|Year : 2003 | Volume
| Issue : 1 | Page : 71-75
The diagnostic significance of enzyme linked immuno-sorbent assay for herpes simplex, varicella zoster and cytomegalovirus retinitis.
Hajib N Madhavan, K Priya
L & T Microbiology Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, India
Hajib N Madhavan
L & T Microbiology Centre, Vision Research Foundation, Sankara Nethralaya, Chennai
Source of Support: None, Conflict of Interest: None
Purpose: To evaluate the diagnostic usefulness of enzyme linked immuno-sorbent assay (ELISA) in single serum samples to associate herpes simplex virus (HSV), varicella zoster virus (VZV) or cytomegalovirus (CMV) with viral retinitis as against polymerase chain reaction (PCR) on intraocular specimens. It was also designed to study the seroprevalence in normal healthy individuals, and the genomic prevalence of HSV, VZV and CMV in patients without an active viral inflammatory process.
Methods: PCR for the detection of HSV, VZV and CMV genomes was done on 33 and 90 intraocular fluids from viral retinal patients and non-viral controls respectively. ELISA was done on 30 and 100 serum samples from viral retinitis patients and normal healthy controls respectively.
Results: PCR did not detect HSV, VZV and CMV genomes except one, in which VZV-DNA was detected. ELISA showed prevalence rates of 28%, 83% and 90% for antibodies against HSV, VZV and CMV respectively in the normal population. In the 30 viral retinitis patients, PCR detected HSV-DNA in 2 (6.7%), VZV-DNA in 7 (23.3%) and CMV-DNA in 6 (20.0%) patients, while ELISA detected antibodies against HSV, VZV and CMV in 13 (43.3%), 24 (80.0%) and 23 (76.7%) patients respectively. ELISA was of value in indirect diagnosis only in 6 (20.0%) as compared to 15 (50.0%) of 30 patients by PCR, this difference was statistically significant (McNemar test, P value = 0.005).
Conclusion: Serology by ELISA is no longer a useful diagnostic tool to associate HSV, VZV and CMV viruses with viral retinitis.
Keywords: Enzyme-linked immuno-sorbent assay, Polymerase chain reaction, Herpes viruses, ELISA in viral retinitis, PCR in viral retinitis
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Madhavan HN, Priya K. The diagnostic significance of enzyme linked immuno-sorbent assay for herpes simplex, varicella zoster and cytomegalovirus retinitis. Indian J Ophthalmol 2003;51:71-5
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Madhavan HN, Priya K. The diagnostic significance of enzyme linked immuno-sorbent assay for herpes simplex, varicella zoster and cytomegalovirus retinitis. Indian J Ophthalmol [serial online] 2003 [cited 2020 Sep 25];51:71-5. Available from: http://www.ijo.in/text.asp?2003/51/1/71/14734
The common aetiological agents of viral retinitis are herpes simplex virus (HSV), varicella zoster virus (VZV) and cytomegalovirus (CMV). Rapid detection of the causative viral agent is a key factor in preservation of sight since it allows for specific therapy.,, Conventional methods for aetiological diagnosis include detection of the virus antigen and virus isolation from intraocular specimens and serological methods of antibody assay by enzyme linked immuno-sorbent assay (ELISA)., The molecular method of nucleic acid detection by polymerase chain reaction (PCR) in the intraocular fluid has been in use during the last few years.,,, However, the high levels of technical facilities and expertise required for virus isolation and PCR are not available in most microbiology laboratories in India. Since serum is more easily obtainable than aqueous humor (AH), ophthalmologists mainly depend on antibody assay on single serum samples to associate one of the above three herpes viruses with retinitis in these patients. Serodiagnosis using paired serum samples is useful for primary herpes virus infections and not in patients with recurrent disease as the antibody levels do not increase remarkably in the latter. We have previously shown that PCR on intraocular fluids from patients with viral retino-choroiditis is an absolutely specific, more sensitive and reliable diagnostic tool than the conventional virological methods for detection of HSV, VZV and CMV. In the present study, we evaluated the diagnostic usefulness of ELISA in single serum samples for association of HSV, VZV or CMV as a causative agent of viral retinitis as against PCR on the intraocular specimens. This study also examined the seroprevalence of these viruses in healthy persons and PCR on the intraocular fluids obtained from patients undergoing cataract surgery and those presenting with non-viral retinal inflammations.
| Materials and Methods|| |
Study patients and specimens
A total of 33 intraocular fluid specimens [27 aqueous humor (AH) and 6 vitreous fluid (VF)] were collected from 30 patients with clinically diagnosed viral retinitis. These patients were apparently healthy and serologically negative for human immunodeficiency virus (HIV). The intraocular fluids were subjected to PCR to detect HSV, VZV and CMV genomes.
