Comparison of PCR, Fluorescent in Situ Hybridization and Blood Cultures for Detection of Bacteremia in Children and Adolescents During Antibiotic Therapy.

The gold standard in microbiological diagnostics of bacteremia is a blood culture in automated systems. This method may take several days and has low sensitivity. New screening methods that could quickly reveal the presence of bacteria would be extremely useful. The objective of this study was to estimate the effectiveness of these methods with respect to blood cultures in the context of antibiotic therapy. Blood samples from 92 children with sepsis were analyzed. Blood cultures were carried out in standard automated systems. Subsequently, FISH (Fluorescent In-Situ Hybridization) and nested multiplex-real-time-PCR (PCR) were performed. Blood cultures, FISH and PCR yielded positive results in 18%, 39.1%, and 71.7% of samples, respectively. Significant differences were found between the results obtained through culture before and after induction of antibiotherapy: 25.5% vs. 9.7%. There was no significant difference in FISH and PCR results in relation to antibiotics. The three methods employed demonstrated significant differences in detecting bacteria effectively. Time to obtain test results for FISH and PCR averaged 4-5 hours. FISH and PCR allow to detect bacteria in blood without prior culture. These methods had high sensitivity for the detection of bacteremia regardless of antibiotherapy. They provide more timely results as compared to automated blood culture, and may be useful as rapid screening tests in sepsis.The gold standard in microbiological diagnostics of bacteremia is a blood culture in automated systems. This method may take several days and has low sensitivity. New screening methods that could quickly reveal the presence of bacteria would be extremely useful. The objective of this study was to estimate the effectiveness of these methods with respect to blood cultures in the context of antibiotic therapy. Blood samples from 92 children with sepsis were analyzed. Blood cultures were carried out in standard automated systems. Subsequently, FISH (Fluorescent In-Situ Hybridization) and nested multiplex-real-time-PCR (PCR) were performed. Blood cultures, FISH and PCR yielded positive results in 18%, 39.1%, and 71.7% of samples, respectively. Significant differences were found between the results obtained through culture before and after induction of antibiotherapy: 25.5% vs. 9.7%. There was no significant difference in FISH and PCR results in relation to antibiotics. The three methods employed demonstrated significant differences in detecting bacteria effectively. Time to obtain test results for FISH and PCR averaged 4­5 hours. FISH and PCR allow to detect bacteria in blood without prior culture. These methods had high sensitivity for the detection of bacteremia regardless of antibiotherapy. They provide more timely results as compared to automated blood culture, and may be useful as rapid screening tests in sepsis.

A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood.

BACKGROUND: The study describes the application of the PCR method for the simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may cause sepsis. Material for the study was sterile blood inoculated with four species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed method is based on nested-multiplex real-time PCR . RESULTS: Analysis of the obtained data shows that sensitivity of nested-multiplex real-time PCR remained at the level of 10(1) CFU/ml for each of the four studied species of microorganisms and the percentage of positive results of the examined blood samples from the patients was 70% and 19% for the microbiological culture method. The designed primers correctly typed the studied species as belonging to the groups of Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi. CONCLUSIONS: Results obtained by us indicated that the designed PCR methods: (1) allow to detect bacteria in whole blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the main groups of microorganisms within a few hours.

[Pseudo-outbreak of MRSA in the pediatric units]. / Pseudoepidemia Staphylococcus aureus o fenotypie MRSA na oddzialach pediatrycznych.

The aim of this paper was to investigate the marked increase noted in the number of MRSA isolates recovered from stool of pediatric units patients. The study involved 37 patients with infection of digestive tract and 1 patient with infection of the central nervous system and took place from 2008 to 2009 years. 3 MRSA isolates collected from staff screening were also included. 44 MRSA isolates were identified using the Vitek automated system. Antimicrobial susceptibility patterns were determined by disk-diffusion method. All isolates were typed by PFGE using SmaI enzyme. Enterotoxin A gene was detected by PCR method. The study confirmed horizontal transmission of epidemic strain of MRSA in the pediatric units, but no connection between clinical symptoms and Staphylococcus aureus not produsing the exotoxin excluded a true outbreak. This pseudo-outbreak emphasizes the importance of cooperation between the microbiology lab and ward personnel for both: competent diagnosis of illness as well as nosocomial infection surveillance and control activities. The key to the described situation was failure to use approciate criteria to diagnose infection. On the other hand it turned out to be a good time for preparing new hygiene procedures and an aggressive educational program for ward staff and parents that promotes best transmission prevention practices.