JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2001, p. 3164–3170 0095-1137/01/$04.00⫹0 DOI: 10.1128/JCM.39.9.3164–3170.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 39, No. 9
Antibody Responses to Recombinant Epstein-Barr Virus Antigens in Nasopharyngeal Carcinoma Patients: Complementary Test of ZEBRA Protein and Early Antigens p54 and p138 R. DARDARI,1 W. HINDERER,2 D. LANG,2 A. BENIDER,3 B. EL GUEDDARI,4 I. JOAB,5 A. BENSLIMANE,6 AND M. KHYATTI1* Institut Pasteur du Maroc,1 Centre d’Oncologie, CHU Averroes Ibn Rochd,3 and Centre d’Immunologie, Faculte´ de Me´decine et de Pharmacie,6 Casablanca, and Institut National d’Oncologie, CHU Avicenne, Rabat,4 Morocco; Biotest AG, Research & Development, Dreieich, Germany2; and Institut de Ge´ne´tique Mole´culaire, Paris, France5 Received 20 November 2000/Returned for modification 20 March 2001/Accepted 30 May 2001
Serological tests based on the antibodies directed against the Epstein-Barr virus early antigen (EA) and viral capsid antigen (VCA), which have been recognized as tumor markers for nasopharyngeal carcinoma (NPC), are routinely used to help in the diagnosis of this malignancy. The detection of these antibodies reveals very low titers, found only in a small proportion of young compared with older NPC patients. This is a problem for the diagnosis of NPC, especially among Maghrebians, among whom young people are also affected, and emphasizes the necessity to search for more reliable markers. The present study reports results of immunoglobulin G (IgG) and IgA responses of NPC patients to recombinant EA antigens p54 (BMRF1) and p138 (BALF2), VCA complex antigens p18 (BFRF3) and p23 (BLRF2), and EBNA antigen p72 (BKRF1). Our results show that IgA-EA-p54 and -p138 (IgA-EA-p54ⴙ138) antibodies have a diagnostic value for detection of NPC (70%), compared with IgA-VCA-p18ⴙ23 and IgA-EBNA-p72, which have limited diagnostic value, especially in young patients. It is also noteworthy that IgA-EA-p54ⴙ138 can detect a high percentage (64%) of NPC cases negative by immunofluorescence. These results, however, clearly show that a single test cannot achieve the objective of detecting all NPC patients, and it seems advisable to combine different tests for the diagnosis of NPC. The combination of IgG-ZEBRA with IgA-EA-p54ⴙ138 improved the sensitivity of detection of NPC to 95% in the overall NPC population. The use of IgA-EA-p54ⴙ138 in combination with IgG-ZEBRA will facilitate detailed studies on the pattern of antibody response, which may result in the development of useful serological markers to guide the treatment of NPC. dling, and difficult to standardize because of the variability of antigen-producing cells as well as the subjective reading of results. This makes their application in mass screening of populations not convenient. Some of the technical difficulties associated with IF have been overcome by the development of specific enzyme-linked immunosorbent assays (ELISAs), which are easily automated, quick to perform, and thus well suited for mass screening programs involving populations at high risk for NPC. The advent of monoclonal antibody technology, gene cloning, and expression of foreign proteins in cells and organisms has greatly facilitated our understanding of the profile of distinct antibodies to individual EBV polypeptides in patients with NPC and other EBV-related diseases (7, 8, 21). It is therefore worthwhile to address the question of whether antibodies against individual polypeptides might be more sensitive and specific than antibodies directed against the whole complex for diagnosing and monitoring patients with NPC. EA and nuclear antigen (EBNA)-specific ELISAs based on recombinant antigens have been successfully used in EBV diagnosis (6, 17). In contrast, the VCA IF assay serologically defines antigens that are more difficult to replace by recombinant proteins. This is related to the complexity of the VCA protein family and the lack of a complete definition of proteins within the VCA complex (18). In this study, we report results of IgG and IgA responses of NPC patients to the recombinant antigens p54 (BMRF1) and
Epstein-Barr virus (EBV) humoral immunology has played a major role in studies dealing with a relationship between this virus and nasopharyngeal carcinoma (NPC) (12, 13, 24). Detection of antibodies to the EBV viral capsid antigen (VCA) and EBV early antigen (EA) in sera by indirect immunofluorescence (IF) assays was one of the earliest tests developed. To date, the IF assays still serve as the “gold standard” of EBV serodiagnosis (10, 11, 13). These tests showed the importance of antibodies directed against some of the serologically defined EBV antigens in the diagnosis of EBV-associated diseases. They also help in the clinical management of patients with EBV-associated malignancies. Diagnostically relevant antibodies that have been identified by a number of investigators over the years are immunoglobulin G (IgG) and IgA antibodies directed against EA and VCA. The IgA-EA test, which is routinely used in many laboratories throughout the world, is one of the more specific EBV-associated NPC diagnostic tests available. Moreover, detection of anti-IgA antibodies by IF is suitable for the identification of patients with occult NPC, and the identification of populations at high risk for the development of this cancer (3, 12, 19, 25, 30, 31). However, the IF assays are time-consuming, not suitable for automatic han-
