The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.The microbiological spectrum of infectious endophthalmitis shows that the percentage of isolates that are fungi is 8 to 18.5% (2,7,12,22,23) and in keratitis the rate is 16 to 35.9% (8,42). Clinical diagnosis of these ocular infections is confirmed by obtaining intraocular (aqueous or vitreous) specimens or corneal scrapings. However, standard microbiological tests are positive in only 54 to 69% of endophthalmitis cases (13,22,23) (by culture) and 80% (8) of keratitis cases (by Gram and Giemsa stains and culture). In fungal infections, even when positive, results usually take longer than a week because these organisms are difficult to identify and/or are slow-growing. Early diagnosis and rapid intervention is a critical element for an effective treatment of ocular infections. This has led to the development of culture-independent diagnostic tests such as PCR. PCR-based detection methods with universal primers for bacterial DNA in ocular samples (5,16,20,21,26,27,34,36,40) have recently been developed. For detection of fungal pathogens, multicopy gene targets have been evaluated for increasing the sensitivity (33, 39) and universal fungal PCR primers have been developed for broadening the range of detectable fungi (9,14,18,31,37). Studies on fungal DNA detection in ocular ...

We report the presence of long stretches of tandem repeats in the genome of the halophilic Archaea Haloferax mediterranei and Haloferax volcanii. A 30 bp sequence with dyad symmetry (including 5 bp inverted repeats) was repeated in tandem, interspersed with 33-39 bp unique sequences. This structure extends for long stretches--1.4 kb at one location in H. mediterranei chromosome and about 3 kb in the H. volcanii chromosome. The tandem repeats (designated TREPs) show a similar distribution in both organisms, appearing once or twice in the H. volcanii and H. mediterranei chromosomes, and once in the largest, probably essential megaplasmid of each organism but not in the smaller replicons. Sequencing of the structures in both H. volcanii replicons revealed an extremely high sequence conservation in both replicons within the species, as well as in the different organisms. Homologous sequences have also been found in other more distantly related halophilic members of the Archaea. Transformation of H. volcanii with a recombinant plasmid containing a 1.1 kb fragment of the TREPs produced significant alterations in the host cells, particularly in terms of cell viability. The introduction of extra copies of TREPs within the vector significantly alters the distribution of the genome among the daughter cells, as observed by DAPI staining. Although the precise biological role cannot be completely ascertained, all the data conform with the tandem repeats being involved in replicon partitioning in halobacteria.

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PurposeThe aims of this study were to assess the utility of polymerase chain reaction (PCR) in diagnosing fungal keratitis in the last decade in our center and to review the molecular diagnosis of mycotic keratitis.MethodsA retrospective nonrandomized investigation was undertaken at Vissum Corporación Instituto Oftalmologico de Alicante to evaluate 27 corneal samples of 20 patients with proven fungal keratitis from January 2000 to December 2009. Corneal samples (21 corneal scrapings, 5 biopsies, and 1 cornea) were evaluated by Gram stain or calcofluor stain, culture, and PCR. The detection and molecular identification were carried out by DNA amplification and sequencing of the internal transcribed spacer and 5.8S rRNA region from the corneal samples.ResultsPCR detected all the samples that were positive by conventional methods. Four samples were positive by PCR and showed negative results by culture and stain. Combination of microscopy and culture gave positive results in 21 of the 27 samples of patients with mycotic keratitis. Stains showed a 66.7% of positive results, culture showed 59.3%, and PCR showed 92.6%. The time taken for PCR assay was 4 to 8 h whereas positive fungal cultures took 1 to 35 days. Identification at species level by molecular methods was possible in all cases except one. Identification at species level by conventional methods only was possible in eight cases.ConclusionsPCR not only proved to be an effective rapid method for the diagnosis of fungal keratitis but was also more sensitive than stain and culture methods. Fungal PCR must be added as the screening diagnosis test when an early mycotic keratitis is suspected. Molecular identification is the gold standard technique for the identification of corneal fungal pathogens.