Role of Serology in the Diagnosis of Toxoplasmic Lymphadenopathy

Transcription

1 REVIEWS OF INFECTIOUS DISEASES VOL. 9, NUMBER 4 JULY-AUGUST by The University of Chicago. All rights reserved /87/ $02.00 Role of Serology in the Diagnosis of Toxoplasmic Lymphadenopathy Robert G. Brooks;t Robert E. McCabe,t and Jack S. Remington From the Department of Immunology and Infectious Diseases, Research Institute, PaloAlto Medical Foundation, Palo Alto; and the Division ofinfectiousdiseases, Department ofmedicine, Stanford University Medical Center, Stanford, California Serologic results obtained in92cases of toxoplasmic lymphadenopathy diagnosed bylymph node biopsy were reviewed, and guidelines for serologic diagnosis of this disease were established. When tests were first performed within six months of onset of lymphadenopathy, single high titers of IgGtoxoplasma antibodies (suggestive of acute infection) were found with the Sabin-Feldman dye testandthe direct agglutination testin 930/0 and 760/0 of cases, respectively. Observations of significant rises in titer were uncommon because of the late acquisition of sera. Within the first six months aftertheonset of lymphadenopathy, IgM toxoplasma antibody was demonstrable by the double-sandwich IgM enzyme-linked immunosorbent assay in 88% of cases and by the IgM-immunofluorescent antibody test in 78%. 1\venty percent of patients who had serum samples drawn more than 12 months afteronsetof lymphadenopathy still had IgM toxoplasma antibodies demonstrable bythe enzyme-linked immunosorbent assay. No patient first tested six or more months after onset of lymphadenopathy was positive in the IgM-immunofluorescent antibody test. These results provide the basis for recommendations on the use of serologic tests for the diagnosis of acute toxoplasmic lymphadenopathy. Acute acquired toxoplasma infection is a common cause of asymptomatic lymphadenopathy in immunocompetentadults [1]. Although specific criteria for diagnosis of toxoplasmosis by lymph node biopsy havebeen described,biopsy resultsmay be nonspecific or confused with those obtained in other diseases [2-6], and diagnosis by serologic methods is preferable. Time of onset of lymphadenopathy is a reliable marker of acuteness of the infection [7-10]. Thus, patients with acute toxoplasmiclymphadenopathy diagnosed by biopsy constitute a population in which different serologic tests can be evaluated for their ability to detect this form of toxoplasmosis in otherwise normal, immunocompetent adults. Received for publication August 28,1986, and in revised form January 5, Dr. Brooks is the recipient of an Edith J. Milo Memorial fellowship and an Edward H. Heller Memorial Fund Fellowship. Dr. McCabe is the recipient of an Edith J. Milo Memorial Fellowhip and grant no. AI from the National Institute of Allergy and Infectious Diseases. Present address: Department of Medicine, Orlando Regional Medical Center, Orlando, Florida t Present address: Section of Infectious Diseases, Department of Medicine, VeteransAdministration Medical Center, Martinez, California Please address requests for reprints to Dr. Jack S. Remington, Research Institute, Palo Alto Medical Foundation, 860 Bryant Street, Palo Alto, California We studied sera from 92 patients with acute toxoplasmiclymphadenopathydiagnosedbybiopsy. We compared traditional methods of testingfor antibodies to Toxoplasma (the Sabin-Feldman dye test [Dr] and the IgM-indirect immunofluorescent antibody [IgM-IFA] test) with newer methods (the direct agglutination [Agg] test and the double-sandwich IgM enzyme-linked immunosorbent assay [DS-IgM ELISA]), and weestablished guidelinesfor interpretation of the test results. Materials and Methods During the period from July 1973through January 1984, we reviewed 114cases of toxoplasmic lymphadenopathy diagnosed by biopsy in immunocompetent patients. The histologiccriteria for toxoplasmic lymphadenopathy used in this study have been described [2]. Serologictests for toxoplasma infection wereperformed within sixmonths of the clinicalonset of lymphadenopathy in 92 (80010) of these patients. The latter group of patients forms the basis of this study. A prerequisite for inclusion was the measurement of both IgG and IgM toxoplasma antibodies in the first serum sample. Patients wereexcluded if (1) their first serum sample was not obtained within six months of the onset of clinical lymphadenopathy, (2) the date of onset of their clin- 775