* Corresponding author. Mailing address: Laboratoire de Virologie, Institut Pasteur du Maroc, 1, Rue Abou Kacem Ez-Zahraoui, B.P. 120-Casablanca, Morocco. Phone: 212-22-26.94.24/27.57.78/27.52.06. Fax: 212-22-26.09.57. E-mail:
[email protected]. 3164
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p138 (BALF2) and p18 (BFRF3) and p23 (BLRF2) of the EA and VCA complexes, respectively. Seroreactivity to p72 (BKRF1), representing the EBNA complex, was analyzed concomitantly. We then compared the sensitivity and specificity of these antigens alone or in combination with IgG antibodies directed to the EBV transactivator protein ZEBRA (BZLF1), an immediate-early protein responsible for the switch between EBV latency and replication. The study focused on NPC, especially in young patients who show a high frequency of serological nonresponders by IF (i.e., IgA-EA and -VCA) (2). MATERIALS AND METHODS Patients and controls. One hundred sera were collected from patients with histologically proven NPC, admitted to the two public oncology centers of Morocco: the National Institute of Oncology in Rabat and the Oncology Center in Casablanca. Young patients were defined as those between 10 and 30 years old, and adult patients were defined as those over 30 years old. The control group consisted of 77 healthy individuals and 28 patients with cancers other than NPC (larynx, tongue carcinoma, and Hodgkin’s disease). Healthy individuals consisted of randomly selected blood donors, laboratory staff, and healthy parents of patients awaiting renal transplantation. Healthy controls were matched for sex and age. EBV-specific serology. All sera were titrated for IgG and IgA antibodies to VCA and EA by using the IF assay (14, 15). The P3HR-1 cell line was used to detect VCA antibodies, while Raji cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (40 ng/ml) and n-butyrate (17 mM/ml) were used for EA. Titers represent the highest serum dilutions at which clear IF staining of the positive target cells was observed. Detection of IgG and IgA antibodies to the EBV-VCA-p18ⴙ23, EA-p54ⴙ138, and EBNA1-p72 recombinant proteins by ELISA. The following recombinant antigens were used within an anti-EBV ELISA system (Biotest AG, Frankfurt am Main, Germany). VCA-p18⫹23 is an autologous fusion protein expressed in Escherichia coli, the N-terminal portion of which consists of full-length p23 (amino acids 1 to 162), followed by the carboxy half of p18 (amino acids 105 to 176). VCA-p18 (BFRF3) is a small capsid antigen, highly immunogenic in humans, and contains the essential B-cell epitopes presented by the carboxy region. VCA-p23 (BLRF2) is a small capsid antigen that demonstrated VCA-like antibody profiles in serological analysis. Both VCA components were connected by a 3-amino-acid linker as described previously (18). The EA-p54 and -p138 antigens were used separately as a mixture to test IgG and IgA antibodies. EA-p54 (full-length BMRF1) corresponds to a dominant immunogen of the EA-Diffus complex. EA-p138 (truncated BALF2) is the major DNA-binding protein. This highly reactive antigen is not detectable in an EA IF assay based on chemically induced Raji cells. EBNA1-p72, the major antigen of the EBNA complex, comprises the Cterminal part of the protein, but does not contain the glycine-alanine copolymer, a structural feature of EBNA1, which shows cross-reactivities with certain autoantibodies and cytomegalovirus. Detection of IgG antibodies to the EBV-ZEBRA protein by ELISA. Recombinant ZEBRA protein was purified from E. coli transformed by the recombinant pET3c plasmid containing the BZLF1 gene under the control of the T7 RNA polymerase promoter. The anti-ZEBRA titer of a serum was determined with an ELISA involving plates coated with 30 ng of purified protein per well. An EBV-negative serum and an NPC serum were used as negative and positive controls, respectively, in each microtiter assay (22). The titer of the positive control was set at 9,600. The tests were repeated when a substantial deviation from this value was observed. The EBV-ZEBRA-specific ELISA titers were defined by the amount of anti-ZEBRA antibody in the serum measured by the endpoint dilution method as previously described (29). The endpoint dilution was defined as the intersection between the dilution curve of a given serum and the negative/positive cutoff curve. Determination of cutoff values. The cutoff values were calculated as the mean optical density (OD) of negative controls plus 3 standard deviations (SD) for IgA-EBNA-p72, 2 SD for IgA-EA-p54⫹138, and 1 SD for IgG-EA-p54⫹138 and IgA-VCA-p18⫹23. Clinical sensitivity and specificity. Sensitivity of the tests was defined as the percentage of NPC individuals detected positive, and specificity was defined as the percentage of negatives scored among healthy individuals.
