Background Herpes Simplex Virus (HSV) keratitis is a leading cause of corneal blindness. Definitive laboratory diagnosis is essential for timely management. Collection of corneal scrapings in patients with advanced epithelial keratitis and corneal thinning poses perforation risks; tear fluid is a feasible and convenient alternative but has not been widely evaluated for HSV detection.
Methods Tear fluid alone (229) or along with corneal scrapings (153) from patients of suspected herpetic keratitis was tested for HSV-1 antigen by indirect immunofluorescence assay, virus isolation in Hep 2 cells and PCR to amplify the 111 bp region of the thymidine kinase (tk) coding gene and the 144 bp region from the DNA polymerase coding gene of HSV.
Results HSV 1 antigen was detected in 31/229 (13.53%) tear specimen and 35/153 (22.87%) corneal scrapings in immunofluorescence assay; virus was isolated from 12/229 (5.2%) tear and 17/153 (11.11%) corneal scrapings, and PCR was positive for both the genes in 32/229 (13.97%) tear specimen and 56/153 (36.66%) corneal scrapings.
Conclusion Corneal scrapings yielded a significantly better HSV positivity than tears in both the PCR assay (p<0.0005) and immunofluorescence assay. PCR was much more sensitive than immunofluorescence and virus isolation. However, tears should be tested for definitive laboratory diagnosis of HSV infection whenever corneal scraping collection is not possible.
- corneal scrapping
- herpes simplex virus
- laboratory diagnosis
- PCR assay
- diagnostic tests/investigation
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- corneal scrapping
- herpes simplex virus
- laboratory diagnosis
- PCR assay
- diagnostic tests/investigation
Herpes simplex virus (HSV) is a leading cause of keratitis and endothelitis worldwide.1 The diagnosis of herpes simplex keratitis (HSK) is frequently based on clinical features, which often are not confirmatory.1 Until recently, laboratory methods that helped in achieving a diagnosis included virus isolation using tissue culture and antigen detection methods such as the immunofluorescence assay (IFA)2 3
Virus isolation, though considered a gold standard for diagnosis of viral infections, is relatively time-consuming and often depends upon viable infectious material that needs to be speedily transferred to a special virology laboratory for processing.2 The immunofluorescence technique is adversely influenced by false-positive and false-negative results, small sample size and subjective variations in the interpretation of data.2 These shortcomings have been overcome by the introduction of more rapid and sensitive molecular techniques such as the PCR assay for detecting viral DNA in corneal scrapings and sometimes tears from patients suffering from HSK.4–6 Although corneal scraping remains the clinical specimen of choice for laboratory diagnosis, the clinician is often faced with the problem of collecting scrapings from patients who develop persistent epithelial defect and reduced corneal thickness.4 7 Laboratory testing of tears may provide a definitive diagnosis, though this needs widespread evaluation.4
The purpose of the present study was to assess the relative efficacies of indirect immunofluorescence assay, virus isolation and PCR assay in confirming the diagnosis of HSK and to evaluate the suitability of tear specimens as a substitute for corneal scrapings.
Materials and methods
A total of 229 clinically suspected cases of viral keratitis, especially Herpes Simplex Virus Keratitis (HSK), reporting to the Dr R P Centre for Ophthalmic Sciences, New Delhi, India between February 2007 and January 2010, were included in the study.
Collection of specimens
After informed consent, tear specimens were collected from the lower conjunctival fornix with Schirmer strips (Flogel Carboxy methyl cellulose ophthalmic sterile strips, Madhu Instruments, Delhi, India) and borosilicate glass capillaries (3.5 mm diameter×70–80 mm length) (Borosil, Mumbai, India). The samples were pooled and divided into three parts. After informed consent, corneal scrapings were collected from 153 patients using the standard procedure and divided into three parts.
A smear was prepared from both specimens on clean glass slides for immunofluroescence assay. The second part from both specimens was placed into Tris-EDTA (TE) buffer for PCR assay, and the third part of both was placed in Dulbecco Minimum Essential Medium (MEM) (Invitrogen, Carlsbad, California) with antibiotics for tissue culture isolation. The specimens were stored at −70°C until processed for virus isolation and PCR assay, and the immunofluorescence slides were stored at −20°C.
