Detection and gram discrimination of bacterial pathogens from aqueous and vitreous humor using real-time PCR assays.

PURPOSE: To develop and apply real-time PCR protocols to the detection and classification of the Gram status of bacterial pathogens in aqueous and vitreous humor collected from clinically suspected intraocular infections. METHODS: The analytical specificity of two PCR assays, SYBR Green 16S rDNA-Based Universal PCR (SGRU-PCR), and a Multiplex Gram-Specific TaqMan-Based PCR (MGST-PCR), was determined with 31 clinically important pathogens, including 20 Gram-positive and 11 Gram-negative. Analytical sensitivity was determined with a 10-fold dilution of Staphylococcus epidermidis and Escherichia coli DNA. Assays were further tested on aqueous (n = 10) and vitreous humor (n = 11) samples collected from patients with clinically diagnosed intraocular infections. RESULTS: DNA was amplified from all control bacterial isolates when using SGRU-PCR. MGST-PCR correctly classified the Gram status of all these isolates. The SGRU-PCR limit of detection of S. epidermidis and E. coli DNA was 100 fg/µL (E = 0.82 and 0.86; r(2) = 0.99) and for MGST-PCR, 1 pg/µL (E = 0.66 and 0.70; r(2) = 0.99. For clinical intraocular samples, positivity of culture was 47.6% and for real-time PCR assays, 95.2%. Gram classification was achieved in 100% of MGST-PCR-positive samples. Among microbiologically negative samples, real-time PCR assays were positive in 90% of cases. The false-positive rate in control aqueous was 3.2%, and control samples of vitreous were negative. CONCLUSIONS: The real-time PCR assays demonstrated good correlation, with culture-proven RESULTS: With the use of these methods, bacterial detection was improved from 47.6% to 95.3%, demonstrating them to be sensitive, rapid tests for diagnosis of bacterial endophthalmitis.

[Morphological assessment of different amniotic membrane epithelial denuding techniques]. / Avaliação morfológica de diferentes técnicas de desepitelização da membrana amniótica humana.

PURPOSE: To evaluate the morphological features of the amniotic membrane denuded by different techniques. METHODS: Human amniotic membrane was collected at the time of delivery, fixed in increasing concentrations of glycerol (0-50% in DMEM) and preserved at -80 degrees C until the time of use. The study consisted of 4 groups: intact epithelium (control) and denuded by trypsin (2 mg/mL at 1:250), dispase (1.2 U/mL in Mg2+ and Ca2+ free Hank's balanced salt solution) or ethylenediaminetetraacetic acid (EDTA), 0.02%. Specimens were submitted to electron (scanning and transmission) microscopy analysis. RESULTS: Scanning electron microscopy disclosed intact epithelium in the control group and its absence in the amniotic membranes denuded by trypsin and dispase. In those denuded by ethylenediaminetetraacetic acid there were areas with and without epithelium. When assessed by transmission electron microscopy, the epithelium was intact and firmly adhered to the basement membrane by hemidesmossomes in controls and in parts of ethylenediaminetetraacetic acid group. There were only collagen fibers in the dispase- and trypsin-treated groups. CONCLUSIONS: Trypsin and dispase treatment of the amniotic membrane may cause complete denuding of the epithelium and basement membrane whereas ethylenediaminetetraacetic acid may leave some intact epithelium-areas and partially destroy the basement membrane in others.

Pulsed-field gel electrophoresis in the identification of the origin of bacterial keratitis caused by Pseudomonas aeruginosa.

PURPOSE: To report the use of pulsed-field gel electrophoresis (PFGE) in the investigation of the origin of infectious keratitis. METHODS: A 19-year-old girl presented with infectious keratitis in the left eye. She was a soft contact lens wearer, and was noncompliant with the proper cleaning methods proposed by the manufacturer. Microbiological assessment was performed by means of culture and PFGE. RESULTS: The patient was treated successfully with topical cefalotin and gentamicin drops. Cultures were positive for Pseudomonas aeruginosa in the cornea and conjunctiva, and in the contact lens, its case and the cleaning solution. PFGE showed identical patterns of banding in each. CONCLUSION: In this case of bacterial keratitis, PFGE proved to be very useful in identifying how the contamination occurred.

Tuberculosis of the cystic duct lymph node.

Tuberculosis of the cystic duct lymph node associated with cholelithiasis is rare. We report a case of a 40 year-old woman with this pathology. She presented with anorexia, biliary colic, postprandial fullness and fever. Imaging studies revealed cholelithiasis and several visible portal lymph nodes. Cholecystectomy was performed and histopathological examination showed tuberculosis of the cystic duct lymph node without affecting the gallbladder. The presence of gallstones and lymphadenopathy in computed tomography, associated with persistent fever and symptoms that resemble cholecystitis, should cause suspicion of tuberculosis. However, diagnosis is usually achieved by microscopic appearance of caseating granulomas and isolation of Mycobacterium tuberculosis. The treatment in this case consisted of cholecystectomy and antitubercular chemotherapy.

Falciform ligament abscess: report of a case.

Falciform ligament abscess is rare. We report a case of a 65-year-old man who presented with right upper quadrant abdominal pain, postprandial fullness, and fever. Computed tomography disclosed a cylindrical mass in the anterior abdomen that aroused suspicion of a hepatic abscess. At laparoscopic surgery, an abscess of the falciform ligament was found and drained. Two months later, the patient developed recurrence of the abscess secondary to acute calculous cholecystitis. Abscess drainage and cholecystectomy were performed. The presence of right uppper quadrant abdominal pain, epigastric tenderness, fever, leukocytosis, and a mass in the anterior abdomen should arouse suspicion of falciform ligament abscess. Its treatment consists of abscess drainage.