3165
Statistical analysis. The Mantel-Haenszel 2 and Student’s tests were used to compare the significance of differences of the trend in seropositivity rates and the mean titers and mean OD values of various antibodies between NPC patients and controls, between young and adult NPC patients, and between young controls and older controls. Pearson’s correlation analysis was performed to determine correlations among different types of antibodies as detected by IF and ELISA tests.
RESULTS Immunoreactivity of sera from NPC patients, healthy donors, and patients with other tumors to VCA-p18ⴙ23, EAp54ⴙ138, and EBNA-p72 antigens. One hundred sera from NPC patients, 77 sera from healthy donors, and 28 sera from patients with tumors other than NPC were tested for their IgG and IgA immunoreactivities to EA, VCA, and EBNA recombinant antigens (Table 1). IgG antibodies specific for EAp54⫹138 were detected in 86% of NPC sera, compared with only 10% in healthy donors and 22% in patients with other tumors (P1 ⬍ 0.001, P2 ⬍ 0.001). IgA antibodies against EAp54⫹138 were detected in 70% of NPC sera, in only 3% of healthy donor sera, and in 11% of sera from patients with other tumors (P1 ⬍ 0.001, P2 ⬍ 0.001). A significant difference was also observed in the mean OD value of both IgG and IgA antibodies against EA-p54⫹138 between NPC patients and healthy donors (1.246 versus 0.573, P1 ⫽ 0.00031; 1.518 versus 0.521, P1 ⫽ 0.0003) and between NPC patients and patients with other tumors (1.246 versus 0.533, P2 ⫽ 0.00041; 1.518 versus 0.485, P2 ⫽ 0.0004). IgG-VCA-p18⫹23 antibodies were detected in more than 97% of sera from both NPC patients and the two control groups (i.e., healthy donors and patients with other tumors), with a significant difference in the mean titer of these antibodies between the NPC patients and the two control groups. In fact, the mean titer of IgG-VCA-p18⫹23 antibodies was 1,815 in NPC patients, compared with 673 in healthy individuals and 863 in patients with other tumors (P1⫽0.001, P2⫽0.0019). IgA antibodies against VCA-p18⫹23 were detected in only 30 and 26% of patients with NPC and patients with other tumors, respectively, compared to 6% in healthy individuals, with no significant difference in the mean OD values of these antibodies between NPC patients and the control groups. The comparison of the positivity rate of IgA antibodies against EBNA-p72 showed a very significant difference between NPC patients and the two control groups. In fact, 42% of NPC patients were positive for these antibodies compared to 3% of healthy donors and 0% of patients with other tumors (P1 ⬍ 0.001, P2 ⬍ 0.001). A significant difference was also observed in the mean OD value of IgA-EBNA-p72 between NPC patients and healthy donors (0.608 versus 0.275, P1⫽0.01) (Table 1). Comparison of the immunoreactivities of NPC sera to the EBV recombinant antigens between young and older patients showed that IgG-EA-p54⫹138 antibodies were detected in 70% of young NPC patients and 81% of older ones (Table 2). IgA antibodies against EA-p54⫹138 were found in 63 and 77% of young and adult patients, respectively. IgG-VCA-p18⫹23 antibodies were detected in 100% of young and older NPC patients, at the same mean titer (1,323 versus 1,405). In contrast, a significant difference between young and older NPC patients was observed in the positivity rate of IgA-VCA-
0.0010/ 0.00019
a Sensitivity is defined as the positivity rate of different markers in NPC patients, and specificity is defined as the negativity rate of different markers in healthy individuals. The P1 value represents the positivity rate and mean OD value between NPC patients and healthy individuals, and the P2 value represents the positivity rate and mean OD value between NPC patients and patients with other tumors. b 95% CI, 95% confidence interval for P ⬍ 0.05. c NS, not significant.
