Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction

INTRODUCTION: Early diagnosis of wound colonisation or prediction of wound sepsis provides an opportunity for therapeutic intervention. There is need for qualitative and quantitative tests that are more rapid than bacterial culture. Pseudomonas aeruginosa results in high morbidity and mortality rates, is inherently resistant to common antibiotics, and is increasingly being isolated as a nosocomial pathogen. We developed three PCR-based methods to detect and quantify P aeruginosa in wound biopsy samples: conventional PCR, enzyme-linked immunosorbent assay (ELISA)-PCR, and RTD-PCR with rapid thermal cycling (LightCycler(™) technology), all based on the amplification of the outer membrane lipoprotein gene oprL. We compared the efficacy of these methods to bacterial culture by quantitatively measuring levels of P aeruginosa in serial dilutions, in reconstituted skin samples and 21 burn wound biopsy samples. MATERIALS AND METHODS: Serial 10-fold dilutions were made from an overnight P aeruginosa culture and plated out onto Luria-Bertani and cetrimide agar plates. The agar plates were incubated overnight at 37°C, and the colonies were counted in order to estimate the number of CFU per dilution tube. A sample was taken from each dilution tube as a template for the three PCR-based methods. Serial P aeruginosa dilutions (see above) were added to uninfected cadaveric skin. The reconstituted biopsy samples were homogenized using a tissue tearer and DNA was extracted using XTRAX DNA buffer. The DNA was resuspended in distilled water. A sample was taken as a template for the PCR-based methods. Twenty-one burn wound biopsy samples were taken from nine patients with suspected P aeruginosa burn wound infection. The biopsy samples were longitudinally divided into two pieces. From one piece, DNA was extracted (using XTRAX DNA buffer) and used as a template for PCR-based techniques (see above). The other piece was homogenized, in physiological water, using a tissue tearer. Serial 10-fold dilutions of the suspension were spread on Luria-Bertani and cetrimide agar plates. Colony counts were performed after overnight incubation at 37°C. The PCR mixture contained sterile distilled water, PCR buffer, deoxynucleotide mixture or digoxigenin labelling mix, MgCl(2), diluted template, primers PAL1 and PAL2, and AmpliTaQ DNA polymerase. The amplification was performed in a GeneAmp(®) PCR System 2400. An aliquot of the reaction mixture was put on an agarose gel for electrophoresis and visualisation of the PCR product. An image of the gel was made using a digital camera. Image analysis software was used to calculate the band mass of the experimental bands. An aliquot of the digoxigenin labelling reaction was denatured and then hybridized with the biotinylated capture probe PrL. Some of the resultant solution was transferred to a well of a streptavidin-coated microtitre plate (MTP) and incubated for 3 h at 45°C. The solution was discarded. Peroxidase conjugated antidigoxigenin was added and the MTP was incubated for 30 min at 37°C. The solution was discarded and ABTS substrate was added. The MTP was incubated for 30 min at 37°C. Absorbance was read at 405 nm. The RTD-PCR mixture contained PCR grade sterile water, diluted template DNA, primers PAL1 and PAL2, 3' fluorescein (FL)-labelled probe oprL-FL, 5' LC Red 640-labelled and 3' phosphorylated probe oprL-LC, MgCl(2), and LC DNA Master Hybridisation Probes, containing Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, and MgCl(2). The amplification was performed in a LightCycler(™). The fluorescence signal of LC Red 640 was measured during the annealing phase. The measured fluorescence data was processed with analysis software. RESULTS AND DISCUSSION: The three methods showed a good concordance with the culture results. Conventional PCR was at least 100 times less sensitive than bacterial culture and had a low dynamic range (2 logs). With a lower detection limit of 10(3) CFU/g tissue, ELISA-PCR was ten times more sensitive than conventional PCR. The dynamic range, however, did not increase. ELISA-PCR is very time consuming (8 h). The RTD-PCR produced a linear quantitative detection range of 7 logs with a lower detection limit of 10(3) CFU/g tissue. More important, however, was that the time from sample collection to result was less than 1 h. Two biopsy specimens scored significantly higher in ELISA-PCR and RTD-PCR than in bacterial culture. This could indicate that DNA from dead bacteria was amplified. One out of ten culture positive biopsy samples was found negative by all PCR-based methods. Topical antimicrobial agents possibly inhibited PCR. These results show that RTD-PCR has potential for the rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment. Further study is required to assess the reliability of this new technology, and its impact on patient outcome and hospital costs

