Identification of Curvularia lunata by polymerase chain reaction in a case of fungal endophthalmitis.

Curvularia is a dematiaceous mold that infects plant species and is found in the soil. In humans, it is known to cause keratitis after trauma to the eye. We report the findings of persistent fungal endophthalmitis in a 74-year-old female patient who had undergone prior cataract surgery. Mold identification and antifungal susceptibilities were done on 2 separate samples of vitreous fluid and they were found to be consistent with Curvularia lunata by the use of PCR amplification methods.

Rapid diagnosis of Mycobacterium tuberculosis infection in children using interferon-gamma release assays (IGRAs).

Diagnosis of tuberculosis (TB) in children by the tuberculin skin test (TST) poses a diagnostic challenge for physicians due to its low specificity and cross-reactivity with nontuberculous mycobacteria and bacille Calmette-Guerin (BCG). Although interferon-gamma release assays (IGRAs) have been shown as novel TST alternatives for diagnosis of latent TB infection (LTBI) in adults, their effectiveness is less clear in children. The present study examined QuantiFERON-TB Gold (QFT-G) responses and IFN-gamma production capacity of TST-positive children, younger children ≤5 years. A total of 517 children of whom 434 were TST positive ranging in age from 1 month to 18 years were evaluated by the QFT-G. Of the 517 children, 434 (84%) were TST positive, 25 (5.8%) of whom were found to be QFT-G positive and 25 (5.4%) with an indeterminate response. Of the 517 children, 355 (68.7%) were previously BCG immunized and 310/355 (87.3%) were TST positive including 18/27 (66.7%) QFT-positive children. Adequate IFN-gamma production by purified TB peptides or mitogen was observed in 92.8% of children, 29.6% of whom were <5 years. This study shows that the QFT-G assay is useful for diagnosis of LTBI. The finding of 5.8% positive QFT-G in 434 TST-positive children underscores the superior specificity of the QFT-G than the TST and its greater cost effectiveness in preventing unnecessary and potentially toxic treatment in children. The study suggests that the majority of positive TST in children represent false-positive reactions and supports the use of IGRAs for diagnosis of LTBI in children, including those <5 years of age.

Comparing micellar, hemolytic, and antibacterial properties of di- and tricarboxyl dendritic amphiphiles.

Homologous dicarboxyl dendritic amphiphiles-RCONHC(CH(3))(CH(2)CH(2)COOH)(2), 4(n); and ROCONHC(CH(3))(CH(2)CH(2)COOH)(2), 5(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were synthesized. Critical micelle concentrations (CMCs) in aqueous triethanolamine solutions and at pH 7.4 were measured along with hemolytic activity (effective concentrations, EC(10)) in phosphate-buffered saline (PBS). LogCMC showed a linear dependence on chain length (n); the longest chain in each series had the lowest CMC-in triethanolamine: 4(21), 180µM and 5(22), 74µM and at pH 7.4: 4(21), 78µM and 5(22), 33µM. These two series, 4(n) and 5(n), and three series of homologous tricarboxyl dendritic amphiphiles-RCONHC(CH(2)CH(2)COOH)(3), 1(n); ROCONHC(CH(2)CH(2)COOH)(3), 2(n); RNHCONHC(CH(2)CH(2)COOH)(3), 3(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were tested for growth inhibition of Staphylococcus aureus strain ATCC 6358 and methicillin-resistant S. aureus (MRSA) strain ATCC 43330 by microdilution in 0.1-strength brain heart infusion broth (BHIB). Amphiphiles 4(19), 4(21), 5(18), and 5(20) showed the strongest antibacterial activity (2.2-3.4µg/mL) against S. aureus (vancomycin, MIC=0.25µg/mL). These four plus 1(21), 2(20), 2(22), and 3(20) exhibited the strongest antibacterial activity (1.7-6.8µg/mL) against MRSA (vancomycin, MIC=0.25µg/mL). The MICs of these amphiphiles against six clinical MRSA were similar to those against the ATCC strain. In PBS, EC(10)s of the most active homologues ranged from 7 to 18µg/mL and 18 to 220µg/mL for di- and tricarboxyl dendritic amphiphiles, respectively. To assess the potential safety of using dendritic amphiphiles as drugs, measurements of micellar and hemolytic properties were conducted in the same medium (full-strength BHIB) that was used for antibacterial activity. The CMCs (9-36µg/mL, ∼18-72µM) of ten amphiphiles were measured by microdilution (log2 progression) with dye-covered beads. The EC(10)s were similar to those in PBS. The MICs of most amphiphiles (14-72µg/mL) and vancomycin (1.1-2.2µg/mL) against both S. aureus and MRSA increased significantly compared to the MICs measured in 0.1-strength BHIB. The one exception, 5(18), had an MIC against S. aureus of 1.1µg/mL compared to vancomycin (2.2µg/mL). With CMC (9-18µg/mL) and EC(10) (16µg/mL) values higher than the MIC, 5(18) was discovered as a lead for further development.

Improved real-time multiplex polymerase chain reaction detection of methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C polymorphisms using nearest neighbor model-based probe design.

The disorders of folate metabolism caused by methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms may lead to several disease states including coronary heart disease, venous thrombosis, and several types of cancer. We have developed a real-time multiplex single-tube polymerase chain reaction procedure on the LightCycler for the detection of the two most commonly occurring variants, 677C>T and 1298A>C, in the MTHFR gene. An improved probe design, based on the nearest neighbor model for nucleic acid-probe duplex stability, resulted in a better separation (DeltaTm approximately 10 degrees C) of melting peaks of the wild-type and mutant alleles than that by the existing method (DeltaTm approximately 3 degrees C) for specimens heterozygous for the 1298A>C polymorphism. Of the 333 blood specimens analyzed by this procedure, we did not find any samples that gave ambiguous results. The specimens with homozygous mutation for one polymorphism were of the wild type for the other variant. The assay was validated by the comparison of the genotyping results of 50 blood specimens from the LightCycler polymerase chain reaction with the conventional restriction fragment length polymorphism procedures. There was 100% concordance of the test results obtained by the two techniques. This assay is reliable, economical, and can be performed by less trained technologists compared with the procedure performed by the conventional restriction fragment length polymorphism technique.