2 776 Brooks. McCabe. and Remington icallymphadenopathy wasnot recorded, and/or (3) they had previously had an episode of lymphadenopathy suspected or known to be due to Toxoplasma. Patients were referred from a wide geographic radius, with most areasof the United States represented. The mean age of the patients was 29 years. There were 58 female and 34 male patients. Serology Serologic tests were performed on samplesof serum within 72 hr of receipt in our laboratory. Sera were then stored at -70 C. When multiple serum samples from a singlepatient were available, only those samples collected four weeks or more apart were tested in parallel. A serial rise in titer of two tubes or more was considered significant. A micromodification of the or was performed with fourfold dilutions of sera [11]. The Aggtest was performed as described by Desmonts and Remington [12]. In brief, a 50-J,11 volume of 0.2 M 2 mercaptoethanol was added to each well of a U shaped microtiter plate. Twofold dilutions of sera (starting with 1:20)were then prepared. A 50-J,11 volume of antigen (formalin-fixedwhole Toxoplasma gondii organisms)wasthen added to each well. The pattern of agglutination was read after 16hr of incubation at room temperature. The IgM-IFA test wasperformedas previously described [13]. Since rheumatoid factor [14] and antinuclear antibodies [15] cause false-positive results in the IgM-IFA test, serawere screened for their presence and were not utilizedif either test waspositive. Before 1980, serawere testedin twofolddilutionsbeginningat 1:10; thereafter, fourfold dilutions beginning at 1:16 were used. Because we have observed loss of IgM reactivity, as measuredby the IgM-IFA test, in several serafrozen for long periods(authors' unpublished observations), only sera tested within one month of receipt were included in the analysis of IgM-IFA test results. The DS-IgM-ELISA [16] was performed by the single-dilution method described by Siegel and Remington [17]. The walls of microtiter plates were coated, postcoated, and washed as originally described for the serial dilution method for DS-IgM-ELISA [16]. Test sera werediluted 1:100, and a l00-j,11 volume was added to each of three wells. Sonicated toxoplasmaantigen and then alkalinephosphataseconjugated F(ab)2'fragments of the IgG fraction of rabbitantiserumto T. gondiiwere addedto eachwell. The results were read in a Micro-ELISAreader set to measure absorbance at 405 nm (Dynatech Laboratories, Alexandria, Va.). Singlehigh- and mediumtiteredsera- assigned reference titersof 10.0 and 5.0, respectively - and a negative serum were tested on each plate as controls. In our laboratory, titers of ~2.0 are considered to represent significant amounts of IgM toxoplasma antibody. Statistics Data on all sera were anlayzedby month after onset of lymphadenopathy. Sera from weeks 1-4 were designated as month 1; weeks 5-8 as month 2; and so on. Because of relatively few data points after month 6, sera from months 7-12 were grouped and analyzed together, as were seraobtainedone through four years after onset. All serologic data were entered in a statisticalanalysis system (SAS) computer program. For purposes of analysis, or titers of <1:8,Aggtitersof <1:20, and IgM-IFA titersof <1:16 were recorded as 0; or titers of >1:64,000 and Agg titers of >1:40,000 were recorded as 64,000 and 40,000, respectively. The reciprocal of the titer was used for calculation of geometric means and standard errors of the means with the PROC MEANS function of the SASprogram [18]. Rank-sum analysis and Kruskal-Wallis tests were used for assessment of differences in serologic test results [18]. Differences were considered significant when the value of P was ~.05. Results Serologic Results for First Sera Obtained After Onset of qrmphadenopathy The serologic test results for the first serum sample obtained from each of the 92 patients are shown in figures 1 and 2. The IgG antibody responses measured by or in the patients' first serum samplesare shown in figure la. The mean or titer of 1:5,949 found in seratestedat month 1after onset of lymphadenopathy increasedto a maximum mean titer of 1:7,862 at month 2 and declined thereafter. The ranges of the mean or titers were similar at all months and were not significantlydifferent. Levels of IgG toxoplasmaantibody measuredin these sera by the Agg test are shown in figure lb. Mean titers