Thirty single serum samples were collected from these 30 patients with clinically diagnosed viral retinitis. The serum samples were obtained soon after collection of intraocular fluid during the active stage of the disease. The serum samples were tested by ELISA for the detection of antibodies against HSV, VZV and CMV.
Control patients and specimens
Ninety intraocular fluid specimens (30 AH from cataract extractions, 20 VF from patients with diabetic retinopathy, 20 AH and 20 VF from culture-proven bacterial or fungal endophthalmitis) collected from 90 patients were included as controls to determine the prevalence of herpes viral DNA in the intraocular fluids of patients without clinical evidence of an active viral inflammatory process.
Hundred serum samples from normal healthy voluntary blood donors (Blood Bank, Voluntary Health Services, Chennai) negative for HIV, Hepatitis B Virus (HBV) and Hepatitis C virus (HCV) and VDRL nonreactive were included as controls for ELISA to determine the seroprevalence of HSV, VZV and CMV in the normal population.
Polymerase chain reaction (PCR)
DNA extraction from the specimens was done as described by us earlier., In brief, 200 Ál of lysis buffer (40mM Tris-Cl [pH 8.0], 10mM EDTA, 0.5% SDS) and 1 Ál of proteinase K (20mg/ml) were added to 50 Ál of the intraocular fluid specimen, the mixture vortexed thoroughly, incubated at 55░C for 2 hours and standard phenol-chloroform extraction performed. The extracted DNA was subjected to PCR for the detection of HSV, VZV and CMV. (Primers used custom synthesised by Bangalore Genei Ltd., India). PCR was done as described by us earlier for the detection of HSV, VZV and CMV genomes., The amplified product was then subjected to gel electrophoresis using 2% agarose gel.
Enzyme linked immuno-sorbent assay (ELISA)
Both anti-IgG and anti-IgM antibodies were assayed against HSV, VZV and CMV using ELISA kits from BIOKIT (Barcelona, Spain) for HSV and CMV, and ELISA kits from Immuno-Biological Laboratories (Hamburg, Germany) for VZV. Tests were done according to the manufacturer's instructions.
The McNemar test and 95% confidence interval were used to analyse statistical significance.,
| Results|| |
Polymerase chain reaction (PCR)
Intraocular fluids from controls
PCR did not detect HSV or CMV genomes in any of the 90 intraocular control fluid samples. VZV-DNA was detected in 1 (1.1%) of 90 control samples. This was the vitreous fluid from a culture-proven case of bacterial endophthalmitis.
Intraocular fluids from clinically diagnosed viral retinitis patients
PCR detected HSV-DNA in 2 (6.7%), VZV-DNA in 7 (23.3%) and CMV-DNA in 6 (20.0%) of 30 patients. Overall, herpesviral aetiology was attributed in 15 (50.0%) of 30 patients.
Enzyme linked immuno-sorbent assay (ELISA)
Serum samples from controls
The bar diagram in [Figure - 1] depicts the overall prevalence of serum anti-IgG and anti-IgM antibodies against HSV, VZV and CMV in 100 normal healthy blood donors. The prevalence rate of both anti-IgG and anti-IgM antibodies was 3%, 2% and 7% against HSV, VZV and CMV respectively. Anti-IgM antibodies alone were prevalent in 2% and 1% against HSV and CMV respectively, while anti-IgG antibodies alone against HSV, VZV and CMV were prevalent in 26%, 83% and 89% respectively. The overall prevalence rates of anti-HSV-1, anti-HSV-2, anti-VZV and anti-CMV antibodies (IgG or IgM or both) were 27%, 1%, 83% and 90% respectively in the normal population. The age-wise distribution of the prevalence rates of antibodies against HSV, VZV and CMV is shown in [Table - 1]. The 95% confidence interval shows that the prevalence rates of anti-HSV, anti-VZV and anti-CMV antibodies in such a normal population would fall into a range of 19-37%, 74-89% and 82-95% respectively.
Serum samples from clinically diagnosed viral retinitis patients
ELISA detected anti-HSV antibodies in 13 (43.3%), anti-VZV antibodies in 24 (80.0%) and anti-CMV antibodies in 23 (76.7%) of 30 patients. Anti-IgM antibodies along with anti-IgG antibodies to one of the three viruses were detected in 6 (20.0%) of 30 patients. Anti-IgM antibodies alone were not detected in any of the serum samples.
