Evaluation of the HB&L System for the Microbiological Screening of Storage Medium for Organ-Cultured Corneas.

Aims. To compare HB&L and BACTEC systems for detecting the microorganisms contaminating the corneal storage liquid preserved at 31°C. Methods. Human donor corneas were stored at 4°C followed by preservation at 31°C. Samples of the storage medium were inoculated in BACTEC Peds Plus/F (aerobic microorganisms), BACTEC Plus Anaerobic/F (anaerobic microorganisms), and HB&L bottles. The tests were performed (a) after six days of storage, (b) end of storage, and (c) after 24 hours of preservation in deturgescent liquid sequentially. 10,655 storage and deturgescent media samples were subjected to microbiological control using BACTEC (6-day incubation) and HB&L (24-hour incubation) systems simultaneously. BACTEC positive/negative refers to both/either aerobic and anaerobic positives/negatives, whereas HB&L can only detect the aerobic microbes, and therefore the positives/negatives depend on the presence/absence of aerobic microorganisms. Results. 147 (1.38%) samples were identified positive with at least one of the two methods. 127 samples (134 identified microorganisms) were positive with both HB&L and BACTEC. 14 HB&L+/BACTEC- and 6 BACTEC+/HB&L- were identified. Sensitivity (95.5%), specificity (99.8%), and positive (90.1%) and negative predictive values (99.9%) were high with HB&L considering a 3.5% annual contamination rate. Conclusion. HB&L is a rapid system for detecting microorganisms in corneal storage medium in addition to the existing methods.

Improvement of human corneal endothelium in culture after prolonged hypothermic storage.

PURPOSE: To evaluate whether the organ culture method for human cornea preservation may be applied to corneas stored for several days at 4 degrees C. METHODS: The cell density, viability, and morphology of corneal endothelium were examined in 140 human corneas stored at 4 degrees C for the minimal time required for transport to the bank and for the preliminary controls of cornea status (1.6 +/- 1.1 days) and in 46 corneas preserved at 4 degrees C for 6.1 +/- 1.9 days in Optisol-GS. The evaluation was repeated after 19.7 +/- 9.1 days of incubation at 31 degrees C in a culture medium containing 2% newborn calf serum. RESULTS: After the hypothermic storage the corneal endothelium had a mean density of 2475 +/- 159 cells/mm2 without significant difference between the short and the long-term incubation. Several corneas of the two groups showed signs of endothelium degeneration and were positive to trypan blue test. After the incubation at 31 degrees C, the corneas with endothelial degeneration decreased by 52.2% and those positive to trypan blue decreased by 21.7%. Polymorphism (enlarged endothelial cells) increased from 9.6% to 14.5% of the corneas. The remodeling of the endothelium led to a 6.7% decrease in cell density. These results were similar after short-term and long-term storage at 4 degrees C. CONCLUSIONS: Organ culture was effective in improving corneal endothelium when the hypothermic storage was prolonged to the upper temporal limit for this procedure (7-10 days). These results may encourage the possibility of an eye bank to allocate the available cornea pool, thus decreasing the risk of discarding precious material.

Detection and characterization of Helicobacter pylori from patients with gastroduodenal diseases.

Polymerase chain reaction and cytotoxin assays were performed to identify as Helicobacter pylori type I (cagA+/tox+) or type II (cagA-/tox-) 56 (59.6%) strains from 94 patients. Of these patients 64 were affected by nonulcer dyspepsia (NUD), 10 by gastric ulcer (GU), 19 by duodenal ulcer (DU), and 1 by both GU and DU. H. pylori strains were tested for cagA using two sets of primers; target sequences were detected in 40-42/56 (71.4-75%) depending on the set of primers used, while cytotoxin-producing strains (tox +) were 26/56 (46.4%). Tox+ strains were isolated in 13/32 (40.6%), 2/7 (28.6%), and 11/17 (64.7%) in NUD, GU, and DU patients, respectively. However, the different percentage between cagA+ strains from NUD patients (13/32; 40.6%) and patients with ulcerative diseases (13/23; 54.2%) is not statistically significant (p = 0.462). Because the two sets of primers employed for amplification of cagA target sequences give different results, we concluded that cagA alone could not be taken as predictive factor for severity of gastroduodenal disease. It has been found that H. pylori type I is associated with duodenal ulcer disease.

Detection of Mycoplasma genitalium and Chlamydia trachomatis DNAs in male patients with urethritis using the polymerase chain reaction.

The use of PCR assays as a fast and reliable method is constantly improving and easing microbiological diagnosis. We used a polymerase chain reaction (PCR) assay designed to detect Mycoplasma genitalium and Chlamydia trachomatis in urethral swab samples of 56 males with urethritis and 44 asymptomatic patients as a control group. The PCR assay provides an amplification of target sequence within MgPa (M. genitalium protein attachment) gene. Results indicated that M. genitalium was present in 6 (10.7%) patients with urethritis and none in the control group. Eleven of 56 (17.8%) patients were positive for Chlamydia trachomatis when tested by an outer membrane protein primer-based PCR. The amplified DNA fragments were homogeneous as shown by restriction enzyme analysis and found to be consistent with the published sequences. The PCR assay employed was as reliable as the cultural method in detecting C. trachomatis in the urethral swabs of patients with urethritis (100% of sensitivity when compared with the cultural method) and it has been revealed as an essential method for detection of M. genitalium.