Seroreactivity against Epstein-Barr virus (EBV) among first-degree relatives of sporadic EBV-associated nasopharyngeal carcinoma in Indonesia

Epstein–Barr virus (EBV) infection and family history are significant risk factors associated with undifferentiated nasopharyngeal carcinoma. The presence of aberrant immunoglobulin A (IgA) antibodies against specific EBV antigens in healthy individuals can be predictive of the disease. Very limited reports explored the EBV IgA antibody presence within families of sporadic cases of nasopharyngeal carcinoma. This study aimed to determine whether EBV IgA was observed more frequently among family members of sporadic cases of nasopharyngeal carcinoma compared to community controls and evaluated the non‐viral factors as determinants of antibody level. First‐degree relatives of nasopharyngeal carcinoma patients (n = 520) and case‐matched community controls (n = 86) were recruited. Sera from all individuals were tested in standardized peptide‐based EBV IgA ELISA. Data on demographic variables and other exogenous factors were collected using a questionnaire through face‐to‐face interviews. A similar frequency of EBV IgA (cut‐off value/CoV 0.354) was observed in the first‐degree relatives of cases and in community controls (41.2% vs. 39.5%, P = 0.770). However, with a higher antibody level (OD₄₅₀ = 1.000; about three times standard CoV), the relatives showed significantly higher frequency (36.9% vs. 14.7%, P = 0.011). When adjusted for all exogenous factors, the strongest factors associated with seropositivity are being a father (odds ratio/OR = 4.36; 95% confidence interval/CI = 1.56–12.21) or a sibling (OR = 1.89; 95% CI = 1.06–3.38) of a case of nasopharyngeal carcinoma. The higher level of EBV IgA seroreactivity in first‐degree relatives of sporadic cases of nasopharyngeal carcinoma compared to the general population supports the use of EBV IgA ELISA for screening among family members. J. Med. Virol. 84:768–776, 2012. © 2012 Wiley Periodicals, Inc.     

Significance of specific Epstein-Barr virus IgA and elevated IgG antibodies to viral capsid antigens in nasopharyngeal carcinoma patients

The feasibility of using elevated Epstein-Barr virus (EBV) specific-IgG antiviral capsid antigen (VCA) and IgA anti-VCA antibody levels as an aid in diagnosis of nasopharyngeal carcinoma (NPC) was analyzed by determination of serum antibody titers to EBV in 54 NPC patients, 114 healthy blood donors, and 40 family members by the immunoperoxidase assay (IPA). No significant difference was found in the prevalence rate of EBV IgG anti-VCA antibodies (titer greater than or equal to 20) between the patient group and the control and family groups (100% vs 92% and 90%, respectively). The prevalence rate of elevated EBV IgG anti-VCA titers (greater than or equal to 80, greater than or equal to 160, greater than or equal to 320, greater than or equal to 640) was significantly higher in the NPC patients than in controls. For example, at an IgG titer of greater than or equal to 320, the prevalence rate was 82% in the NPC patient group and 1.7% in the controls (P less than 0.0001). The prevalence of EBV IgA anti-VCA antibodies (greater than or equal to 10) was significantly higher in the NPC patients than in control and family groups (82% vs 6.1% and 0%, respectively). The prevalence rate for elevated EBV IgA anti-VCA (greater than or equal to 20) was found to be significantly higher (P less than 0.0001) in NPC patients than in the control group (70% vs. 1.7%). A significantly high proportion (P = 0.0004) of NPC patients who had serum EBV IgA anti-VCA titers of less than 20 had elevated IgG titers to VCA greater than or equal to 320 (21% vs 1.7% among controls). It appears that testing for IgG antibodies at a serum dilution of 1:320 and for IgA antibodies at a dilution of 1:20 by the IPA technique comprises the best combination for the differentiation between NPC patients and health controls (91% vs 3.4%), and it is suggested that these be used as screening markers for NPC patients.     