0.035 0.01/⫺ 0 100 ⬍0.001/ ⬍0.001 0.556–1.298 0.927 0.201 NS/NS 26 78 ⬍0.001/ NS 531–1,195 863
0.485 0.353–0.617 100 0.119 0 0.0003/ NSc 0.0004 11 89 ⬍0.001/ ⬍0.001 0.533 0.424–0.642 0.176 0.00031/ 0.00041 Patients with other diseases (28) ⫹ 22 ⫺ 78 P1/P2 value ⬍0.001/ ⬍0.001
0.275 0.025 3 97 ⫺0.143–3.589 1.723 0.140 6 94 428–918 673 97 3 0.174–0.868 0.521 0.115 3 97 0.425–0.721 0.573 0.123 10 90 Healthy donors (77) ⫹ ⫺
0.608 0.099 42 58 1,437–1,728 1.728 0.306 30 70 1,287–2,343 1,815 1.301–1.735 100 0 1.518 0.161 70 30 0.881–1.611 1.246 0.100
95% CI Mean OD %
86 14 ⫹ ⫺
NPC patients (100)
Mean OD %
Mean OD
95% CI
%
Mean titer
95% CI
%
Mean OD
95% CI
%
IgA-EBNA-p72 IgA-VCA-p18⫹23
b
Result for:
IgG-VCA-p18⫹23 IgA-EA-p54⫹138 IgG-EA-p54⫹138 Serum type (n)a
TABLE 1. Reactivities of recombinant VCA-p18⫹23, EA-p54⫹138, and EBNA-p72 fragments in sera from NPC patients compared with those of healthy donors patients with other diseases
J. CLIN. MICROBIOL.
0.528–0.688
DARDARI ET AL.
95% CI
3166
p18⫹23 antibodies (19% versus 52%, P3 ⬍ 0.01) and IgAEBNA-p72 antibodies (35% versus 61%, P3 ⬍ 0.02). No significant difference was observed between young and older NPC patients in the mean OD value of IgG and IgA anti-EAp54⫹138, IgA-VCA-p18⫹25, and IgA-EBNA-p72 (Table 2). A significant difference in the positivity rates and the mean OD values of IgG and IgA anti-EA-p54⫹138 antibodies was observed between young NPC patients and young controls (1.227 versus 0.573, P1 ⫽ 0.0013, 1.354 versus 0.521, P1 ⬍ 0.001). The mean OD value was more than twofold higher in NPC patients than in healthy controls. The same result was observed in a comparison of adult NPC patients with adult healthy donors (1.296 versus 0.568, P2 ⫽ 0.001, 1.656 versus 0.699, P2 ⬍ 0.001) (Table 2). Comparison of sensitivity and specificity between young and older NPC patients showed that the sensitivity of IgA-EAp54⫹138 was 63% in young NPC patients, and its specificity was 97%; whereas in patients over 30 years old, the respective values were 77 and 97% (Table 2). IgA-VCA-p18⫹23 and IgA-EBNA-p72 showed a low sensitivity for detection of NPC in young NPC patients compared to older ones. In fact, only 19 and 35% of young NPC patients were positive for IgA-VCAp18⫹23 and IgA-EBNA-p72, respectively, compared to 52 and 61% of patients over 30 years old. In contrast, IgA-EBNA-p72 shows a high specificity for detection of NPC in the two age groups (⬎97%) (Table 2). Comparison between ELISA and IF for the detection of IgG and IgA to VCA, EA, and EBNA. As illustrated in Table 3, 64% of NPC sera negative for IgA-EA by IF were positive for IgA-EA-p54⫹138, and 7% that were negative for IgA-EAp54⫹138 were positive for IgA-EA by IF. A positive correlation was observed between the mean OD value of IgA-EAp54⫹138 antibodies as detected by ELISA and the mean titer of IgA-EA as detected by IF (RR ⫽ 0.229, P ⬍ 0.01). Of NPC sera negative for IgG-EA by IF, 75% were positive for IgGEA-p54⫹138, and only 9% of sera that were negative for IgG-EA-p54⫹138 were positive for IgG-EA by IF. Our results also showed that only 24% of sera negative for IgA-VCA by IF presented IgA to