users. Ann Intern Med 118:350355, 1993. Grimprel E, Sanchez P J, Wendel GD, et al.:Use of polymerase chain reaction and rabbit infectivity testing to detect Treponema pallidum in amniotic fluid, fetal and neonatal sera, and cerebrospinal fluid. J Clin Microbiol 29:17111718, 1991. Hay PE, Clarke JR, Taylor-Robinson D, et al.: Detection of treponemal DNA in the CSF of patients with syphilis and HIV infection using the polymerase chain reaction. Geoitourin Med 66:428-432, 1990. Hicks CB, Bensnn PM, Lupton GP, Tramont EC: Semnegative secondary syphilis in a patient infected with human immtmodeficiency virus (HIV) with Kaposi Sarcoma. Ann Intern Med 107:492, 1987. Hook EW, Roddy RE, Lukehart SA, et al.: Detection of Treponema pallidum in lesion exudate with a pathogen-specific monoclonal antibody, J Clin Microbiol 22:241, 1985. Hutchinson CM, Rompalo AM, Reichart CA, et al.: Characteristics of patients with syphilis attending Baltimore STD clinics. Multiple highrisk subgroups and interactions with human immunodeficiency virus infection. Arch Intern Med 151:511-516, 1991. Hutchinson CM, Hook EH Ill, Baker-Zander SA, et al.: Altered clinical presentation of early syphilis in patients with human immunodeficiency vires infection. Ann Intern Med 121:94-99, 1994. Lukehart SA, Hook EW Ill, Baker-Zander SA, et al.: Invasion of the central nervous system by Treponema pallidum: Implications for diagnosis and therapy. Ann Intern Med 109:855-862, 1988. Lukehart SA: Immunology and pathogenesis of syphilis. In: Quinn TC, Gallin Jl, Fauci AS (eds). Sexually Transmitted Diseases, Vol.8 in Advances in Host Defense Mechanisms. New York, Raven Press, 1992. Marra CM, Critchlow CW, Hook EW, et al.: The role of CSF treponemal antibodies in the diagnosis ofasymptomatic neurosyphilis. Arch Neurol. 52:68, 1995, Matlow AG, Rachlis A: Syphilis serology in human immunodeficiency vitals-infected patients with symptomatic neurosyphilis: Case report and review. Rev Infect Dis 12:703-707, 1990. Radolf JD. Norgard MV, Brandt ME, et al.: Lipo0197-1859D4/$0.00 + 7.00

166 CLINICAL IMMUNOLOGY Newsletter proteins of Borrelia burgdorferi and Treponema pallidum activate cachectin/tumor necrosis factor synthesis. Analysis using a CAT reporter construct. J Immunol 147:1968-1974, 1991 17. Radolf JD, Norgard MV, Schuiz WW: Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum. Proc Natl Acad Sci USA 86:2051-2055, 1989. 18. SanchezPJ, Wendel GD, Grimprel E, et al.:

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Evaluation of molecular methodologies and rabbit infectivity testing for the diagnosis of congenital syphilis and neonatal central nervous system invasion by Treponema pallidum. J Infect Dis 167:148-157, 1993. 19. Tikjob G, Russel M, Petersen CS, et al.: Seronegative secondary syphilis in a patient with AIDS: Identification of Treponenxt pallidtml in biopsy specimen. J Am Acad Dermatol 3:506-508, 1991.

20. Walker EM, Zampighi GA, Blanco DR, Miller JN, Lovett MA: Freeze-fracture analysis demonstrafes rare protein in the outer membrane of Treponema pallidum subsp, pallidum. J Bacteriol 173:5585-5588, 1991.

Borrelia burgdorferi, 12:153, 154, 155, 157-158 cross-reactions with T. pallidura, 12:160-161 Bruton's tyrosine kinase, mutations in, molecular pathogenesis of X-linked agammaglobulinemia and, 6:69, 70-76

cytotoxicity, cell-mediated, antibody-dependent, against HIV positive target cells, 8:106-110

21. Wicher K, Noordhoek GT, Abbroscato F, Wicher V. Detection of Treponema pallidum in early syphilis by DNA amplification. J Clin Microbiol 30:497-500, 1992.

I n d e x to V o l u m e 1 4 acute leukemia, 1lq23, recent progress and new questions, 3/4:40-41 acute lymphoblastic leukemia T-cell, 3/4:33, 34-37, 48-51 transcription factor deregulation in, Ttg-1 gene and, 3/4:48-51 1lq23 translocations in, 3/4:40, 41 acute nonlymphoblastic leukemia, 11q23 translocations in, 3/4:40 acute pmmyelocytic leukemia, molecular genetics and pathophysiology, 3/4:37-39 adenosine deaminase deficiency, 1:2, 5, 6 new therapies for, 10/11:, 133, 134-137 adjuvants, 9:127 antibody effects of, 9:127-128 induction of call-mediated immunity effects of, 9:128-129 agarnmaglobulinemia, X-linked, 2:19, 26 molecular pathogenesis of, mutations in Bruton's tyrosine kinase and, 6:69, 70-77 AIDS. see also HIV infection CD8 T-cell in, 7:85, 89-95 Amblyomma americanum, 5:65 Antennapedia-Bithorax complex, genes of, 3/4:41 antibodies, see also specific antibody adjuvant effects on, 9:127-128 VZV, 9:117, 118, 119 antibody-dependent cell-mediated cytotoxicity against HIV posilive target cells, 8:106-110 antigens, see also specific antigen borrelial, 12:155 apoptosis, autoimmunity and, 10/11:140-144 ataxia-telangi ectasia, 2:29-30 autoimmune disease, non-MHC T lymphocyles in, 1: 12 autoimmunity, apoptosis and lympbopoiesis and, 10/11:140-144 B-cells immunodeficiencies, combined with T-cell immunodeficiencies, 2:28-29 lymphopeiesis, 2:25 in X-linked hyper-lgM irnmunodeficiency, 6:78 Baer R, 3/4:33, 34-37 Bartonella, 5:58 Bash RO, 3/4:33, 34--37 Baum LL, 8:101,106-110 Baum M, 7:89-95 bcl-2-immunoglobulin transgenic mouse, pathogenesis of follicular lymphoma and, 3/4:52--56 Benach JL, 12:153, 154-156 bone marrow transplantation haploidentical, for adenosine deaminase deficiency, 10/11:135 for primary immunodeficiencies, 1:2-6 for x-linked severe combined immunodeficiency, 6:80 Borreha, surface-exposed epitopes of, immune response against, 12:156-161