Characterization of ELISA detection of broad‐spectrum anti‐Epstein–Barr virus antibodies associated with nasopharyngeal carcinoma

Epstein–Barr virus (EBV) infection is associated with undifferentiated nasopharyngeal carcinomas (NPC). A distinct seroreactivity pattern to EBV is predictive of subsequent risk of sporadic and familial nasopharyngeal carcinomas. There are currently no accepted screening tools for guiding the clinical management of individuals at high‐risk for nasopharyngeal carcinomas, particularly unaffected relatives from nasopharyngeal carcinoma multiplex families. Therefore, the reproducibility of a panel of largely synthetic peptide‐based anti‐EBV antibody ELISAs was evaluated and their ability to distinguish nasopharyngeal carcinoma cases from controls was explored. IgG and IgA antibodies against 6 different EBV antigens (10 assays, total) were tested on sera from 97 individuals representing the full spectrum of anti‐EBV seroprevalence (i.e., healthy individuals with no known EBV seroreactivity, healthy individuals with known EBV seroreactivity, and nasopharyngeal carcinoma cases). Each specimen was tested in triplicate to assess within‐batch and across‐batch variation, and the triplicate testing was repeated on two separate days. Reproducibility was assessed by the coefficients of variation (CVs) and intraclass correlation coefficients (ICCs). All markers were detectable in 17% or more of samples. For all but one marker, the overall, within‐batch, and across‐batch CVs were below 15%, and the ICCs were above 70% for all but three markers. Sensitivity of these markers to detect prevalent nasopharyngeal carcinomas ranged from 22% to 100%, and among unaffected controls, most distinguished those with and without known seropositivity. In conclusion, a large number of EBV markers can be measured reliably in serum samples using peptide‐based anti‐EBV ELISAs. J. Med. Virol. 85:524–529, 2013. Puiblished 2012. This is a US government work, and, as such, is in the public domain of The United States of America.     

Elevated Epstein-Barr virus seroreactivity among unaffected members of families with nasopharyngeal carcinoma

Serum antibodies to Epstein–Barr virus (EBV) antigens can be used to predict the risk of nasopharyngeal carcinoma (NPC). To investigate whether EBV seropositivity rates were higher among healthy family members from multiplex and sporadic families with NPC (i.e., families with multiple or single cases) compared to the general population, a study was conducted on 2,665 unaffected individuals from 140 multiplex and 413 sporadic families. The titers of the IgA antibody to the EBV capsid antigen (VCA‐IgA) were compared to those of 904 controls from the general population. The VCA‐IgA titer was correlated among sibling pairs to a high significance in both family types (P < 0.0001 and P = 0.0005 for the multiplex and the sporadic families, respectively); parent–offspring pairs also showed significant correlation (P < 0.0001 and P = 0.0002, respectively); and spouse pairs were correlated, but at lower significance levels (P = 0.0790 and P = 0.0040, respectively). When compared to the controls, among first‐degree relatives in the multiplex families, the age‐ and gender‐adjusted odds ratio (OR) was 2.06 (95% confidence interval 1.56–2.71), 3.55 (2.24–5.64), and 2.25 (1.57–3.23) for siblings, parents, and children, respectively. In the sporadic families, the adjusted OR was 1.55 (1.21–2.00) and 2.08 (1.51–2.86) for siblings and parents, respectively. The adjusted P‐value of spouses lost significance in the multiplex families, but remained significant in the sporadic families (P = 0.0146). In conclusion, EBV seropositivity rates were elevated among unaffected family members in both multiplex and sporadic families with NPC. J. Med. Virol. 83:1792–1798, 2011. © 2011 Wiley‐Liss, Inc.     

Analyses of Epstein-Barr virus latent membrane protein-1 in Malaysian nasopharyngeal carcinoma: high prevalence of 30-bp deletion,Xho1 polymorphism and evidence of dual infections

