Co-expression of p53 and bcl-2 may correlate to the presence of epstein-barr virus genome and the expression of proliferating cell nuclear antigen in nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) has been well documented in the aetiology of nasopharyngeal carcinoma (NPC), although its role as well as the genetic basis in the genesis of NPC have not been elucidated. The p53 gene mutations are infrequently found in NPC, but the expression of p53 protein, as well as bcl-2 oncoprotein, has been reported in a high percentage of cases, and also in association with EBV. Proliferating cell nuclear antigen (PCNA) has also been shown to be increased in NPC, suggesting its association among the overexpression of p53 and bcl-2 oncoprotein. We undertook this study to evaluate the correlation among these abnormalities in the development of NPC. The expression of p53 protein, bcl-2 oncoprotein, and the level of PCNA were investigated by immunohistochemistry in 53 patients with NPC. Twenty tissue samples from these patients were studied for p53 gene mutations by single strand conformation polymorphism (SSCP) and DNA sequencing as well as EBV genomes by polymerase chain reaction. Among the 53 specimens, 42 (79%) showed expression of p53 protein and 40 (75%) gave positive result for bcl-2 oncoprotein. A significant association was found between p53 expression and bcl-2 oncoprotein (P=0.002; Fisher's exact test) with 68% of the patients showing coexpression of both markers. The PCNA labelling index in the 53 patients varied from 5% to 80%. High PCNA labelling index was frequently found in the patients with overexpression of p53 protein and bcl-2 oncoprotein. The PCNA index in patients with p53 expression was significant higher than in those without p53 expression (P=0.002). Of the 20 patients, p53 mutations were found in four cases. EBV genomes were detected in 14 cases of which 12 cases showed overexpression of both p53 and bcl-2 and one case with only p53 expression and one case with bcl-2 expression. EBV genomes were detected in two cases with p53 mutations. We conclude that EBV is the important etiologic factor in NPC which may be involved in p53 and bcl-2 overexpression. The mutant p53 protein is correlated to deregulation of PCNA. p53 mutations participate in a small proportion of the tumorigenesis.

Epstein-Barr virus DNA in nasopharyngeal carcinoma in Thai patients at Ramathibodi Hospital, Bangkok.

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in Thailand. The association of Epstein-Barr virus (EBV) and NPC, especially undifferentiated type, has been documented. There is, however, conflicting evidence with regard to the squamous cell type. We have therefore investigated EBV-DNA in all the three types of NPC to assess the association of EBV and NPC in Thai patients. EBV-DNA was detected in formalin-fixed paraffin-embedded tissues from the patients of Ramathibodi Hospital, Bangkok using polymerase chain reaction. A primer pair that amplified EBV nuclear antigen gene was used in the reactions and the amplification products were hybridized with a specific EBV probe. EBV-DNA was identified in 24 of 28 of tissue samples from patients with undifferentiated NPC, 25 of 40 samples from patients with squamous cell NPC and 3 of 4 samples with nonkeratinizing NPC. None of 12 nasopharyngeal tissue samples without NPC contained detectable EBV-DNA. Our results indicated a strong association of EBV with undifferentiated as well as non-keratinizing NPC. EBV-DNA was demonstrated in most cases of squamous cell NPC but the association of EBV in this type of carcinoma was not as frequent as in the other two types of NPC.

Evaluation of a locally developed direct immunofluorescence test for chlamydial infections.

The high cost of diagnostic tests for chlamydial infections limits their use which may result in under estimation of the incidence of chlamydial infections. This study was an attempt to reduce the cost of the test by developing an immunofluorescence test for C. trachomatis using monoclonal antibody to major outer membrane protein of C. trachomatis. Urethral swabs were obtained from patients with symptoms of urethritis. The developed immunofluorescence test was compared with culture method and a commercial immunofluorescence test kit (BioMerieux). Compared with the culture method, the sensitivity, specificity, predictive value of positive and predictive value of negative of the developed test were 79, 85, 61 and 93 per cent respectively. The results obtained from the comparison with commercial test kit showed an agreement of 88 per cent. The developed test was positive in 32 per cent of specimens while the commercial test was positive in 24 per cent. The commercial test kit showed excellent reactions and it contained monoclonal antibody to lipopolysaccharide of Chlamydiae in addition to monoclonal antibody to major outer membrane protein which can lead to stronger immunofluorescence staining. The locally developed test, however, costs much less without compromising the results.

A comparison of passive haemagglutination test and immunofluorescent test for antibody to native deoxyribonucleic acid.

Antibody to double-stranded DNA is a specific marker for systemic lupus erythematosus. The recommended method for detection of this antibody is immunofluorescence. Haemagglutination was developed and the results of antibody detection were evaluated with those obtained by immunofluorescence. Human group O erythrocytes were treated with glutaraldehyde and coated with DNA from calf thymus. Testing in 169 active and inactive SLE sera, 59 sera were positive and 91 sera were negative by both methods. Five sera were negative by haemagglutination but positive by immunofluorescence. Fourteen sera with low haemagglutination titer were negative by immunofluorescence. The correlation between the results obtained by both methods were highly significance with contingency coefficient of 0.61 and correlation coefficient between the results of 78 sera positive by both or either method was 0.74 (p less than 0.001). Sixty-three sera from blood donors and seventy sera from pregnant women were negative by the two techniques. PHA is simpler, quicker and can be assayed in laboratories without the use of fluorescent microscope. It can be established as a very useful alternative test to immunofluorescence.

Development of enzyme-linked immunosorbent assay for hepatitis B e antigen.

Hepatitis B e antigen (HBeAg) in sera indicates infectivity and when found during pregnancy, indicates a need for vaccination against hepatitis B virus. A sensitive test for HBeAg is needed in all hospitals but this test is expensive. Local development of enzyme-linked immunosorbent assay (ELISA) for HBeAg and its antibody (anti-HBe) was considered necessary and it was successfully conducted. The developed test was compared with ELISA test for HBeAg and anti-HBe manufactured by Organon Teknika (205 routine specimens and 103 sera positive for HBsAg) and Roche Diagnostic (160 routine specimens). The locally made and imported kits showed overall agreement of 97.5 to 98.1 per cent and the locally made test was always slightly more sensitive. The local test was also rapid, reproducible, and specific. The development lead to self reliance on ELISA test for HBeAg and anti-HBe.

Evaluation of latex cryptococcal agglutination test in cryptococcal meningitis.

There is a growing demand for laboratory diagnosis of cryptococcal meningitis, which is partly due to the increasing incidence of AIDS in Thailand. Presently, latex cryptococcal agglutination test (LCAT) is the most sensitive and specific test for laboratory cryptococcal meningitis. However, the test is very expensive and not readily available. LCAT must be developed locally to meet the need in Thailand. Rabbit antibody to C. neoformans was raised and used to sensitize latex particles used in LCAT. The developed LCAT was compared with a reference LCAT. The locally made LCAT was almost identical to the reference LCAT in sensitivity and specificity. It was extensively compared with the culture and India ink examination, in 73 cerebrospinal fluid specimens from cryptococcal meningitis and 155 specimens from other diseases. LCAT was found specific and more sensitive than fungal culture and India ink examination. LCAT is now extensively used in Thailand and recommended by Thai experts for use in all general hospitals. It is a simple, sensitive, specific, rapid and inexpensive tool for both diagnosis, prognosis and follow-up of cryptococcal meningitis.