3 Toxoplasmic Lymphadenopathy: Serology (201 YAl, 'y (51 /, (29) '---...I I-----'------L...--~~J> )12 of 1:11,053 at month 1 increased to a peak titer of 1:15,600at month 3 and declined thereafter. Differences between individual months, however, did not reach statistical significance. Results ofigm antibody tests on initial serum samples are shown in figure 2. IgM-IFA titers (figure 2A) peaked at month 2 (mean titer, 1:335) and then decreased at months 3 and 4. Only one patient was first tested five months after onset of lymphadenopathy (titer, 1:64) and one patient six months after onset oflymphadenopathy (titer, <I :16)(data not shown). In the DS-IgM-ELISA (figure 2B), a peak mean titer of5.8 was reached at month 3. This titer did not differ significantly from titers noted at months 1,2, and 4. There were few data points after the fourth month to be evaluated. L..--_L..--_L I_-----L_-----I.~!---L...t,L--J ) 12 MONTH FOLLOWING CLINICAL ONSET OF LYMPHADENOPATHY Serologic Results for Follow-Up Serum Samples In figures 1 and 2 are also shown the serologic test results obtained when initial and follow-up samples werecombined. The 194sera wereobtained from one week to four years after onset oflymphadenopathy. The mean DT titer of 1:9,403 at month 3 (figure la) decreased to 1:1,516 for sera obtained more than one year after onset oflymphadenopathy. There were no statistically significant differences when the mean titers obtained for any ofthe months within the first year after onset of lymphadenopathy were compared. The mean titer for sera obtained more than one year after onset was significantly lower than the mean titer for each individual month within the first year. In the Agg test (figure IB), a peak titer of

4 778 Brooks, McCabe, and Remington A 500 a:: w 400 ~ ~ LL J c::x: U a:: a. u IJJ 100 a:: 0 B a:: w ~ 4.0 ~ (19) 2 (81 3 (17) 4 ;1~)'2-,_,.l A (;Q)r-'" "ii6lt',y -', (261 L...-_..l.--_~_...L..-_...L..-_...L-._--L--Ir--L-,t ~ >12 MONTH FOLLOWING CLINICAL ONSET OF LYMPHADENOPATHY 1:13,682 was obtained at month 3. The titer decreased to a mean of 1:6,899 for sera obtained more than one year after onset. Mean titers for months 1-12 did not differ significantly from one another. The mean titer for sera obtained more than one year after onset of lymphadenopathy was significantly lower than the titer at months 2-6 but not significantly different from that at month 1or months In the DS-IgM-ELISA (figure 2B), titers clearly decreased with time from a peak observed one to three months after onset of lymphadenopathy to lower values at six months or later. Mean DS-IgM ELISA titers in sera obtained more than one year after onset of lymphadenopathy were significantly lower than titers at all time points other than month 6. Five (50070) of 10patients with serum samples obtained between seven and 12 months after onset of lymphadenopathyand four (20070) of 21 patients with serum samples obtained more than 12months after lymphadenopathy had titers of ~2.0. Wedid not analyze data obtained with all samples combined in the Figure 2. Results of IgM-indirect immunofluorescent antibody test (A) and double-sandwich IgM ELISA (B) for the first serum (solid line) and for all sera (broken line) obtained after the onset of lymphadenopathy. Bar indicates SEM. Numbers of observations are shown in parentheses. IgM-IFA test since a decrease in titers with storage was observed in some samples of sera. Sensitivity of Different Serologic Tests for Acute Toxoplasmic Lymphadenopathy Since in some instances patients are not seen by a physician for weeks to months after they first note the onset oflymphadenopathy and in others the physician considers the diagnosis oftoxoplasmic lymphadenopathy late (e.g., after lymph node biopsy), we considered it of interest to examine the proportion of patients who had a given titer when their first serum sample was obtained within six months of onset of lymphadenopathy (tables 1-4). Sensitivity (i.e., the number of sera with a given cutoff titer) decreased progressively as the titer chosen for the prediction of acute infection was raised. The degree of sensitivity was if a cutoff titer of ~1:1,024 was used in the DT (table 1).Use of a DT cutofftiter of ~1:2,048 to define acute toxoplasmic lymphade-

