ABSTRACT
Seven herpes simplex type-1 (HSV-1) isolates from herpes simplex keratitis (HSK) cases clinically resistant to acyclovir (ACV) were analyzed for the mechanism of ACV resistance in them. The purpose of the study was to focus the attention of ophthalmologists on the frequency of occurrence of ACV resistance in HSK and to characterize such a phenomenon. We employed in-vitro plaque reduction assay, thymidine kinase assay, polymerase chain reaction, single-strand confirmation polymorphism analysis and sequencing to detect any mutation(s) in thymidine kinase gene in this analytical study. Four of the seven HSV-1 isolates proved ACV resistant by plaque reduction assay and three of them showed reduced thymidine kinase activity. Altered mobility pattern indicative of mutation within 335 base pair PCR product bracketing the suggested homopolymer mutational hotspot (7 Guanosine) was detected in 2 of these 3 isolates. DNA sequencing showed a deletion at nucleotide position 336 from the tk gene transcription start in both the isolates. This mutation has generated the first TGA stop codon 27 nucleotides downstream in the tk open reading frame. Our study also suggests the need of clinical/molecular surveillance of ACV resistance in HSV types in a given geographic location for better management of HSV infections.
Subject(s)
Acyclovir/pharmacology , Antiviral Agents/pharmacology , Herpesvirus 1, Human/drug effects , Keratitis, Herpetic/virology , Base Sequence , Developing Countries , Drug Resistance, Microbial/genetics , Herpesvirus 1, Human/isolation & purification , Humans , India/epidemiology , Keratitis, Herpetic/epidemiology , Microbial Sensitivity Tests , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Sequence Analysis, DNA , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , Viral Plaque AssayABSTRACT
BACKGROUND: Herpetic ocular disease is a major cause of blindness. Rapid and accurate diagnosis is essential for prompt, proper treatment. We evaluated the usefulness of detection of herpes simplex virus (HSV) DNA by polymerase chain reaction (PCR) in the laboratory diagnosis of herpetic keratitis. METHODS: A retrospective study was conducted involving 234 patients who attended the cornea clinic at the Regional Ophthalmic Institute, Chennai, India, between March 1995 and September 1997. Inclusion in the study was based on clinical diagnosis of herpetic keratitis. Oligonucleotide primers directed against the HSV-I thymidine kinase gene were used, yielding a 110 base pair amplicon. The utility of PCR analysis was assessed against other diagnostic markers: HSV isolation on cell culture, HSV antigen detection by indirect immunofluorescence, detection of anti-HSV IgG by enzyme-linked immunosorbent assay (ELISA) and detection of HSV-specific tear secretory IgA (sIgA) by ELISA. These tests showed overall sensitivity values of 22.4%, 39.8%, 30.4% and 20.3% respectively. RESULTS: In epithelial keratitis all 35 specimens from which virus was cultured were positive by PCR. PCR gave a positive result in 23 (82.1%) of the 28 specimens in which HSV antigen was detected and in 4 (57.1%) of the 7 specimens that showed HSV-specific IgG. In addition, PCR detected HSV DNA in 5 of the 30 cases in which these three tests gave a negative result. PCR of two pooled tear samples (collected 1 week apart from the same patient) from 40 patients with stromal keratitis gave a positive result in 12 cases (30%). In stromal keratitis the sensitivity of PCR in detecting HSV DNA in tear samples was 85.7% with culture, indirect immunofluorescence and detection of anti-HSV IgG as the gold standard, and 80% with detection of sIgA as the gold standard. INTERPRETATION: The results confirm the good correlation with the clinical picture that can be obtained with PCR analysis. They also highlight the diagnostic utility of PCR in detecting HSV DNA in tear samples. This is particularly important in herpetic stromal keratitis, in which collection of corneal scrapings is not advised and, hence, conventional techniques such as virus isolation and antigen detection become difficult.
Subject(s)
Keratitis, Herpetic/diagnosis , Polymerase Chain Reaction/methods , Antibodies, Viral/analysis , Antigens, Viral/analysis , Corneal Stroma/immunology , Corneal Stroma/virology , Cross-Sectional Studies , DNA Primers/chemistry , DNA, Viral/analysis , Developing Countries , Enzyme-Linked Immunosorbent Assay , Epithelium, Corneal/immunology , Epithelium, Corneal/virology , Fluorescent Antibody Technique, Indirect , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/immunology , Herpesvirus 1, Human/isolation & purification , Humans , Immunoglobulin A, Secretory/analysis , Immunoglobulin G/analysis , India , Keratitis, Herpetic/virology , Predictive Value of Tests , Reproducibility of Results , Retrospective Studies , Sensitivity and Specificity , Tears/immunology , Tears/virologyABSTRACT
Corneal scrapings collected from 70 patients were used to assess the diagnostic value of indirect immunofluorescence (indirect IF) procedure in comparison with routine virus culture (RVC) for the diagnosis of Herpes simplex virus induced keratitis (HSK). Virus specific antigen was detected by indirect IF in 22 (31.42%) cases. In contrast, only 20% (14) of the cases had positive viral isolation which sometimes took as long as a week to show a cytopathogenic effect (CPE). It is concluded that antigen detection by indirect IF is a rapid, specific and sensitive technique for demonstrating HSV-1 antigen in corneal scrapings from HSK patients and a useful laboratory tool not only for diagnosing HSK but also for monitoring efficiency of anti HSV treatment for HSK.
Subject(s)
Cornea/virology , Herpesvirus 1, Human/isolation & purification , Keratitis, Herpetic/diagnosis , Animals , Antigens, Viral/analysis , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Fluorescent Antibody Technique, Indirect , Herpesvirus 1, Human/growth & development , Herpesvirus 1, Human/immunology , Humans , Keratitis, Herpetic/virology , Vero Cells , Virus CultivationABSTRACT
A case of uveitis with microfilariae in the anterior chamber is reported. The organism was recovered from the aqueous and identified as Brugia malayi.
