ABSTRACT
PURPOSE: Most common viruses causing ocular infections are Herpes Simplex Viruses (HSV) type 1 and type 2, Cytomegalovirus (CMV), Varicella-zoster Virus (VZV), and few strains of Adenovirus. Diagnosis of these infections through clinical manifestations and using conventional methods has a number of limitations. The purpose of this study was to develop a multiplex Polymerase Chain Reaction (PCR) for simultaneous detection of all pathogenic viruses from ocular infections. METHODS: Ten uniplex PCRs were standardized, two each for HSV type 1 (HSV-1) and type 2 (HSV-2), CMV, VZV, and Adenovirus. Various multiplexing combinations of above PCRs were put to finalize targets and reaction conditions enabling diagnosis of all in a single reaction. The uniplex and multiplex PCRs were run for known positive and negative controls, and samples from clinically suspected patients and healthy controls. RESULTS: Out of the 170 samples from suspected ocular infections, 24.7% were positive by uniplex PCR and 22.9% were correctly identified by multiplex PCR. None of the samples negative by uniplex PCRs was positive by the multiplex PCR. The sensitivity and specificity of multiplex PCR compared to the commonly used uniplex PCRs as gold standard was 92.86% and 100%, respectively. The prevalence of different viral pathogens was 13.5% for HSV-1, followed by 5.9% for Adenovirus, 2.4% for VZV, 1.8% for HSV-2, and 1.2% for CMV. CONCLUSION: The establishment of multiplex PCR has found immediate application in diagnosing ocular viral pathogens in a single reaction, thus saving time, manpower, and resources by fivefold.
ABSTRACT
Presence of Chlamydia trachomatis in endocervix was determined in 2466 women attending a tertiary care hospital in New Delhi, India over a period of 16 years, using a monoclonal-based direct immunofluorescence assay, tissue culture isolation, and a conventional PCR assay. Chlamydia antigen could be detected in 391 out of 2466 (15.85%) of patients studied; in 27.27% women with PID, 16.74% women with cervicitis, 16.03% women with infertility, and 12.06% women with adverse pregnancy outcomes, respectively. There was a statistically significant decreasing trend in Chlamydia antigen positivity between the years 1994-1999 and 2000-2004; the apparent decline in antigen positivity between the years 2000-2004 and 2005-2010 was not statistically significant. Antigen detection assay detected equal number of positives as the PCR assay; tissue culture isolation demonstrated lower positivity. In a few representative specimens from cervicitis patients, genotyping was done using RFLP pattern analysis of C. trachomatis MOMP gene amplified by PCR assay, all of these belonged to Chlamydia trachomatis serovar E.