Laboratory methods in the detection of herpes simplex virus (HSV) in keratitis--a 9-year study including polymerase chain reaction (PCR) during last 4 years.

Conventional methods of fluorescent antibody test (FAT) and virus isolation (VI) and molecular method of polymerase chain reaction (PCR) were compared for the detection of HSV in keratitis during a 9-year period. Of 186 corneal scraping specimens, 108 were subjected to FATand VI in the pre-PCR period (initial 5 years) while 78 to FAT, VI and PCR in the PCR period (latter 4 years). HSV was detected by FAT in 44/186 (23.7%), VI in 18/186 (9.7%) and PCR in 27/78 (34.6%) specimens. Overall, HSV was diagnosed in 56/186 (30.1%) specimens. PCR has increased the clinical sensitivity by 12.8%, which is statistically significant (McNemar test, P-0.002). VI should be replaced by PCR. FAT though less sensitive should always be employed as a routine to give an early diagnosis, the results of which could be further confirmed, if necessary, by PCR, which is a more sensitive and specific diagnostic tool.

Preparation of amniotic membrane for ocular surface reconstruction.

We describe the preparation and preservation of human amniotic membrane required for transplantation in the management of ocular surface diseases. Informed consent is obtained and the donor is screened to exclude risk of transmissible infections such as human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus, and Treponema pallidum infections. Ideally, the media and washing solutions needed for the preparation of amniotic membrane are prepared only a week to 10 days prior to use and not stored in the freezer weeks ahead. The AM obtained under sterile conditions after elective caesarian section is washed free of blood clots and chorion. With the epithelial surface up, amniotic membrane is spread uniformly without folds or tears on individually sterilized 0.22 micron nitrocellulose membranes of the required sizes. The prepared filter membrane with the adherent amniotic membrane is placed in the preservative medium and stored at -80 degrees C. The membranes are released when the repeat serology for HIV after the window period has excluded virus infection in the donor. Depending on consumption they may be used up to 6 months after preparation, though many have recommended storage for an indefinite period. Since the amniotic membrane has only incomplete expression of HLA antigens and amniotic epithelial cells do not express them, it is not rejected after transplantation. The presence of several cytokines in the amniotic membrane promotes epithelialization with reduction of fibrosis during healing.