Evaluation of DNA Extraction Methods and Their Clinical Application for Direct Detection of Causative Bacteria in Continuous Ambulatory Peritoneal Dialysis Culture Fluids from Patients with Peritonitis by Using Broad-Range PCR

BACKGROUND: The aims of this study were to compare several DNA extraction methods and 16S rDNA primers and to evaluate the clinical utility of broad-range PCR in continuous ambulatory peritoneal dialysis (CAPD) culture fluids. METHODS: Six type strains were used as model organisms in dilutions from 10(8) to 100 colony-forming units (CFU)/mL for the evaluation of 5 DNA extraction methods and 5 PCR primer pairs. Broad-range PCR was applied to 100 CAPD culture fluids, and the results were compared with conventional culture results. RESULTS: There were some differences between the various DNA extraction methods and primer sets with regard to the detection limits. The InstaGene Matrix (Bio-Rad Laboratories, USA) and Exgene Clinic SV kits (GeneAll Biotechnology Co. Ltd, Korea) seem to have higher sensitivities than the others. The results of broad-range PCR were concordant with the results from culture in 97% of all cases (97/100). Two culture-positive cases that were broad-range PCR-negative were identified as Candida albicans, and 1 PCR-positive but culture-negative sample was identified as Bacillus circulans by sequencing. Two samples among 54 broad-range PCR-positive products could not be sequenced. CONCLUSIONS: There were differences in the analytical sensitivity of various DNA extraction methods and primers for broad-range PCR. The broad-range PCR assay can be used to detect bacterial pathogens in CAPD culture fluid as a supplement to culture methods.

Prevalence of Plasmid-mediated Quinolone Resistance and Its Association with Extended-spectrum Beta-lactamase and AmpC Beta-lactamase in Enterobacteriaceae / 대한진단검사의학회지

BACKGROUND: We investigated the prevalence of plasmid-mediated quinolone resistance and its association with extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase in Enterobacteriaceae. METHODS: A total of 347 non-duplicated isolates of Enterobacteriaceae were collected between August and October 2006 from 2 hospitals. Qnr determinant screening was conducted using PCR amplification, and all positive results were confirmed by direct sequencing. Qnr-positive strains were determined on the basis of the presence of ESBL and AmpC beta-lactamase genes. RESULTS: The qnr gene was detected in 47 of 347 clinical Enterobacteriaceae isolates. Among the 47 qnr-positive strains, Klebsiella pneumoniae (N=29) was the most common, followed by Escherichia coli (N=6), Enterobacter cloacae (N=6), Citrobacter freundii (N=5), and Enterobacter aerogenes (N=1). These isolates were identified as qnrA1 (N=6), 8 qnrB subtypes (N=40), and qnrS1 (N=1). At least 1 ESBL was detected in 38 of the 47 qnr-positive strains. Qnr-positive strains also showed high positive rates of ESBL or AmpC beta-lactamase, such as TEM, SHV, CTX-M, and DHA. DHA-1 was detected in 23 of 47 qnr-positive strains, and this was co-produced with 1 qnrA1 and 22 qnrB4. Strains harboring MIR-1T and CMY were also detected among the qnr-positive strains. Antimicrobial-resistance rates of qnr-positive strains to ciprofloxacin, levofloxacin, norfloxacin, nalidixic acid, and moxifloxacin were 51.1%, 46.8%, 46.8%, 74.5%, and 53.2%, respectively. CONCLUSIONS: The qnr genes were highly prevalent in Enterobacteriaceae, primarily the qnrB subtypes. They were closely associated with EBSL and AmpC beta-lactamase.

Septic Peripheral Embolism in Left Leg fromAggregatibacter aphrophilus Endocarditis / 대한임상미생물학회지

Aggregatibacter aphrophilus is a facultatively anaerobic gram-negative coccobacillus or bacillus that grows with no dependence on X factor and variable requirement for V factor. The organism is normal flora in the human oral cavity and upper respiratory tract and, rarely, causes invasive infections such as bacteremia, endocarditis, brain abscess, or osteomyelitis. We report a case of septic peripheral embolism in left leg from A. aphrophilus endocarditis. A 49-year-old man with known hypertension presented with acute muscle pain in the left leg. On physical examination, a regular heartbeat with a pansystolic murmur was heard. There were decreased pulses in the left popliteal and dorsalis pedis arteries and coldness of the left foot, although sensory and motor functions were intact. Angiography revealed an embolus in a branch of the left femoral artery. He underwent emergency embolectomy, and gram-negative bacilli grew in the embolus cultures. The same microorganism was isolated in two pairs of blood culturs and subsequently identified as A. aphrophilus. Transthoracic echocardiography revealed mitral regurgitation and multiple vegetations on the mitral valve. The patient was treated with a third-generation cephalosporin for 4 weeks and mitral valve replacement in view of the diagnosis of infective endocarditis and septic peripheral embolism.

The Optimal Wavelength of Spectrophotometric Broth Microdilution Antifungal Susceptibility Test for Flucytosine and Three Azoles / 대한진단검사의학회지

BACKGROUND: There is no guideline for the appropriate wavelength at which to measure the optical density (OD) value in broth microdilution antifungal susceptibility testing, although a spectrophotometric reading method is commonly used. The present study aimed to analyze the difference in the OD values over the range of visible light and to ascertain the optimal wavelength for the spectrophotometric method of microdilution testing. METHODS: We measured the OD of background blank controls of broth medium, antifungal agents, and inocula of five type strains using a Synergy HT multi-detection microplate reader at 5-nm intervals from 380 nm to 760 nm. We also estimated the OD differences between the 50% of growth control and blank control. RESULTS: The OD of the blank control showed a parabola shape with two peaks and steadily decreased at longer wavelengths. The curves of the antifungal agent were similar to those of blank controls, and the influence of each antifungal agent on the OD was minimal. For the difference in OD between 50% of growth control and the blank control, the curve was the opposite of the blank control, and the OD increased steadily at the wavelengths above 600 nm. CONCLUSIONS: The range between 600 nm and 700 nm was the optimal wavelength for broth microdilution antifungal susceptibility testing, although any wavelength within the visible light spectrum can be used.