Nasopharyngeal carcinoma, a malignancy associated closely with Epstein-Barr virus (EBV), is prevalent among Chinese of Southern China origin. Epidemiological studies indicate a high prevalence of EBV in Asia with viral isolates having typical characteristics of the putative viral oncogene, latent membrane protein 1 (LMP-1), such as the loss of the Xho1 restriction site in Exon 1 and the 30-bp deletion in Exon 3. The EBV LMP-1 gene from throat washings of 120 nasopharyngeal carcinoma patients and 14 healthy individuals were analyzed. Similar analyses were also carried out on 30 and 12 postnasal space biopsies from nasopharyngeal carcinoma patients and healthy individuals, respectively. The 30-bp deletion was detected in 20% of nasopharyngeal carcinoma throat washes and in 100% of nasopharyngeal carcinoma postnasal space biopsies. Interestingly, 16% of the nasopharyngeal carcinoma biopsies possessed both the deleted and the undeleted variants, suggestive of dual infections. The notion of dual infections in nasopharyngeal carcinoma was further supported by the coexistence of both "F" and "f" (BamH1F region) EBV variants in 11% of the nasopharyngeal carcinoma biopsies. All of the throat washes and biopsies from the healthy controls showed the undeleted variant. The loss of the Xho1 restriction site was found with higher frequency both in throat washes and biopsies from patients with nasopharyngeal carcinoma. The discrepancy in the frequency of the 30-bp deletion between throat washes (20%) and postnasal space biopsies (100%) was an indication that this deletion is specific for viral isolates from primary tumour sites.     

Detection of IgA antibodies to Epstein-Barr virus-associated antigens by ELISA

The detection of IgA antibodies to the Epstein-Barr virus (EBV)-associated viral capsid antigen (VCA) and early antigens (EA) is of diagnostic and prognostic importance for patients with nasopharyngeal carcinoma (NPC). An ELISA for the determination of serum IgG antibodies to these antigens has been developed which uses the double antibody method. 136 sera obtained from healthy donors and patients with non-EBV related tumors and lymphomas were tested by ELISA; only 3 sera, from patients with chronic lymphatic leukemia, hairy cell leukemia and Burkitt-like lymphoma, contained antibodies of IgA class to VCA and EA. Ninety-five sera from patients suspected of having NPC were tested. IgA anti-VCA was found in 28 sera (29.5%), 12 of which also contained IgA anti-EA. The assays described are suitable for diagnosis and follow-up of patients with EBV-associated nasopharyngeal carcinoma. Furthermore, isolated EA components may be tested for their reactivity with IgA antibodies, as was shown for the 60 kDa polypeptide associated with the EA complex.     

Use of bacterially expressed EBNA-1 protein cloned from a nasopharyngeal carcinoma (NPC) biopsy as a screening test for NPC patients

EBV serological tests have been used for many years as accessory diagnostic predictors of nasopharyngeal carcinoma (NPC). To increase the sensitivity and specificity of the NPC detection rate, a novel enzyme-linked immunosorbent assay (ELISA) was established using a bacterially-expressed GST-EBNA-1 protein, containing the EBNA-1 sequence cloned from an NPC patient. Serum samples were collected from age- and gender-matched patients with NPC, community control subjects and hospital control patients and tested using this ELISA. The positivity rates were 78.7% (247/314) in NPC, 11.5% (28/244) in hospital controls and 3.8% (10/263) in the community control group. These serum samples were also tested for IgA anti-VCA antibodies and their ability to neutralize EBV DNase and the sensitivities of the anti-VCA antibody and DNase-neutralization tests also were analyzed. The optimum combination is VCA plus EBNA-1, which can identify 92.5% (287/310) of NPC patients, and shows a specificity of 92.7% (242/261) for normal individuals.     

Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, tonsillar carcinoma and control groups

In the sera of 17 patients with nasopharyngeal carcinoma (NPC) and of 19 patients with tonsillar carcinoma (TC) the titres of IgA, IgG and IgM antibodies to EBV VCA (viral capsid antigen) and of IgG antibodies to EBV EA (early antigen) were determined by the indirect immunofluorescence (IF) method. Significant difference was observed in the frequency of IgA antibodies to EBV VCA and IgG antibodies to EBV EA between NPC patients and controls. There was also a significant difference between the frequency of IgM antibody to EBV VCA and EBV EA antibody titres in TC patients and controls. The geometric mean titre (GMT) of IgG antibodies to EBV VCA was significantly higher in the NPC and TC patients as compared to controls.     