5 Toxoplasmic Lymphadenopathy: Serology 779 Table 1. Use of the dye test to predict acute toxoplasmic lymphadenopathy by titer and month after onset of lymphadenopathy. No. (%) positive at indicated cutoff titer Month after onset (no. of patients) ~1:1,024 ~1:2,048 ~1:4,096 ~1:8,OOO ~1:16,OOO 1 (31) 30 (97) 23 (74) 17 (55) 8 (26) 2 (6) 2 (28) 26 (93) 23 (82) 21 (75) 14 (50) 6 (21) 3 (18} 17 (94) 15 (83) 11 (61) 5 (28) 2 (11) 4 (lo) 9 (90) 7 (70) 7 (70) 7 (70) 5 (50) 5 (3) 2 (67) 1 (33) 1 (33) 1 (33) 0(0) 6 (2) 2 (100) 2 (100) 1 (50) 1 (50) 1 (50) Total (92) 86 (93) 71 (77) 58 (63) 36 (39) 16 (17) nopathy resulted in a considerable loss of sensitivity. Analysis ofagg test titers (table2) yielded similar data and conclusions for titers of ~1:2,560. In the IgM-IFA test (table 3), relatively few patients had titers of ~1:64 in the first three months of lymphadenopathy. Sensitivity decreased markedly if only titers of ~1:256 were considered indicative of acute toxoplasmic lymphadenopathy. In the DS IgM-ELISA (table 4), of patients had titers of ~2.0during the first six months after onset of lymphadenopathy. Only of patients had a titer of ~5.0, and all ofthese titers wereobserved in the first four months after onset of lymphadenopathy. Sensitivity of Fourfold or Greater Rises in DT or Agg Test Titers A fourfold or greater rise in titer in sequential sera is considered indicative of acute infection. We analyzed DT and Agg test titers in sera obtained four or more weeks after the original serum sample (table 5). Ten (19070) of 53 patients had fourfold or greater rises in DT titer. Six ofthese rises were noted in follow-up sera obtained in the third month after onset of lymphadenopathy. In the Agg test, a fourfold or greater titer rise was demonstrable in 17(40010) of 42 patients. Discussion The purpose ofthis study was to determine the sensitivity of different serologic tests in the diagnosis of acute toxoplasmic lymphadenopathy. Lymphadenopathy is the most frequent clinical manifestation of acute infection with Toxoplasma [1, 19,20]. In outbreaks of infection with Toxoplasma, onset of clinical symptoms that include lymphadenopathy occurs within seven to 21 days after the presumed time of infection [7-9, 21]. Thus, patients with toxoplasmic lymphadenopathydiagnosedby biopsy provide a reference population for whom the time of infection can be estimated with some degree of accuracy. Diagnosis of acute toxoplasma infection by serologic methods would obviate the need for invasive and costly procedures, such as biopsy of lymph nodes or other tissue; furthermore, in the majority of cases, serologic testing may be the only feasible method for diagnosis. Table 2. Use of the agglutination test to predict acute toxoplasmic lymphadenopathy by titer and month after onset of lymphadenopathy. Month after No. (0/0) positive at indicated cutoff titer onset (no. of patients) ~1:2,560 ~1:5,120 ~1:lO,240 ~1:20,480 1 (23) 14 (61) 11 (48) 8 (35) 6 (26) 2 (25) 19 (76) 17 (68) 13 (25) 6 (24) 3 (17) 16 (94) 13 (76) lo (59) 6 (35) 4 (9) 7 (78) 6 (67) 6 (67) 3 (33) 5 (2) 2 (100) 2 (100) 2 (100) 2 (100) 6 (2) 1 (50) 1 (50) 0(0) 0(0) Total (78) 59 (76) 50 (64) 39 (50) 23 (29) Table 3. Use of the IgM-indirect immunofluorescent antibody test to predict acute toxoplasmic lymphadenopathy by titer and month after onset of lymphadenopathy. No. (%) positive at indicated Month after cutoff titer onset (no. of patients) ~1:16 ~1:64 ~1:256 1 (26) 20 (77) 17 (65) 7 (27) 2 (19) 17 (89) 15 (79) 6 (32) 3 (8) 7 (88) 7 (88) 4 (50) 4 (5) 3 (60) 3 (60) 2 (40) 5 (1) 1 (100) 1 (100) 0(0) 6 (1) 0(0) 0(0) 0(0) Total (60) 47 (78) 43 (71) 17 (28)