High-risk genital papillomaviruses and degree of dysplastic changes in the cervix: A prospective study by fast multiplex polymerase chain reaction in Belgium

The epidemiologic studies with the least selection bias do not support the hypothesis that HPV types 16 and/or 18 are strongly associated with cervical cancer. In this preliminary report, we describe our findings regarding type 16, 18, and 33 detection rates in 323 normal and 71 dysplastic or neoplastic cervical scrapes using fast multiplex PCR. This modified PCR technique has been shown to be the most sensitive, specific, and reproducible DNA detection method for large epidemiologic studies. The results indicate a high relative risk of increasingly severe cervical abnormality associated with the presence of high-risk HPV DNA. The analysis of the prevalence and age data according to CIN status by nonparametric statistic tests highlights the importance of other factors inversely correlated with age in the cervical transformation process.SCOPUS: ar.jFLWNAinfo:eu-repo/semantics/publishe

Prevalence of high risk genital papillomaviruses in the Belgian female population determined by fast multiplex polymerase chain reaction

Because in situ/filter hybridisation is not sensitive enough and because classical polymerase chain reaction (PCR) protocols are generally not sufficiently reproducible and specific, there is little accurate information on the prevalence of human papillomaviruses (HPV) 16, 18, and 33 infections in women without dyskaryotic changes of the cervix. In our hands, our Fast Multiplex PCR protocol has always been the most sensitive, specific, and reproducible DNA detection assay in all the microbiological and haematological applications we attempted (Vandenvelde C, Verstraete M, Van Beers D [1990]: Journal of Virological Methods 30:215-228; Vandenvelde C, Scheen R, Corazza F, Van Beers D [1991a]: Journal of Experimental and Clinical Hematology 33:293-297; Vandenvelde C, Scheen R, Van Beers D, Fondu P [1991b]: Journal of Experimental and Clinical Hematology 30:25-29). Using this new technique, cervical scrapes from 336 Belgian women attending the cervical cancer screening clinic were examined for the presence of these three high-risk genital papillomaviruses. Positive results were confirmed using another set of HPV-specific primers. Exactly one sixth of our population was found positive for one or more of these HPVs. Types 33 and 16 were significantly more prevalent than type 18. The nonparametric statistical analysis of the data suggest that some risk factors such as particular sexual habits, that are inversely related to age, must exist.SCOPUS: ar.jFLWNAinfo:eu-repo/semantics/publishe

Oral contraceptives and papillomavirus persistence in normal cervix [3]

SCOPUS: le.jinfo:eu-repo/semantics/publishe

Le syndrome respiratoire aigu post-transfusionnel (TRALI): Complication émergente ou principale des transfusions?

Transfusion-Related Acute Lung Injury or TRALI is one of the most frequent severe transfusion reactions (one third of Non Hemolytic Febrile Transfusion Reactions). It presents as a generally mild form of acute respiratory distress syndrome occurring 1 to 2 hours after transfusion of a plasma-containing blood component. This transfusion reaction appears, most of the time, in patients with focal inflammation and is of good prognosis when adequately diagnosed and treated. TRALI prevention rests on donor selection for plasma donation, hemovigilance system effectiveness and restrictive transfusion strategy.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Low-dose aprotinin for reduction of blood loss after cardiopulmonary bypass [2]

SCOPUS: le.jinfo:eu-repo/semantics/publishe

Fast Multiplex polymerase chain reaction on boiled clinical samples for rapid viral diagnosis

An assessment of optimal conditions for rapid simultaneous amplification of multiple human papillomavirus (HPV) sequences has been made using Thermus aquaticus DNA polymerase. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of well characterized cell lines. In our hands, Fast Multiplex PCR (FM-PCR) the technique of running multiple PCR reactions simultaneously with minimum incubation time at each temperature, was highly sensitive (amplification factor = 5 x 109 after 50 cycles), specific (100%) and reproducible (100%) for several microbiological applications. Diagnosis was generally obtained in less than 5 h after sampling. The results show that, after optimization of assay conditions, efficiency and specificity of Multiplex PCR depends exclusively on the primers design and concentration of the primers.SCOPUS: ar.jinfo:eu-repo/semantics/publishe