Antibody against the Epstein-Barr virus BHRF1 protein,a homologue of Bcl-2, in patients with nasopharyngeal carcinoma

The Epstein-Barr virus (EBV) open reading frame BHRF1, a homologue of the oncogene bcl-2, was cloned from a patient with nasopharyngeal carcinoma (NPC) and overexpressed in Escherichia coli. The resulting recombinant BHRF1 fusion protein, with an apparent molecular weight of 35 KD, was used as antigen in an immunoblotting assay for IgG antibody in human sera. Anti-BHRF1 antibody was detected in 57 (61.3%) of 93 patients with NPC, 5 (5.7%) of 87 patients with nonmalignant diseases of the nasopharynx, and in 1 (1.3%) of 78 healthy blood donors. The positivity rate in these nonmalignant patients was 4.4 times that of the normal controls. Negative results were observed in four patients with infectious mononucleosis and patients with other cancers, including 4 with esophageal cancer, 11 with lung cancer, 10 with lymphoma, 13 with gastric carcinoma, 10 with cervical carcinoma, and 10 with other head and neck cancers. Antibody neutralizing EBV DNase and IgA antibody to viral capsid antigen (VCA) were assayed in parallel. The results showed that 7.5% of the NPC patients were negative for anti-DNase and anti-VCA antibodies and EBV infection could be detected by the anti-BHRF1 antibody alone. The demonstration of anti-BHRF1 antibody in most NPC sera strongly supports the hypothesis that the EBV BHRF1 protein is expressed in most NPC patients and its specific antibody can be a useful marker for the diagnosis of NPC. J. Med. Virol. 56:179–185, 1998. © 1998 Wiley-Liss, Inc.     

Evaluation of commercial EBV RecombLine assay for diagnosis of nasopharyngeal carcinoma

BACKGROUND: In recent years a number of Epstein-Barr virus (EBV) proteins were defined as being immunodominant for either IgM, IgG or IgA immune responses, yielding promising markers for diagnostic serology. Specific reactivity patterns to these proteins have been described for infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), various types of lymphoma, and healthy EBV carriers. OBJECTIVES: To compare the NPC-related diagnostic value of EBV RecombLine test (Mikrogen, Germany) with a standardized immunoblot assay [Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM, et al. Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004;190:53-62] and to define the diagnostic value of individual EBV marker proteins in a population with high incidence of NPC. RESULT: Sera from Indonesian NPC patients taken at primary diagnosis (n=108) were analyzed for IgG and IgA reactivity and compared with regional healthy blood donors (n=62), non-NPC patient controls (n=10) and IM patients (n=10). Most NPC patients and controls showed strong IgG reactivity to VCA-p18, -p23, and EBNA1, limiting their diagnostic use. Few (<20%) healthy donors and patient controls showed IgG reactivity to EA proteins p47/54 and p138, yielding combined sensitivity/specificity and PPV/NPV values of 92.6%/98.3% and 99.0%/88.1%, for diagnosing NPC. NPC sera showed significantly more EBV reactive IgA antibody (>80% positive) than controls (<10% positive), although being less broadly reactive and significantly less strong compared to IgG. For IgA best results were observed for RecombLine EBNA1 with sensitivity/specificity and PPV/NPV values of 92%/89% and 93.4%/85.9%, respectively. CONCLUSION: In high incidence NPC regions with low incidence IM yet high prevalence of EBV infection, both RecombLine IgG and IgA tests provide a useful alternative to the more complex cell-extract based immunoblot assay as confirmation test for NPC diagnosis in particular when using EA and EBNA1 as discriminators in IgG and IgA testing, respectively.     

Antibody response to Epstein-Barr virus antigens in patients with chronic viral infection

We tested antibody titres against Epstein-Barr virus (EBV) antigens in patients suffering from chronic viral disease and compared them with those determined in sex- and age-matched healthy controls. Patient sera showed signs of active EBV infection [antibodies against early antigen (EA) and/or viral capsid antigen (VCA) in the IgM or IgA classes] significantly more frequently than the control group. Correspondingly, geometric mean titres (GMT) of antibodies against all viral antigens were elevated in the patients. The strongest association with EBV was observed in patients whose clinical symptoms closely resembled infectious mononucleosis: 92% of the subjects in this subgroup possessed anti-EA and 41 and 25% had IgM and IgA anti-VCA antibody, respectively. In patients with signs of lymphoproliferation only and in those suffering from frequent respiratory infections the association with EBV was less marked but still significant. Patients with transient defects in humoral and cellular immunity mounted higher titres against VCA in the IgG class than those without immune defects.     