VCA-p18⫹23, and 61% of sera negative by ELISA were positive by IF. Sensitivity complementation of recombinant antigens EAp54ⴙ138 and ZEBRA protein. Taking into account the sensitivity and specificity of all of the recombinant proteins tested, IgA-EA-p54⫹138 proved more sensitive and specific than both of the other antigens (i.e., IgA-VCA-p18⫹23 and IgA-EBNAp72) and the IF assay for detection of NPC (Table 4). To compare the sensitivity and specificity of IgA-EA-p54⫹138 alone or in combination with IgG-ZEBRA for detection of NPC, data were considered only for those sera examined by all tests. The serological analysis of IgG antibodies against the EBV-ZEBRA protein showed that these antibodies were detected in more than 90% of NPC patients irrespective of their age, compared to only 3% found in healthy donors. The combination of IgA-EA-p54⫹138 with IgG-ZEBRA improved the sensitivity to 95%. However, the improvement in sensitivity did not significantly affect specificity (97%). DISCUSSION In this study, the seroreactivities to the recombinant proteins of EBV-encoded antigens expressed in different phases of virus
3167 DIAGNOSIS OF NPC WITH ANTI-ZEBRA AND ANTI-EA-p54⫹138 VOL. 39, 2001
TABLE 2. Reactivities of recombinant VCA-p18⫹23, EA-p54⫹138, and EBNA-p72 proteins in sera from young and older NPC patients Result for:
Mean OD
1.186–1.79
95% CI
0 100
35 65
0.540 0.112
Mean OD
0.432–0.688
95% CI
IgA-EBNA-p72
1.488 0.281
⫺0.143–3.589
0.486–0.826
IgA-VCA-p18⫹23 95% CI
19 81
1.723 0.140
0.656 0.093
IgG-VCA-p18⫹23 Mean titer
599–2,047
6 94
61 38
IgA-EA-p54⫹138
1,323
428–918
NS
1.711–2.517
0.275 0.026
IgG-EA-p54⫹138 95% CI
100 0
673
⬍0.01
2.114 0.253
3 97
Serum type (n)a Mean OD
1.061–1.647
97 3
52 48
⫺1.155–2.637
%
1.354 0.309
0.174–0.868
NS
302–2,148
0.771 0.255
%
95% CIb
63 37 0.521 0.115
NS
1,405
11 89
%
Mean OD
1.099–1.355
3 97
100 0
526–1,016
%
1.227 0.240 0.425–0.721
1.348–1.969
771
%
70 30 0.573 0.123
1.656 0.135
100 0
Young individuals ⱕ 30 yr old NPC patients (54) ⫹ ⫺ 10 90
77 23
0.352–1.046
Healthy donors (35) ⫹ ⫺ 0.0013
1.14–1.452
0.699 0.121 ⬍0.001
NS
⬍0.001
NS
NS
NS
NS
NS
NS
⬍0.001
⬍0.01
NS
NS
NS
0.008
⬍0.01
⬍0.02
⬍0.001
NS
NS
0.05
⬍0.001
⬍0.001
1.296 0.086
3 97
⬍0.001
NS
NS
⬍0.001
P1 value
81 19
0.42–0.716
⬍0.001
NS
NS
⬍0.001
Older individuals ⬎30 yr old NPC patients (46) ⫹ ⫺ 0.568 0.139
P2 value NS
NS
0.017
3 97
P3 value NS
Healthy donors (42) ⫹ ⫺
P4 value
a Sensitivity is defined as the positivity rate of different markers in young and older NPC patients, and specificity is defined as the negativity rate of different markers in young and older healthy individuals. P1, value for positivity rate and mean OD value between young NPC patients and young healthy individuals; P2, value for positivity rate and mean OD value between older NPC patients and older healthy individuals; P3, value for positivity rate and mean OD value between young and older NPC patients; P4, value for positivity rate and mean OD value between young and older healthy donors. 95% CI, 95% confidence interval for P ⬍ 0.05. NS, not significant. b
c
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J. CLIN. MICROBIOL.
