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Diagnostic value of enzyme linked immuno-sorbent assay for cytomegalovirus disease. K Priya, HN MadhavanL and T Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, India., India
Correspondence Address: Source of Support: None, Conflict of Interest: None PMID: 12432189
BACKGROUND: Since interpretation of results of enzyme linked immuno-sorbent assay (ELISA) for diagnosis of Cytomegalovirus (CMV) infection in India is difficult, its diagnostic value required evaluation. AIMS: To evaluate the diagnostic value of ELISA against polymerase chain reaction (PCR) in CMV disease. SETTINGS AND DESIGN: Results of ELISA test for CMV antibodies in CMV-DNA PCR positive and negative patients and normal healthy blood donors were analysed. METHODS AND MATERIAL: Anti-CMV antibodies were assayed by ELISA on the sera of 26 CMV PCR positive and 21 PCR negative patients and 35 normal healthy blood donors. STATISTICAL ANALYSIS: Chi square and Fischer exact test were used for statistical analysis. RESULTS: Anti-CMV antibodies (IgG or IgG and IgM) were present in 20 (76.9%) of 26 PCR positive and 13 (61.9%) of 21 PCR negative patients. ELISA was negative in six (23.1%) of 26 PCR positive patients. Of the 28 paediatric patients, ELISA was positive in 14 (73.7%) of 19 PCR positive and three (33.3%) of nine PCR negative patients showing a statistically significant difference (Chi square test, P value 0.038). Among the 19 patients having complications after organ transplant, ELISA showed anti-CMV antibodies in six (85.7%) of seven PCR positive and 11 (91.7%) of 12 PCR negative patients showing no significant difference. CMV-DNA was not detected in the buffy coat of 35 sero-positive blood donors. CONCLUSION: ELISA has no diagnostic value in the detection of CMV activation although it may help in the differential diagnosis of CMV infection in the paediatric age group. Keywords: Antibodies, Viral, analysis,Case-Control Studies, Chi-Square Distribution, Comparative Study, Cytomegalovirus, immunology,isolation &purification,Cytomegalovirus Infections, diagnosis,DNA, Viral, analysis,Enzyme-Linked Immunosorbent Assay, methods,Female, Human, Male, Polymerase Chain Reaction, methods,Probability, Reference Values, Sensitivity and Specificity,
Human cytomegalovirus (CMV) infections are usually subclinical and the disease may be of diverse severity.[1] Rapid aetiological diagnosis helps in the institution of specific therapy. Laboratory diagnostic methods include virus antigen detection, virus isolation in diploid cell cultures, antibody assays by enzyme linked immuno-sorbent assay (ELISA) and polymerase chain reaction (PCR).[1],[2],[3]],[4] In India, ELISA is the most common method used but interpretation of its results are difficult. Therefore, we evaluated the diagnostic value of ELISA between PCR positive and PCR negative groups of patients with congenital and acquired CMV infections and post-organ transplant complications.
The investigations were done on clinical specimens referred from the neighbouring hospitals to the clinical microbiology laboratory, Sankara Nethralaya, Chennai. PCR was done on blood, urine or nasopharyngeal aspirates obtained from 47 patients clinically suspected to have CMV disease and were divided into positive and negative groups for CMV-DNA. In the 26 PCR positive patients (congenital disease - 16, acquired disease - 3, post-organ transplant - 7) the specimens tested were 14 blood, 9 urine and 3 nasopharyngeal aspirates. In the 21 PCR negative patients (congenital disease - 7, acquired disease - 2, post-organ transplant -12) the specimens tested were 15 blood and 6 urine samples. Serum samples were collected from these patients on the same day of collection of specimen for PCR during the acute stage of the disease and tested by ELISA for anti-CMV antibodies. Heparinised peripheral blood from 35 normal healthy voluntary blood donors (Blood Bank, Voluntary Health Services, Chennai) tested negative for human immunodeficiency virus (HIV), Hepatitis B virus (HBV) and Hepatitis C virus (HCV) and VDRL non-reactive were used as control specimens. The plasma was used for ELISA and the buffy coat was used for PCR. The DNA extraction and PCR was done as standardised and described by us earlier.5 PCR protocol consisted of nested primers coding for the morphological transforming region II of CMV which were custom-synthesised by Bangalore Genei, India. Anti-CMV IgG and IgM antibodies were assayed using the ELISA kits of BIOKIT (Barcelona, Spain) as per the manufacturer’s instructions. The ELISA units (EU) were calculated using the high positive (HP) and low positive (LP) and negative controls and results were expressed as HP and LP indicating the amount of anti-CMV antibodies present in the sera. The Chi square test and Fischer exact test were used for the analysis of statistical significance.6
The results of ELISA in 47 patients belonging to both the PCR positive and negative groups expressed as HP and LP indicating the presence of anti-CMV antibodies in the serum samples is shown in [Table - 1]. ELISA detected anti-CMV antibodies (IgG or both IgG and IgM) in 20 (76.9%) of 26 PCR positive patients and 13 (61.9%) of 21 PCR negative patients. Among the 33 patients with anti-CMV IgG antibodies, 20 (60.6%) were PCR positive and 13 (39.4%) were PCR negative. Anti-CMV IgM antibodies were detected only along with IgG antibodies in five (19.2%) of 26 PCR positive and one (4.8%) of 21 PCR negative patients. Comparing the results of the EU expressed as HP and LP, thus indicating the amount of anti-CMV antibodies with PCR results, HP anti-CMV IgG antibodies was present in 20 (76.9%) of 26 PCR positive and 10 (47.6%) of 21 PCR negative patients, which was