Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR.

A protocol for the rapid detection of fungal DNA in ocular samples, derived from three species, Candida albicans, Aspergillus fumigatus, and Fusarium solani, has been developed. Two novel panfungal primers complementary to 18S rRNA sequences present in all three species were designed. Panfungal PCR was followed by three nested PCRs utilizing species-specific primers. PCR sensitivity ranged from 50 to 100 fg of free DNA and between one and two C. albicans organisms. In addition, we also developed a rapid and reliable DNA extraction protocol. This protocol minimized DNA loss during extraction, whilst removing compounds from vitreous and aqueous fluids that have previously been shown to have inhibitory effects on PCR. Preliminary results obtained after testing the protocol on three patient samples support culture results and medical history. However, one patient was PCR positive but culture negative, suggesting that the sensitivity of this protocol may exceed that of traditional culture techniques. This system, therefore, constitutes an additional protocol that may significantly aid patient management in cases where fungal endophthalmitis is suspected.

Detection of and discrimination between gram-positive and gram-negative bacteria in intraocular samples by using nested PCR.

A nested PCR protocol has been developed for the detection of and discrimination between 14 species of gram-positive and -negative bacteria in samples of ocular fluids. First-round PCR with pan-bacterial oligonucleotide primers, based on conserved sequences of the 16S ribosomal gene, was followed by a gram-negative-organism-specific PCR, which resulted in a single 985-bp amplification product, and a multiplex PCR which resulted in two PCR products: a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram-positive bacteria only). All products were detected by gel electrophoresis. The sensitivity of the assay was between 10 fg and 1 pg of bacterial DNA, depending on the species tested, equivalent to between 24 and 4 live bacteria spiked in water. The identification was complete in 3.5 h. The molecular techniques were subsequently applied to four samples of intraocular fluid, (three vitreous and one aqueous) from three patients with clinical signs of bacterial endophthalmitis (test samples) and two samples of vitreous from a patient with chronic intraocular inflammation (control samples). In all culture-positive samples (two of three vitreous and one of one aqueous), a complete concordance was observed between molecular methods and culture results. PCR correctly identified the gram stain classification of the organisms. The bacterial etiology was also identified in a culture-negative patient with clinical history and signs highly suggestive of bacterial endophthalmitis. Furthermore, control samples from a patient with chronic intraocular inflammation remained PCR negative. In summary, this protocol has demonstrated potential as a rapid diagnostic test in confirming the diagnosis of infection and also determining the Gram status of bacteria with high specificity and sensitivity.

Multifocal choroiditis: clinicopathologic correlation.

Many of the white dot syndromes are considered to have a granulomatous pathogenesis. The histopathologic characteristics of this case of multifocal choroiditis seen within 15 months of apparent clinical onset show that the white dot lesions were nongranulomatous perivascular choroidal infiltrates, consisting mainly of B lymphocytes. Early choroidal neovascularization was also seen.

Suppurative corneal ulceration in Bangladesh. A study of 142 cases examining the microbiological diagnosis, clinical and epidemiological features of bacterial and fungal keratitis.

Suppurative keratitis is an important preventable cause of blindness, particularly in the developing world. This study analyses 142 cases of suppurative keratitis referred to Chittagong Eye Infirmary Bangladesh. Some 53.5% of cases were bacterial and 35.9% were fungal. The five most common pathogens were: Pseudomonas sp. 24%, Streptococcus pneumoniae 17%, Aspergillus sp. 13%, Fusarium sp. 7% and Curvularia sp. 6%. Gram stain and culture results were consistent in 62.6% of cases. Previous antibiotic treatment was a significant factor for failure of culture isolation and less so for Gram stain failure. On Gram stain, 55.9% of pseudomonal cases were missed, but only 2% of fungal cases were missed. Over all, Gram stain had a sensitivity of 62% and positive predictive value of 84% for bacterial cases, and 98% and 94% for fungal cases, respectively. Fungal ulcers were typically filamentous, but an antecedent history of trauma was not common. The most frequent injury was due to rice grains, but the inoculum appeared to be introduced during eye washing with contaminated water. Pseudomonal ulcers occurred most frequently in the monsoon season, and Fusarium cases were seen only in the hot, dry season.