antibodies, which are not detected by IF with chemically induced Raji cells. Our results on the IgG immunoresponse to VCA polypeptides agree with those of earlier studies showing a powerful IgG reaction with the small VCA proteins p18 and p23 (26– 28). In fact, these two recombinant proteins (i.e., p18 and p23) have been recently described as having a high sensitivity for detection of EBV infection (18). VCA-p18-p23 proved to be a useful diagnostic ELISA antigen, yielding high IgG sensitivity among primary infections and in detecting previous infections (18). In our study, IgG-VCA-p18⫹23 antibodies were detected in more than 97% of members of the three groups studied (i.e., NPC patients, healthy donors, and patients with other tumors). However, the mean titers of these antibodies were significantly higher in the NPC group than in healthy donors and patients with other tumors. Several earlier investigations showed a significant decline in anti-EBV VCA antibody titers after radiotherapy, while a persistently elevated or rising IgG or IgA antibody titer could signal either a locally recurrent or systemic disease (12, 20). The detection of IgG-VCA-p18⫹23 antibodies at high titers in the NPC group, reported in the present study, may be considered an important finding. Thus, more investigations should be undertaken to evaluate this marker in the posttherapeutic surveillance of this malignancy. The VCA complex protein, the antigen most commonly used for serological diagnosis, is composed of more than 30 different proteins. However, the relevant target molecules for IgA antibodies in NPC patients are still poorly defined (1, 4, 27). Our results show that only 30% of NPC patients were positive for IgA-VCA-p18⫹23 antibodies compared with 6% of healthy individuals. Comparison of the immunoreactivities of NPC sera with this fusion protein between young and older NPC patients showed that only 19% of young patients were positive for IgA-VCA-p18⫹23 compared with 52% of older patients. The results observed in adult NPC patients are in agreement with those of van Grunsven et al., who reported that 61% of adult NPC patients from Hong Kong presented IgA antibodies to VCA-p18 (27). The detection of IgA-VCA-p18⫹23 in 30% of our overall NPC population, as opposed to 61% of adult NPC patients from Hong Kong, may be explained by the fact
TABLE 3. Comparison between ELISA and IF detection of IgG and IgA against VCA and EA antigens in NPC patients % Detected by ELISAa IF
IgG-VCA ⫹ ⫺
IgG-VCAp18⫹23
IgA-VCAp18⫹23
IgG-EAp54⫹138
IgA-EAp54⫹138
⫹
⫺
⫹
⫺
⫹
⫺
⫹
⫺
100 0
0 100 39 24
61 76 92 75
9 25 93 64
7 36
IgA-VCA ⫹ ⫺ IgG-EA ⫹ ⫺ IgA-EA ⫹ ⫺
a RR ⫽ 0.229, P ⬍ 0.01. RR represents the correlation coefficient, as determined by Pearson’s correlation test, for determination of correlation between the titer of IgA-EA antibodies detected by IF and ELISA.