Specific IgA antibody to Epstein-Barr viral capsid antigen: a better marker for screening nasopharyngeal carcinoma than EBV-DNA detection by polymerase chain reaction

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. To assess whether EBV DNA detection by polymerase chain reaction (PCR) or presence of specific serum antibody to viral capsid antigen (VCA) was a better marker for screening NPC, nasopharyngeal tissues and blood samples from 58 NPC patients and 24 non-NPC patients (23 with laryngotracheal stenosis and 1 with chronic tonsillitis) were tested for the presence of EBV DNA and serum specific VCA antibodies, respectively. EBV DNA was detected in 56 (96.5%) of NPC patients and 15 (62.5%) of non-NPC controls, with predominantly EBV type A in both groups. On the other hand, specific VCA IgA antibody was detected in the majority of NPC patients: 52 (89.7%) while only 4 (16.7%) were detected in non-NPC controls. Therefore, specific VCA IgA antibody may serve as a better marker for screening NPC than EBV DNA detected by PCR.     

Use of bacterially expressed GST/EBNA-1 fusion proteins for detection of antibodies in sera from patients with nasopharyngeal carcinoma and healthy donors

Epstein-Barr virus nuclear antigen-1 (EBNA-1) is a protein expressed consistently in EBV infected cells and in EBV related malignant tissues. Antibodies against EBNA-1 may therefore possibly be used as a marker for disease screening. Western blot analysis of serum antibodies was performed using GST (glutathione-S-transferase) fusion proteins containing different regions of EBNA-1 as antigens. Serum samples were collected from 38 patients with nasopharyngeal carcinoma (NPC) and 38 healthy individuals in Taiwan. All samples were found IgG positive for EBNA-1 when a truncated protein GST/E1 (70-102, 325-641) was used as the antigen. Thirty-three out of 38 NPC sera (86.8%) were positive for IgA antibody against EBNA-1. The positive rate was higher in comparison with IgA antibody against VCA (65.7%) or antibody against DNase (60.5%). Only 2.6% of sera from normal individuals were positive for an IgA response against EBNA-1. The major antigenic determinants for NPC serum IgA response were between amino acid(aa) 390 to aa 459 when different portions of EBNA-1 were used as antigens. The results suggest that IgA response against EBNA-1 could be used in combination with other EBV serology markers for NPC screening.     

Measurement of EBV-IgG anti-VCA avidity aids the early and reliable diagnosis of primary EBV infection

Current serological methods for the diagnosis of Epstein-Barr virus (EBV) infection still differentiate poorly between primary infection and reactivation. This is particularly true when IgG and IgM antibodies are present simultaneously and only a single serum sample is provided for analysis. The demonstration of the IgG avidity state has the potential to distinguish recent from past or reactivated infection. An analysis of the kinetics of avidity maturation of anti-VCA antibodies in primary EBV infection was undertaken with longitudinally collected sets of sera from 28 well-characterised EBV cases and in sera from 35 cases with previous EBV infection and recent primary infection due to HIV, CMV, or hepatitis A. Antibodies directed against the viral capsid antigen (VCA) and Epstein-Barr nuclear antigen (EBNA-1) were sought, using a commercial enzyme immunoassay (EIA). In parallel with standard IgG anti-VCA detection, serum was incubated with 8 M urea to disrupt low-avidity complexes to allow calculation of the percentage avidity. In cases with primary EBV infection, the mean avidity rose from 54% at 6 weeks to 82% by 28 weeks after the onset of symptoms, but remained lower than that of the control sera (96%). The addition of the avidity measurement improved the sensitivity of IgG and IgM anti-VCA testing in diagnosis of primary EBV infection from 93% to 100%. The specificity of IgM anti-VCA testing alone was poor, with 14 of 35 cases (49%) demonstrating false-positive results, but it improved to 97% by the demonstration of high-avidity IgG anti-VCA. The combination of negative IgG anti-EBNA and low-avidity IgG anti-VCA had a sensitivity and specificity of 100%. The routine addition of IgG anti-VCA avidity estimation to diagnostic EBV serology is recommended.     

