Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis.

BACKGROUND: Microbiological diagnosis of sepsis relies primarily on blood culture data. This study compares four diagnostic methods, i.e. those developed by us: nested, multiplex, qPCR (qPCR) and FISH with commercial methods: SeptiFast (Roche) (SF) and BacT/ALERT® 3D blood culture system (bioMérieux). Blood samples were derived from adult patients with clinical symptoms of sepsis, according to SIRS criteria, hospitalized in the Intensive Care Unit. RESULTS: Using qPCR, FISH, SF, and culture, microbial presence was found in 71.8%, 29.6%, 25.3%, and 36.6% of samples, respectively. It was demonstrated that qPCR was significantly more likely to detect microorganisms than the remaining methods; qPCR confirmed the results obtained with the SF kit in all cases wherein bacteria were detected with simultaneous confirmation of Gram-typing. All data collected through the FISH method were corroborated by qPCR. CONCLUSIONS: The qPCR and FISH methods described in this study may constitute alternatives to blood culture and to the few existing commercial molecular assays since they enable the detection of the majority of microbial species, and the qPCR method allows their identification in a higher number of samples than the SF test. FISH made it possible to show the presence of microbes in a blood sample even before its culture.

Comparison of methods for isolation of bacterial and fungal DNA from human blood.

The study aimed at optimization of DNA isolation from blood of representatives of four microbial groups causing sepsis, i.e., Gram negative: Escherichia coli, Gram positive: Staphylococcus aureus, yeast: Candida albicans, and filamentous fungus: Aspergillus fumigatus. Additionally, the five commercial kits for microbial DNA isolation from the blood were tested. The developed procedure of DNA isolation consisted of three consecutive steps, i.e., mechanical disruption, chemical lysis, and thermal lysis. Afterward, DNA was isolated from the previously prepared samples (erythrocyte lysis) with the use of five commercial kits for DNA isolation. They were compared paying heed to detection limit, concentration, DNA purity, and heme concentration in samples. The isolation of DNA without preliminary erythrocyte lysis resulted in far higher heme concentration than when lysis was applied. In the variant with erythrocyte lysis, two of the commercial kits were most effective in purifying the DNA extract from heme. Designed procedure allowed obtaining microbial DNA from all four groups of pathogens under study in the amount sufficient to conduct the rtPCR reaction, which aimed at detecting them in the blood.

[Occurrence of human intestinal parasites in selected populations of Cracow region in the years 2000-2006 on the basis of parasitological stool examinations performed in the Laboratory of Parasitology of the District Sanitary-Epidemiological Center]. / Wystepowanie pasozytów jelitowych czlowieka w wybranych populacjach na terenie Krakowa w latach 2000-2006 na pod- stawie badan parazytologicznych kalu przeprowadzonych w Laboratorium parazytologii Wojewódzkiej Stacji Sanitarno-Epidemiologicznej.

BACKGROUND: Infections with intestinal parasites are the most frequent parasitic diseases in all human populations. According to available epidemiological data enterobiosis, giardiosis and ascariosis are the most prevalent in Poland. The aim of this study was to evaluate the prevalence of human intestinal parasites in three selected populations in Cracow between 2000-2006. MATERIAL AND METHODS: As many as 5383 stool samples were tested with the use of coproscopic methods and ELISA for the presence of Giardia intestinalis coproantigen. RESULTS: In 283 stool samples different species of intestinal parasites were detected. The prevalence of human intestinal parasites was minimal in 2002, with the ratio of 3.30%, while the maximal prevalence was noted in 2005 (8.86%). The mean prevalence of intestinal parasites in 2000-2006 was 5.26%. In this period of time the most prevalent intestinal parasite among children and adults was Enterobius vermicularis (2.35% and 1.84% respectively), and in the population of citizens returning from the tropics were Entamoeba coli (6.98%). Besides, a gradual trend of decrease in the number of patients referred directly to our laboratory for parasitic examination of stool samples was noted over the last years.

[Bacterial and fungal bloodstream infections in patients at the University Hospital of Cracow]. / Bakteryjne i grzybicze zakazenia krwi u chorych w Oddzialach Klinicznych Szpitala Uniwersyteckiego w Krakowie.

In presented material evaluation of changes in sepsis and types of bloodstream infections of hospitalized patients in Wards of the University Hospital in Cracow were examined. Results of 9,138 blood cultures studied in years 1989-1999 were analysed. All of the blood samples were recovered from 4,656 infected adults at Clinics of the University Hospital in Cracow. Microbiological blood examinations were held in system of constant monitoring of isolated cultures applying BacT/Alert--colorimetric system (Organon Teknika). Cultured micro--organisms were identified using commercial biochemical tests (bio-Merieux). During period of research changes of profile of isolated microorganisms was observed. Percentage of blood infections of Enterococcus spp. etiology increased from 2.2% in 1989 to 9.8% in 1997-98 (p = 0.014). Dynamic growth of non-fermentative S. maltophilia bacilli to 5.5% (p = 0.036) and Serratia marcescens to 13.8% (p = 0.042) in 1999 was revealed. Designed according to our research review of fungal flora in years 1989-1999 revealed tendency of systematic growth of invasive candidemia frequency, from 1.1% to 10.4%. Diagnostic and therapeutic profile of Departments was in a strict connection with increase of the number and meaning of the politiological bacteremias (p = 0.036) in total number of systemic infections cases.

Evaluation of the activity of thermostable DNA polymerases in the presence of heme, as a key inhibitor in the real time PCR method in diagnostics of sepsis.

The study aim was evaluation of the usefulness of several thermostable DNA polymerases in real time PCR conducted in the presence of the heme. Our study had the advantage of testing several different polymerases, one of which proved to be the least sensitive to heme activity. We also found that there is no need of supplementing the reaction mixture with protective substances like BSA. Selection of the appropriate polymerase can increase the efficiency of the PCR reaction which is very important for diagnosis of sepsis and for other analyses performed on DNA template isolated from the blood.