Microsporidial keratitis in India: 16S rRNA gene-based PCR assay for diagnosis and species identification of microsporidia in clinical samples

Purpose: To evaluate 16S rRNA-based polymerase chain reactions for the detection and species identification of the microsporidia that cause keratitis. Methods: Of the 5892 cases of microbial keratitis seen between September 2002 and December 2005, 31 (0.5%) microscopically diagnosed cases of microsporidial keratitis were included in the test group; 103 patients with nonmicrosporidial keratitis constituted the control group. A 16S rRNA-based pan-microsporidian PCR was chosen for the detection of microsporidian DNA. Species level identification was made using species-specific primer sets of Encephalitozoon spp (E. cuniculi, E. hellem, and E. intestinalis). Sequencing and BLAST analysis of amplicons obtained with pan-microsporidian primers were performed for validation. Results: The corneal scrapings from 26 of 31 cases in the test group and 2 of 103 cases in the control group showed a 250- to 280-bp amplicon in PCR by pan-microsporidian primers (sensitivity of 83% and specificity of 98%). The amplicons of 13 of 26 test group samples were identified by species-specific PCR: E. cuniculi, n = 7 (549 bp); E. hellem; n = 3 (549 bp); E. intestinalis; n = 1 (520 bp). The two cases in the control group were identified to be E. cuniculi. The remaining 15 cases (test group) were confirmed to be Vittaforma corneae by sequencing and BLAST analysis. All species were confirmed by sequencing and database homology comparison. Conclusions: This study is the first to validate PCR-based assays for detection of microsporidial DNA in corneal scrapings. Pan microsporidian PCR can be a useful adjunct with smear examination in the diagnosis of microsporidial keratitis

Comparison of an immortalized human corneal epithelial cell line with Vero cells in the isolation of Herpes simplex virus-1 for the laboratory diagnosis of Herpes simplex keratitis

BACKGROUND: Herpes simplex keratitis (HSK) is a sight threatening ocular infection often requiring a specific and prompt laboratory diagnosis. Isolation of Herpes simplex virus (HSV-1) in culture provides the most reliable and specific method and is considered as the "Gold Standard" in the laboratory diagnosis of HSK in spite of its low sensitivity. Using "cell lines of corneal origin" for virus isolation may be beneficial under such circumstances, since these cells have been shown to be excellent substrates for the growth of HSV-1 isolated from the cornea. We report a comparative study of a novel human corneal epithelial cell line (HCE) and the Vero cell line in the isolation of HSV-1 from corneal scrapings employing a shell vial assay. METHODS: Corneal scrapings were obtained from 17 patients with a clinical diagnosis of HSK. All the cases were confirmed by virological investigations (PCR and viral antigen detection positive, n = 15, PCR positive, n = 1, Viral antigen positive, n = 1). Scrapings obtained from 10 patients with infectious keratitis of non-viral origin were included as controls. All the scrapings were simultaneously inoculated into shell vials of HCE and Vero cells. Cultures were terminated at 24 h post-infection. Isolation of HSV-1 was confirmed using an indirect immunofluorescence/ immunoperoxidase assay. RESULTS: Virus could be isolated using both or either of the cell lines in 10/17 (58.82%) patients with HSK. HSV-1 was isolated from 10/ 17 (58.82%) and 4/17(23.52%) specimens in HCE and Vero cells, respectively (P = 0.036). None of the controls yielded HSV-1. While all the 10 (100%) strains were isolated in HCE, Vero yielded only 4/10 (40%) strains in the shell vial culture (P = 0.014). CONCLUSIONS: HCE showed a statistically significant difference in the virus isolation rate with respect to Vero cells. HCE may be an excellent alternative cell line for the isolation of HSV-1, especially from corneal scrapings, for the laboratory diagnosis of HSK

The Development of the LV Prasad-Functional Vision Questionnaire: A Measure of Functional Vision Performance of Visually Impaired Children

PURPOSE - To develop a reliable and valid questionnaire (the LV Prasad-Functional Vision Questionnaire, LVP-FVQ) to assess self-reported functional vision problems of visually impaired school children. METHODS - The LVP-FVQ consisting of 19 items was administered verbally to 78 visually impaired Indian school children aged 8 to 18 years. Responses for each item were rated on a 5-point scale. A Rasch analysis of the ordinal difficulty ratings was used to estimate interval measures of perceived visual ability for functional vision performance. RESULTS - Content validity of the LVP-FVQ was shown by the good separation index (3.75) and high reliability scores (0.93) for the item parameters. Construct validity was shown with good model fit statistics. Criterion validity of the LVP-FVQ was shown by good discrimination among subjects who answered "seeing much worse" versus "as well as"; "seeing much worse" versus "as well as/a little worse" and "seeing much worse" versus "a little worse," compared with their normal-sighted friends. The task that required the least visual ability was "walking alone in the corridor at school"; the task that required the most was "reading a textbook at arm’s length." The estimated person measures of visual ability were linear with logarithm of the minimum angle of resolution (logMAR) acuity and the binocular high contrast distance visual acuity accounted for 32.6% of the variability in the person measure. CONCLUSIONS - The LVP-FVQ is a reliable, valid, and simple questionnaire that can be used to measure functional vision in visually impaired children in developing countries such as India

Awareness of eye donation in the rural population of India

Purpose: To determine the "awareness of eye donation" and "willingness to pledge eyes for donation" in the rural population of Andhra Pradesh, southern India. Methods: A total of 7,775 subjects of all ages, representative of the rural population of Andhra Pradesh, participated in the Andhra Pradesh Eye Disease Study (APEDS). Subjects older than 15 years were interviewed regarding awareness of eye donation and willingness to pledge eyes for donation. Results: Age-gender adjusted prevalence of awareness of eye donation in this population was 30.7% (95% CI: 29.5 - 31.9) but only 0.1% (age-gender adjusted prevalence) (95% CI: 0.05 - 0.25) had pledged eyes. On multivariate analysis the awareness of eye donation was significantly less in those subjects 70 years old (OR 0.7; 95% CI: 0.6-0.8), illiterates (OR 0.2; 95% CI: 0.1-0.2), females (OR 0.8; 95% CI: 0.7-0.9), lower socioeconomic status group (OR 0.4; 95% CI: 0.4-0.5) and Christians (OR 0.2; 95% CI: 0.1-0.6). Media comprised the major source of information about eye donation. Of those aware of eye donation, 32.9% were willing to pledge eyes, and 50.6% needed more information to decide whether or not to pledge their eyes. Conclusions: There needs to be more transfer of knowledge if more eyes are to be pledged. One-third of those aware of eye donation have not pledged their eyes, and an additional 50.6% needed more information to decide. This means only about one-fifth of those aware of eye donations have pledged their eyes