infection (i.e., EBNA-p72 in the latent phase, EA-p54⫹138 in the early phase, and ZEBRA in the immediate-early phase of active replication, as well as VCA-p18⫹23 in the late phase) were examined in NPC patients. IgG and IgA antibodies against EA-p54⫹138 polypeptides showed a high sensitivity for detecting NPC (86% for IgG and 70% for IgA), in contrast to IgA-VCA-p18⫹23 (30%) and IgA-EBNA-p72 (42%). Considering the low reactivity observed with IgG and IgA-EAp54⫹138 with sera from patients with tumors other than NPC, these markers show a high specificity for detecting NPC (78% for IgG and 89% for IgA). In fact, of patients with tumors other than NPC, only 22% were positive for IgG-EA-p54⫹138 and 11% were positive for IgA-EA-p54⫹138. Furthermore, these markers can detect a substantial portion of NPC cases negative by IF (75% for IgG and 64% for IgA). This may be due to the sensitivity of the ELISA test, a method that can detect antibodies to particular EBV proteins, such as anti-p138
TABLE 4. Seroreactivities of recombinant EA antigen and ZEBRA protein in sera from NPC patients compared with those from healthy donors Result for: a
Serum type (n)
IgA-EA-p54⫹138 %
IgG ZEBRA
Mean OD
95% CI
b
%
Mean titer
NPC patients (100) ⫹ ⫺
70 30
1.519 0.161
1.301–1.735
92 8
18,566
Healthy donors (77) ⫹ ⫺
3 97
0.522 0.115
0.174–0.868
3 97
700
P1 value a
⬍0.001
0.0003
⬍0.001
⬍0.001
95% CI
12,143–24,989
574–1,374
IgA-EA-p54⫹138 and/ or IgG-ZEBRA (%)
P2 valuec
95 5
NSd
3 97
NS
⬍0.001
Sensitivity is defined as the positivity rate of different markers in NPC patients, and specificity is defined as the negativity rate of different markers in healthy individuals. P1, value for positivity rate and mean OD value between NPC patients and healthy donors. b 95% CI, 95% confidence interval for P ⬍ 0.05. c P2, value for positivity rate between IgA-EA-p54⫹138 and IgG-ZEBRA. d NS, not significant.
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DIAGNOSIS OF NPC WITH ANTI-ZEBRA AND ANTI-EA-p54⫹138
that 25% of our NPC patients are young. These young patients generally show a low reactivity to EBV-specific IgA antibodies (2, 5). Moreover, the observation that IgA-VCA-p18⫹25 antibodies were detected at almost the same percentage in patients with NPC (30%) and in those with tumors other than NPC (26%) indicates that this marker is not specific to NPC. This marker also proved to be less sensitive than the classical IF assay for detecting IgA-VCA antibodies. The discordance between these two assays could be attributed to individual differences in the immune responses to various epitopes. Furthermore, in the IF assay, several possible epitopes of the whole protein may be accessible, including structural epitopes not detected by the ELISA test. It should also be noted that in NPC sera, the IgA and the IgG immunoreactivities are not necessarily against the same epitope. This may be related to aspects of viral antigen processing and presentation and the different immunobiological responses between local IgA immunity in the nasopharynx and B-cell reactivity (23). IgG-EBNA-p72 antibodies have been described as having a high diagnostic significance, as being indicative of past infection, and as typically being negative among persons infected recently (16). However, little is known about IgA-EBNA-p72 immunoreactivity in NPC sera. Our results indicate that IgA responses to EBNA-p72 show a high specificity for detection of NPC in both young (100%) and older (97%) NPC patients. In contrast, this marker showed a low sensitivity for detecting NPC in the overall NPC group. This sensitivity was particularly low in young NPC patients (35%) compared with older ones (61%). This may be a feature of these recombinant antigens, which probably do not hold the epitopes that are necessary for adequate T-cell help in the induction of the IgA response. It may also be due to the generally accepted age-related changes in cellular immune function that result in decreased effectiveness to control latent EBV (9). Taking into account the sensitivity and specificity of all of the recombinant proteins tested, IgA-p54⫹138 proved more sensitive (70%) and specific (97%) than the other antigens and than the IF assay for detecting NPC. The highest specificity was obtained by deciding a cutoff in favor of specificity rather than sensitivity. It is noteworthy, however, that the combination of IgA-EA-p54⫹138 with IgG-ZEBRA improved the sensitivity for detection of NPC to 95%. This improvement in sensitivity did not affect specificity (97%). The availability of recombinant antigens will facilitate detailed studies of the pattern of antibody response, which may result in the development of useful serological markers to guide the treatment of NPC. Furthermore, the development of less expensive diagnostic tests would then facilitate population screening in countries with a high prevalence of NPC. Since early detection improves cure rates, such a test would be beneficial to individuals at an early stage of the disease.
23.
ACKNOWLEDGMENTS
24.
We thank the medical staff at the National Institute of Oncology (INO), Rabat, and the Oncology Center, Casablanca, for invaluable assistance in recruiting patients for the collection of sera. We also thank J. Menezes and M. Hassar for suggestions and comments on the manuscript.
25.
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