6 780 Brooks, McCabe, and Remington Table 4. Use of the double-sandwich IgM ELISA to predict acute toxoplasmic lymphadenopathy by titer and month after onset of lymphadenopathy. Table S. Use of fourfold or greater rises in dye test (DT) or agglutination test (Agg) titers to predict acute toxoplasmic lymphadenopathy. Month after No. (070) positive at indicated cutoff titer No. of patients with onset (no. fourfold or greater increase Month(s) after of patients) ~2.0 ~3.0 ~4.0 ~5.0 in indicated titer onset of 1 (26) 23 (88) 19 (73) 18 (69) 15 (58) lymphadenopathy DT Agg 2 (27) 25 (93) 21 (78) 18 (67) 12 (44) (17) 16 (94) 16 (94) 13 (76) 12 (71) (9) 7 (78) 7 (78) 6 (67) 5 (56) (3) 2 (67) 2 (67) 1 (33) 0(0) 6 (2) 1 (50) 1 (50) 1 (50) 0(0) Total (84) 74 (88) 66 (78) 57 (68) 44 (52) Total 10 (19070)* 17 (40OJo)t Traditional tests for toxoplasma antibodies have focused on measurement of IgG antibody. The Sabin-Feldman dye test is the "gold standard" to which othertests for IgG toxoplasma antibodies have been compared. Titers in the IgG-IFA test agree well with those found in the DT [22]. Within the first six monthsofinfection, of the patientsstudied had a DT titer of ~1:1,024. Use of a cutoff titer of ~1:2,048 resulted in a dramatic decrease in sensitivity of a single titer for detection of acute infection. High titers in the DT, however, are not specific for the acute infection [13,23]. Sixty-two percent ofpatients tested more than 12 months after onset of lymphadenopathystill had a titer of ~1 :1,024. In addition, only of patients whose follow-up sera were available had fourfold or greater rises in their DT titer suggestive of recent infection. This result is probably due to the fact that the DT titer is usually at or near its peak at the time when the first serum sample is obtained. These findings, observed by other investigators as well [10, 13, 23], demonstrate the difficulty of predicting acute toxoplasma infection by a single high titer in the DT. We were surprised to note that of our patients had negative DS-IgM-ELISA results. Although these results may have been truly negative, it is also possible that the acquisition oftoxoplasma infection by these patients was more remote than was estimated by the patients through recall. In addition, some patients may have chronic or recurrent lymphadenopathy due to Toxoplasma ([30] and authors' unpublished observations) in which IgM antibody may not be demonstrable. A patient recently evaluated at our institution had toxoplasmic lymphadenopathyproven by lymph node biopsyon two separate occasions one year apart. Although DT and DS-IgM- ELISA results were initially positive (titers * Seventy-nine serum samples from 53 patients were tested. t Sixty-three serum samples from 42 patients were tested. of 1:1,024 and 6.0, respectively), at the time of the second biopsy they were 1:64 and negative, respectively. The foregoing data provide the basis for our present recommendations on the use of serologic tests in the diagnosis ofacute toxoplasmic lymphadenopathy. We recommend that a test for IgM toxoplasma antibody be performed in all suspected cases of acute toxoplasma infection. If IgM antibody is demonstrated and this finding is supported by a test for IgG toxoplasma antibody, and if the clinical course is compatible with toxoplasma infection, then further serologic tests or biopsies are probablyunnecessary. A positive result in the IgM-IFA test would suggest infection within the preceding three to four months. A negative result in the IgM-IFA test, however, does not rule out recent toxoplasma infection since high titers ofigg antibodymay block a positive response in this test, even early in infection. Removal of IgG antibody by use of staphylococcal protein A [10] or other means [29] may result in a more reliable outcome of the IgM-IFA test. The DS-IgM-ELISA is more sensitive and specific than the IgM-IFA test. In addition, a recent publication has described a method of performing this test within 2 hr [31]. If the DS-IgM-ELISA result is positive and the precise date of onsetof infection is ofconcern to the physician (e.g., in a pregnant patient with lymphadenopathy), the IgM-IFA test may prove useful since its result becomes negative in the majority ofpatients within three to four months. In cases with equivocal serologic test results, follow-up serum samples should be examined for a rise in titer.