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Cajulis RS, 1:7-10 cancer, see specific type of cancer Cassutt K, 8:106-110 CIM cells, 2:26 in HIV infection, 1:12, 13, 14 immunomodulation with, in H]V-1 disease, 7:86, 87 in T-ALL, 3/4:48, 49 CD8 cells, 2:26 in HIV infection, 1:12, 13; 7:85, 86-87, 89-95 in T-ALL, 3/4:48, 49 CD40 antigen, 6:77 CD40 gene, X-linked immunodeficiency and, 6:78 eDNA libraries, in non-Hodgkin's lymphoma-associated t(2;5) translocations, 3/4:45 cell death, programmed, peripheral tolerance and, 10/11 : 141, 143 ceil-mediated immunity defects of, 2:27-28 induction of, adjuvant effects on, 9:128-129 CEM.NKR, 8:109 cerebrospinal fluid, examination in HTLV-I- associated myelopathy, 10/11:149-150 in Lyme neumborreliusis, 12:154-155 in syphilis, 12:165 certification programs in clinical immunology, 10/11:145-148 Cevenini R, 12:156-161 Check IJ, 9:117, 118-119 Chediak-Higashi syndrome, 1:4.2:29 children, primary immunodeficiencies in, 1:2~5 chromosomes abnormalities, malignancies related to, fluorescence in situ hybridization applications to, 8:110-116 translocations, see translocations X, see X-chromosome; X-linked immunodeficiency diseases yeast artificial, 3/4:40; 6:71 chronic lymphocytic leukemia, chromosome anomalies in, 8:113 chronic myelogenous leukemia, 8:110 Clinical Laboratory Improvement Act (CLIA), 10/11:145 common variable immunodeficiency, 2:27 complement fixation, for scrub typhus, 5:62 Cowdria ruminantium, 5:65 Coxiella, 5:59, 60 Cytocetes ondiri, 5:65 cytogenetics, interphase, as potential tool in cytodiagnosis of urinary bladder carcinoma, 1:7-10 cytokines, control of'HI 1 and TIt 1 immune responses by, 9:129 cytometry qualification, 10/11:147-148

© 1994 Elsevier Science Inc.

D'Apote L, 12:156-161 death, in AIDS patients, 7:92-93 Dekaban G, 10/11:148-152 DiGeorge syndrome, 2:27-28 dipstick test, for rickettsial diseases, 5:60, 64 DNA branched method, 7:97-98 detection, in Lyme neuroborreliusis, 12:155 proviral, as marker in HIV-I disease, 7:96-97 Domer PH, 3/4:33, 34, 41)-43 Donnenberg AD, 10/11:140-144 Dormenberg VS, 10/11:140-144 dot-blot immunoassay, for scrub typhus, 5:64 Downing JR, 3/4:44-48 Drosophilia gene, 3/4:41 Dumler JS, 5:65-68

Ehrliclua, 5:57, 65, 66, 67 ehrlichiuses, human, serodiagnosis of, 5:65-68 ELISA (enzyme-linked immunosorbent assay) detection oflgG antibodies to B. burgdorferi in Lyme disease patients by, 12:159, 160 for scrub typhus, 5:62, 63 enzyme replacement therapy, 10/11:134--135 Farencena A, 12:156-161 Filipovich AH, 1:1, 2~5 Fitzgerald-Bocarsly P, 8:101-105 Fletcher MA, 7:85-89, 89-95 flourescent antibody to membrane antigen, 9:118 flow cytometry for myeloma diagnosis, 10/11,137-138 primary immunodeficiency disease evaluation by, 2:25-32 for urinary bladder carcinoma cytodiagnosis, 1:7.8 fluorescence in situ hybridization applications to hematopoietic malignancies, 8:110-116 in urinary bladder carcinoma cytodiagnnsis, 1:7, 8, 9 follicular lymphoma, pathogenesis, bcl-2-immunoglobulin transgenic mice and, 3/4:52-56 Frias-Hidvegi D, 1:7-10 Friedlaoder A, 7:89 95 Friedmann M, 6:79-83 Gamber C, 7:8549 T chain, common, involvement in X-linked severe combined immunodeficiency, 6:79-83 Garcia MN, 7:89-95 gene therapy for adenosine deaminase deficiency, 10/11:135-136 for primary immunodeficiencies, 1:1,2-6 genetic abnormalities, malignancies related to, fluorescence in s itu hybridization applications to, 8:110-116