Antigen detection by immunoflurescence assay
HSV 1 virus antigen detection was done on acetone fixed smears of corneal scrapings and tear specimens using rabbit anti HSV antibodies (Dako, Glostrup, Denmark) and fluorescein isothiocyanate tagged swine antirabbit immunoglobulin (Dako) as second antibodies.8 The slides were observed under a fluorescent microscope (Nikon, Tokyo, Japan). Air-dried HSV1 (ATCCstrain) infected Hep 2 cells were taken as a positive control, and uninfected Hep2 cells served as a negative control.
Tissue culture isolation of HSV
Virus isolation was done by conventional procedures in a confluent monolayer of Hep-2 cells grown in 12-well tissue culture plates in duplicate. Briefly, 200 μl of specimens was inoculated to each well and centrifuged at 3000 rpm for 10 min. After adsorption at 35°C for 1 h, the inocula were replaced with fresh maintenance medium (MEM, supplemented with 2% fetal bovine serum and antibiotics) and incubated further at 35°C. The plates were examined daily under an inverted microscope (Nikon) for development of a cytopathic effect. The results were confirmed by indirect immunofluorescence assay on the infected cells. The ATCC strain of HSV and the sample collection medium were used as positive and negative controls respectively.
DNA was extracted from both the specimens using commercial QI Amp DNA blood kit (Qiagen, Chatsworth, California), as per the manufacturer's instructions.
Polymerase chain reaction (PCR)
PCR assay was optimised to amplify 111 bp region of the HSV 1 Thymidine Kinase gene and the 142 bp region of DNA polymerase gene using published primer sequences and procedures.9 10 Both sets of primers were synthesised using 392ABI nucleotide synthesiser (Applied Biosystems, Foster City, California). The different parameters of the PCR assay were optimised. Amplification was carried out in a 25 μl reaction mixture containing 15 pmol each of forward and reverse primers, 1.5 U of Taq polymerase, 2.0 mm MgCl2 200 μm of dNTPs in 10× PCR buffer (Fermentas) and 5 μl of template DNA in a thermocycler (Gene Amp PCR system 9700, Applied Biosystems) with initial denaturation for 5 min at 94°C followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min, with a final extension for 5 min. DNA from HSV-infected Hep-2 cells and distilled water were used as positive and negative controls.
The product was electrophoresed in 2% agarose gel (Sigma, St Louis, Missouri), stained with ethidium bromide and visualised under a gel documentation system (Syngene, San Diego, California). The amplified products were sequenced commercially (Bangalore Gene, Bangalore, India).The obtained nucleotide sequences were compared with available HSV sequences from databases using a blast analysis program (NCBI, NIH).
In the immunofluorescence assay, the HSV antigen was detectable in epithelial cells as a bright apple green nuclear and cytoplasmic fluorescence. HSV antigen was detected in 31 (13.53%) of 229 tear specimens. In 153 patients, from whom both corneal scrapings and tears were obtained, 35 (22.87%) and 26 (17%) were positive respectively (p=0.19); 23 (15.03%) for the HSV antigen (table 1).
Virus was isolated from 12 (5.3%) of 229 tear samples. In 153 patients, from whom both corneal scrapings and tear were obtained, 17 (11.11%) and 11 (7.1%) were positive respectively (p=0.23); 10 (6.53%) for virus isolation (table 1).
In the PCR assay from positive specimens, both genes were amplified (figure 1). The nucleotide sequences of amplified products showed a complete match with those of prototype strains available in the database. PCR was positive in 32 (13.97%) of the 229 tear specimens. In 153 patients, from whom both corneal scrapings and tear were obtained, 56 (36.6%) and 28 (18.3%) were positive respectively (p=0.003); 26 (16.99%) in the PCR assay (table 1).