statistically significant (Chi square test, P–0.038). Anti-CMV antibodies were not detected in 14 (29.8 %) of these 47 patients i.e. in six (23.1%) of 26 PCR positive patients and eight (38.1%) of 21 PCR negative patients. The results of ELISA in 28 children below 12 years belonging to both the PCR positive and negative groups expressed as HP and LP indicating the presence of anti-CMV antibodies in the serum samples is shown in [Table - 2]. Among the 28 patients below 12 years clinically suspected as having CMV infection, ELISA detected anti-CMV antibodies in 14 (73.7%) of 19 PCR positive patients and three (33.3%) of nine PCR negative patients, which was statistically significant (Fischer exact test, P–0.05). Among the 17 patients with anti-CMV IgG antibodies, 14 (82.4%) were PCR positive and three (17.6%) were PCR negative. This difference is statistically significant (Chi square test, P–0.008). Comparing the results of the EU expressed as HP and LP, thus indicating the amount of anti-CMV antibodies with PCR results, HP anti-CMV IgG antibodies was present in 14 (73.7%) of 19 PCR positive and two (22.2%) of nine PCR negative patients. This difference was statistically significant (Fischer exact test, P–0.01). The anti-CMV IgM antibodies were present only in the sera of three PCR positive patients (two HP and one LP) and not in any of the PCR negative patients. Among the 19 post-organ transplant patients, ELISA detected anti-CMV antibodies in six (85.7%) of seven PCR positive patients and 11 (91.7%) of 12 PCR negative patients. Among the 16 patients positive for anti-CMV IgG antibodies, six (37.5%) were PCR positive and 10 (62.5%) were PCR negative. HP anti-CMV IgG antibodies were present in six (85.7%) of seven PCR positive and eight (66.7%) of 12 PCR negative patients. The anti-CMV IgM antibodies were present only in the sera of one PCR negative patient (LP) and not in the PCR positive patients. CMV-DNA was not detected in the peripheral blood of the 35 normal healthy blood donor control samples but the sera showed the presence of anti-CMV antibodies. Anti-CMV IgG, IgM or both were present in the serum in 8 (22.9%), 5 (14.3%) and 22 (62.9%) of these healthy donors respectively. Of these, HP and LP levels of anti-IgG antibodies were present in 15 healthy donors each and LP levels of anti-IgM antibodies were present in 27 healthy donors. HP level of anti-IgM antibodies were present in none of the healthy donors.
In this study we considered PCR positivity in the clinical specimen as the gold standard to associate CMV with the disease in the patient. The difference in the presence of anti-CMV antibodies in PCR positive and negative groups was not significant in the 47 patients studied. The absence of anti-CMV antibodies in six (23.1%) of 26 symptomatic PCR positive patients correlated well with the findings of Bevan et al[7] and Zhang et al.[8] These results suggest that CMV seronegativity shown by ELISA does not exclude an activation of CMV. Eight (38.1 %) of 21 PCR negative patients were sero-negative and CMV activation may be ruled out in them only because they were also PCR negative. Highly sensitive PCR assays have been shown to detect the latent CMV-DNA in monocyte-enriched peripheral blood samples in normal healthy people. [9],[10] Therefore we used primers that were absolutely specific and highly sensitive (limit of sensitivity was 1fg) for detection of CMV-DNA of activated virus and not the latent virus. This was confirmed by the absence of CMV-DNA by PCR in the peripheral blood (buffy coat) of normal healthy blood donors who were sero-positive. Similar findings of the absence of CMV-DNA in the intraocular fluid inspite of the sero-positivity were shown in immuno-competent patients.[11],[12] Our findings in the patients with organ transplant complications showed no difference in the presence of anti-CMV antibodies in the sera of both PCR positive and negative patients. All these patients were adults. A similar serological response in terms of HP and LP anti-CMV antibodies was found among the normal healthy blood donors. Sero-positivity against CMV antigen in our population has been shown to be over 90% in individuals aged upto 25 years.[13] Our findings and those of other investigators indicate that serology is of low utility in adult patients to diagnose CMV infection. With regard to congenital and acquired clinically suspected CMV disease in the paediatric age group, the presence of anti-CMV antibodies, as IgG HP (EU) was significantly associated with PCR positive and not in PCR negative patients. Further, the presence of anti-IgM CMV antibodies in PCR positive and its absence in PCR negative paediatric patients also was suggestive of the current activity of CMV. Aggarwal et al[14] and Venkitaraman et al[13] had shown the high sero-positivity of CMV in children in various hospital-based studies in our country. Our findings suggest that ELISA may be a helpful method when PCR is not affordable, provided the presence of anti-CMV antibodies is estimated in terms of EU and correlated with the clinical findings along with a differential diagnosis in the paediatric age group. Thus our findings in this study in correlation with other investigators of the developed countries, authentically show that ELISA has no diagnostic significance in the detection of CMV activation, although it may help in the differential diagnosis of CMV infection in the paediatric age group. Our findings also suggest that institution of therapy for CMV infection solely based on ELISA results includes a 60% risk in treating people without CMV activation, and ELISA fails to identify the activation of CMV in 25% of the cases. [Table - 1], [Table - 2]
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