Comparative results of PCR and ELISA in patients with retinitis
[Table - 2] shows the results of ELISA tests done on the single serum specimens collected from 15 patients in whom specific herpes viral aetiology was detected in the intraocular specimens by PCR. The specific anti-viral antibody to the implicated virus was not detected in the sera of patients 1,6 and 7. Anti-CMV IgM antibodies were detected in the sera of patients 10, 12 and 14 (PCR positive for CMV in the intraocular fluid), indicating probable activation of CMV infection. In the remaining 9 patients, antibodies to all the three or two of the viruses were detected without precisely reflecting to the virus implicated in the retinal inflammation. In three of the 15 patients in whom PCR was negative for all the three viruses, ELISA was also negative, while in the others antibodies against two or three of these viruses were detected. Thus PCR was a better method than ELISA statistically (McNemar test, P value = 0.005). Though ELISA assayed a low titre (ELISA units - EU) of specific anti-IgM antibodies in the sera of 6 patients, the presence of that particular viral DNA was implicated in the intraocular fluid in only 3 (patients 10, 12 and 14 - 50.0%) of them as shown in [Table - 2].
| Discussion|| |
In a developing country like India, most ophthalmolo-gists depend on serum antibody assays by ELISA to confirm and specify the herpes viruses with viral retinitis. This study was designed to evaluate the diagnostic utility of ELISA on serum samples in associating a definitive causative herpes viral agent with viral retinitis. Therefore, we needed to determine the seroprevalence of HSV, VZV and CMV in the normal healthy population and compare this with the results of the serology on patients with viral retinitis. A very high seroprevalence of anti-VZV and anti-CMV antibodies, increasing with age, has also been shown by other investigators in various populations of tropical and temperate regions. ,,,,, In our study the prevalence of both anti-HSV-1 and anti-HSV-2 antibodies was as low as 27% and 1% respectively and the prevalence declined with age. This is at variance with other prevalence rates of 40-70% for anti-HSV-1 antibodies and 2-12% for anti-HSV-2 antibodies.,,, Comparing our results in 2002 with those of Venkitaraman et al in 1986 (16 years earlier), it appears that there is a marked reduction in the seroprevalence rate of anti-HSV antibodies from 83% to 28% among the normal healthy south Indian population. In our study the majority of circulating antibodies were anti-IgG antibodies. Anti-IgM antibodies were prevalent in an insignificant number of persons.
In this study, VZV-DNA was detected in one patient with postoperative bacterial endophthalmitis. It is likely that activation of VZV in this patient could have been triggered during the episode of bacterial endophthal-mitis with a spillover of the virus-laden leukocytes into the vitreous cavity. The absence of HSV, VZV and CMV genomes in the intraocular fluids of patients undergoing cataract extraction and patients presenting with non-viral retinal inflammations indicates that the detection of herpes virus DNA by PCR implicates an active infection. Other investigators have also shown similar findings, barring one report by Fox et al who detected CMV-DNA in one of 8 vitreous fluid samples tested from normal persons. In two other reports, ocular fluids obtained from patients undergoing vitreoretinal surgery for non-viral inflammatory conditions did not show the presence of herpes virus DNA by PCR though a high proportion of these patients tested positive for herpes virus antibodies., Therefore, for the purpose of discussion on the diagnostic usefulness of ELISA, we considered PCR as the gold standard. Virus specific anti-IgM antibodies with a presumed active infection were found only in three PCR positive patients. In three others both ELISA and PCR were negative indicating that these three viruses were not the aetiological agents. Thus, ELISA has been of value for an indirect diagnosis only in 6 (20.0%) as compared to 15 (50.0%) of 30 patients by PCR. This difference was statistically significant. In 15 cases where the PCR was negative, the aetiology was not any of the three herpes viruses tested for. Thus, our findings and those of the previous studies,,, demonstrate that the presence of antibodies to the herpes viruses in the peripheral blood does not implicate any of them as a definitive causative agent.
To conclude, serology by ELISA is no longer a useful diagnostic tool to associate any of these three viruses with viral retinitis. The small volume of aqueous humor sample obtained by a simple office procedure could be more appropriately used for antigen detection and for PCR rather than antiviral antibody assays for calculation of the Goldman-Whitmer coefficient. We believe that a positive PCR on intraocular fluid in a patient with intravitreal inflammation should be regarded as a significant finding and should be managed accordingly. It is suggested that regional laboratories could be established to perform PCR tests on intraocular fluids to detect these viruses for aetiological diagnosis of atypical cases.
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[Figure - 1]
[Table - 1], [Table - 2]
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