Immune responses to Epstein-Barr virus lytic proteins in patients with nasopharyngeal carcinoma

Immune responses to three Epstein-Barr virus (EBV) lytic proteins, DNase, thymidine kinase (TK), and BMRF-1 gene products (50/52 kDa diffused early antigen, EA-D complex) were determined in EBV-infected control individuals and patients with nasopharyngeal carcinoma (NPC). Immunofluorescence assays (IFA) were used to detect their humoral immune responses using recombinant EBV lytic proteins expressed in a baculovirus system as antigens. Cell proliferation assays were performed to evaluate their cellular immune responses by monitoring 3H-thymidine incorporation. Seventy patients with NPC and 32 non-cancer controls were analyzed. The results of IFA showed antibody titers to all three EBV lytic proteins to be higher in the patients with NPC especially for the IgA class. Positivity rates of the three IgA antibodies also were higher in the patients with NPC population. Furthermore, the profiles of the IgA antibodies correlated with those to total early antigens (EA) expressed in the early phase and viral capsid antigen (VCA) expressed in the late phase, of EBV replication. The most interesting finding was that antibody titers to the three EBV lytic proteins were associated significantly with metastases of cervical lymph nodes in patients with NPC. As for cellular immunity to the EA-D complex and DNase, weak responses were observed in the cell proliferation assays. Peripheral blood cells from most individuals could not be stimulated to proliferate, except for a few patients with NPC whose antibody titers against the EA-D complex and DNase also were very high.     

Improved sensitivity of detection by avidin-biotin complex (ABC) immunocytochemistry in Epstein-Barr virus serology

Serum antibodies against Epstein-Barr virus (EBV)-determined antigens have traditionally been titrated by the indirect immunofluorescence (IIF) technique. The avidin-biotin complex (ABC) immunocytochemical technique was used to determine the serum levels of IgA against EBV viral capsid antigen (IgA/VCA) and IgA against EBV early antigen (IgA/EA) in sera of 106 nasopharyngeal carcinoma (NPC) patients prior to treatment and 100 normal individuals. The sensitivity of the ABC technique is enhanced by an amplification of the antigen-antibody reaction, which involves the binding of the enzyme-linked ABC to the second biotinylated antibody. There was a good correlation (r = 0.9988) between ABC and IIF-determined IgA/VCA-positive titres, with the ABC technique being more sensitive than IIF in the detection of IgA/VCA in NPC sera: 94% (99/106) and 76% (80/106), respectively. The frequency of IgA/EA reactivity in NPC sera was also markedly increased by immunodetection with the ABC technique as compared with IIF technique: 63% (69/106) and 28% (30/106) respectively. Both the immunocytochemical techniques were equally specific in discriminating between elevated serum titres of IgA/VCA and IgA/EA in NPC sera from normal human sera.     

IgA antibodies against the Epstein‐Barr nuclear antigen1 as a valuable biomarker for the diagnosis of nasopharyngeal carcinoma in Tunisian patients

Serological tests for Epstein‐Barr virus (EBV) have been used for many years as diagnostic predictors of nasopharyngeal carcinoma. It has been shown previously that the conventional immunofluorescence assay has a limited diagnostic value, especially in young patients from North African area. In the search for more reliable immunoglobulin (Ig) G or IgA antibody markers for the diagnosis of nasopharyngeal carcinoma, immunoblot analysis was performed using a full spectrum of EBV proteins. Sera were collected from 108 patients with nasopharyngeal carcinoma and three control groups composed of 18 patients with lymphoma, 18 other patients with autoimmune diseases and 55 healthy EBV carriers. It was observed that the IgA Epstein‐Barr nuclear antigen 1 (EBNA1), IgA early antigen (EA)‐p138 and IgG EA‐p138 antibodies represent the most specific anti‐EBV responses in either young or older patients with nasopharyngeal carcinoma which yield higher positive rates compared to the three control groups. Since the IgA EBNA1 response showed the highest sensitivity value for the detection of nasopharyngeal carcinoma, a novel enzyme‐linked immunosorbent assay (ELISA) was established using a GST‐EBNA1 protein expressed in bacteria, containing the P‐threonine EBNA1 subtype cloned from DNA EBV sequence of C15 xenograft cells. Detection rates were 85.7% and 94.9% in young and older patients with nasopharyngeal carcinoma respectively, while only 3.6%, 11.1%, and 16.6% in healthy EBV carriers, patients with lymphoma and patients with autoimmune diseases, respectively. Thus, IgA EBNA1 ELISA may be useful for early diagnosis and mass screening of nasopharyngeal carcinoma in Tunisia even in young patients. J. Med. Virol. 81:1412–1421, 2009. © 2009 Wiley‐Liss, Inc.     

Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men.

BACKGROUND: It is probable but unproven that Epstein-Barr virus (EBV) has a role in nasopharyngeal carcinoma. We determined whether antibodies against EBV are present before the development of nasopharyngeal carcinoma. METHODS: A total of 9699 men were enrolled between 1984 and 1986. Blood samples were examined for IgA antibodies against EBV capsid antigen and neutralizing antibodies against EBV-specific DNase. During 131,981 person-years of follow-up, 22 pathologically confirmed new cases of nasopharyngeal carcinoma that were diagnosed more than one year after recruitment were ascertained through linkage with the National Cancer Registry of Taiwan. RESULTS: The cumulative risk of nasopharyngeal carcinoma per 100,000 person-years was 11.2 for subjects who tested positive for neither serologic marker, 45.0 for those who had one marker, and 371.0 for those who had both markers. After adjustment for age and the presence or absence of a family history of nasopharyngeal carcinoma, the relative risk of nasopharyngeal carcinoma was 32.8 for subjects with both markers (95 percent confidence interval, 7.3 to 147.2; P<0.001) and 4.0 for subjects with one marker (95 percent confidence interval, 1.6 to 10.2; P=0.003), as compared with subjects with neither marker. The longer the duration of follow-up, the greater the difference in the cumulative incidence of nasopharyngeal carcinoma between seropositive and seronegative subjects. CONCLUSIONS: IgA antibodies against EBV capsid antigen and neutralizing antibodies against EBV DNase are predictive of nasopharyngeal carcinoma.     

Epstein-Barr virus nuclear antigen 1 linear epitopes that are reactive with immunoglobulin A (IgA) or IgG in sera from nasopharyngeal carcinoma patients or from healthy donors.

The entire amino acid sequence of the unique region of the EBNA 1 protein was synthesized as a set of 41 20-residue peptides with an overlap of 10 amino acids. The peptides were tested in the enzyme-linked immunosorbent assay for reactivity with immunoglobulin A (IgA) and IgG in sera from 50 patients with nasopharyngeal carcinoma (NPC) as compared with 36 serum samples from healthy Epstein-Barr virus (EBV)-seropositive donors and 5 serum samples from EBV-negative donors. The most immunoreactive peptide for both IgA and IgG binding was localized to the glycine-alanine repeat domain of the antigen. In the unique regions, 16 immunoreactive peptides were found. Of these, four were reactive with IgG but not IgA and three peptides were reactive with IgA but not IgG in NPC sera. In addition, several IgA and IgG epitopes on the carboxy-terminal region were specifically reactive with NPC sera, but unreactive with sera from healthy EBV-positive donors. The results suggest that EBV serology specific for individual epitopes may provide additional useful information not available by conventional serology with whole antigens or the EBNA complex.     

Epstein-Barr virus serology in the diagnosis of nasopharyngeal carcinoma

The antibody levels to viral capsid antigen (VCA) and early antigen (EA) of Epstein-Barr virus (EBV) in 164 nasopharyngeal carcinoma (NPC) patients from Sarawak, East Malaysia were significantly higher than those in 147 sex, age and ethnically matched healthy controls. As diagnostic markers of NPC, IgG/VCA at reciprocal titers > or =160 was the most sensitive (89%, with 98% specificity), while IgA/EA at > or =5 was the most specific (100%) but the least sensitive (75%). The sensitivity and specificity of IgA/VCA at reciprocal titers > or =10 were 84% and 97%. IgA/VCA has an advantage over IgG/VCA despite the slightly lower sensitivity due to its consistently more distinct fluorescence reaction. The sensitivity and specificity can be marginally improved by a combination of two tests.