Corneal scrapings yielded significantly higher detection rates than in tears in the PCR assay (p=0.003). In addition, the immunofluorescence assay and PCR accurately detected more positives in both tears and corneal scrapings than the cell-culture technique (table 1). Both PCR and the immunofluorescence assay conducted on tear and corneal samples had similar sensitivities and negative predictive values. However, the specificity and the positive predictive value of PCR were much higher in tear samples than with corneal scrapings (90.6% and 37.5%, respectively, vs 71.3% and 30.3%, respectively; table 1), suggesting that PCR assay used for tear fluids in clinically suspected HSK is less likely to yield false-positive results. It was noted that the specificity of the immunofluorescence assay in corneal scrapings was lower (86.7%) than the specificity noted in tears (91.2%), thus emphasising that tear specimens yielded less non-specific results irrespective of the laboratory methods used.
Herpetic keratitis is a leading cause of preventable blindness. Rapid and accurate diagnosis is essential for prompt treatment.6 Using procedures such as viral isolation and immunofluorescence assay, a confirmatory laboratory diagnosis was achieved in only 5–23% of the clinically suspected cases. This indicates the difficulty in establishing an accurate laboratory diagnosis.
PCR-based techniques have been used previously with corneal scrapings and tear samples.5 7 9 11 Sampling of tear fluid does not cause any appreciable discomfort to patients, and tear samples are always preferred to scraping in cases of reduced corneal thickening due to persistent epithelitis.
In the present study, considering the tear and corneal samples together, positive cases in tissue-culture isolation were between 7.2 and 11.1%, and in immunofluorescence between 17 and 22.9%. Similar observations of lower detection in cell culture compared with immunofluorescence were noted by others.6 12 Biriken et al reported that virus-specific antigen by indirect immunofluorescence could be detected in 22 of 70 (31.4%) cases, whereas only 20% of the cases had positive virus isolation.10 This may be due to inherent problems in virus isolation from clinical specimens, which is known to be low. PCR accurately determined more positive cases than cell culture and immunofluorescence in the corneal scraping and showed marginally higher detection rates than immunofluorescence in the tear. This is in agreement with others.10 12 In clinical settings, the chances of detecting viral DNA/antigen are likely to be higher in the corneal scrapings (primary site of infection) than in the tear fluid, which is supposed to contain the shed viruses only.8 We also observed higher positivity rates in the corneal scrapings than in the tears, employing both the PCR assay and the immunofluorescence procedure.
Our results confirmed that both IFA and PCR showed 100% sensitivities compared with virus isolation, and the negative predictive values for both the tests were also 100%, in both corneal scrapings and tear. Sensitivity ranges of immunofluorescence tests for HSV varying between 77% and 86% have been reported.12–14 Considering tissue culture as the conventional gold standard, the sensitivities of both tests in the tear samples were on a par with those in the corneal scrapings. However, it is now well established that virus isolation rates in tissue cultures are quite low.2
The PCR assay showed a higher detection rate from the corneal samples than from tears. It may be possible that the HSV becomes detectable in tear specimens when replication and viral load are high in corneal ulcers, so that they are sufficiently abundant in tear fluid. Nevertheless, PCR and IFA appeared to be more specific in the tear fluid than in corneal scrapings when virus isolation results were used as a gold standard. As discussed above, although theoretically this may be correct, the virus isolation rates in tissue culture are known to be low,2 thus giving this false impression. Therefore, the increased number of PCR positive and culture negative cases may represent not false positives but rather increased detection rates by PCR assay.
Nevertheless, these findings suggest that, in clinical application, a positive PCR result in tear film would be less likely to be a false positive. Thus, it would be preferable to collect tear specimens from patients in whom collection of scraping would not be technically feasible. From a clinical viewpoint, the observations are undoubtedly relevant because appropriate treatment should be promptly advocated without waiting for culture results in cases where tear fluid PCR yields a positive result. Finally, these results are amply substantiated by those of the others5 7 9 in that PCR performed on tear samples would be of great help in diagnosing the majority of atypical epithelial keratitis and majority of stromal keratitis, where clinical criteria alone would not suffice.5
In conclusion, wherever corneal scraping collection is feasible, tears cannot replace scrapings for HSV detection. However, wherever corneal scrapings cannot be collected, tears may act as a substitute for definitive diagnosis.
The authors thank all clinical faculties and residents of the Dr R P Centre for clinical specimens.
Funding Indian Council of Medical Research, New Delhi.
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was provided by the Institutional Ethics Committee, All India Institute of Medical Sciences.
Provenance and peer review Not commissioned; externally peer reviewed.
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