7 Toxoplasmic Lymphadenopathy: Serology 781 These sera shouldbe tested in parallel with twofold dilutions in an attempt to detect such a rise. In our experience the demonstrationoffourfold or greater rises in Dr titers is infrequent because such increases occur early in infection. In contrast, fourfold or greater rises in Agg test titers are frequently demonstrable, and we think that this test is underused for this purpose. IgM antibody titers usually decrease rather than increase unless the sera are obtained very early (e.g., within the first four weeks ofinfection). Whether the data and recommendations presented herein can be extrapolated to immunocompetent adults with suspected toxoplasma infection but without lymphadenopathy is not known. Patients who exhibit lymphadenopathy differ from people with other manifestations oftoxoplasma infection in their immune response to Toxoplasma [32, 33]. Differences in inoculum size, route of infection, form of organism, and virulence of the infecting strain [34] may well result in different humoral responses and thus in different serologictest results. Further studies are necessary to define serologic responses to toxoplasma infection in patients who are immunocompetent but who do not have lymphadenopathy and in patients who are immunosuppressed or pregnant. The Agg test was first described by Fulton and Turk in 1959[24]. Its early use was limited by a lack of both sensitivity and specificity, but improved methodology has made this test an acceptable alternative to the Dr in most clinical situations [12, 25, 26]. We confirmed the earlier peak in the DT than in the Agg test that has been noted by otherinvestigators [12]. As has been reported elsewhere [12], we were not able to determine the stage ofinfection on the basis of differences in DT and Agg test titers. Because of a later rise in Agg test titers, approximately twice as many patients were observed to have a fourfold or greater titer increase in this test as in the Dr. Fourfold or greater rises in Agg test titers often occurred, even many months after the onset oflymphadenopathy, whereas all fourfold or greater rises in Dr titers were seen in the first five months (table 5). Because no single titer in the DT or the Agg test can be used for the diagnosis of recently acquired infection, investigators have focused on means of detecting IgM antibody for diagnosis of the acute infection. We found that of patients had a positive IgM-IFA result when first tested within six months of the onset of lymphadenopathy. As observed previously ([27] and authors' unpublished ob- servations), titers in the IgM-IFA test rapidly diminished afterthree to four monthsofinfection. Failure to demonstrate IgM antibodies by the IgM-IFA test in sera of some patients with acute acquired toxoplasmosis has been noted by other authors [16, 28] and has been attributed to a blocking effect of high titers ofigg toxoplasma antibodies [10, 29]. It is of interest that in the present study the peak level of IgG toxoplasma antibody (as measured in the Dr) and the peak level of IgM toxoplasma antibody (as measured in the IgM-IFA test) were both found at month 2 after the onset oflymphadenopathy. Since IgG antibody blocks IgM antibody, we would have expected a decrease in IgM antibodyto occuras the IgG antibodytiter increased. The failure to find this correlation could reflect differencesin IgG subclasses or epitope specificity at various times after infection. The DS-IgM-ELISA was developed in an effort to improve the sensitivity and specificity oftests for detection of IgM toxoplasma antibody [16]. With the single-dilution modification of the DS-IgM ELISA [17], of our patients had demonstrable IgM toxoplasma antibody. It was noteworthy that of patients who were tested seven to 12months after the onset of lymphadenopathy still had a titer of ~2.0 in the DS-IgM-ELISA whereas only of patients (data not shown) had positive results in the IgM-IFA test during a similar period. Thus, the DS-IgM-ELISA is useful for the diagnosis of acute toxoplasma infection in the population of patients with signs suggestive of toxoplasmic lymphadenopathy, even when these patients present relatively late after the onset of infection. On the other hand, in the general population who have no signs of the infection, it may be difficult to interpret a positive result in the DS-IgM-ELISA. Since IgM toxoplasma antibodies may persist for periods longer than that usually considered to represent the period of acute infection, the usefulness of their demonstration in defining the recent onset of acquired infection may be diminished. Four ofourpatients haddemonstrable IgM antibody more than one year after infection; the titers were relatively low «4.0). Use of higher cutoff titers to increase specificity produced a marked decrease in sensitivity for acute infection. References 1. KrickJA, Remington JS. Toxoplasmosis in theadult- an overview. N Engl J Med 1978;298: Dorfman RF, Remington JS. Value of lymph-node biopsy

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