Live/dead real-time polymerase chain reaction to assess new therapies against dental plaque-related pathologies.

DNA-based methodology for the identification and detection of specific bacteria in dental plaque offers advantages over culturing techniques. One drawback of current molecular techniques like real-time quantitative polymerase chain reaction (RT-QPCR) is that they are not able to distinguish between live or dead bacteria. To overcome this problem an assay was assessed to discriminate between viable or dead bacteria using DNA intercalating substances, propidium monoazide (PMA) and ethidium monoazide (EMA) in combination with RT-QPCR. The assay was tested on oral pathogens: Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. To determine the effectiveness of EMA and PMA, different concentrations (from 5 to 100 µg ml(-1)) of the substances were added to viable or heat-killed suspensions of both organisms (ranging from 10(8) to 10(4) colony-forming units ml(-1)). Afterwards, PMA was tested on mixtures of varying ratios of viable and dead cells. After DNA extraction, RT-QPCR was performed using species-specific primers. Both compounds inhibited PCR amplification from dead cells. The EMA treatment resulted in the largest signal decrease but EMA also inhibited DNA amplification from viable cells. For this reason, PMA was selected for use in further experiments. It was shown to be efficient in allowing selective PCR detection of only viable cells in mixtures containing both viable and dead cells. The amount of amplified DNA corresponded to the percentage of viable cells in the sample. The developed assay will potentially be useful for assessing bacterial loads remaining after disinfection protocols without interference by non-viable bacteria.

Rapamycin suppresses the expansion and differentiation of porcine neonatal pancreas cell clusters.

BACKGROUND: The role of rapamycin in pancreas stem cells remains to be clearly elucidated. Herein, we evaluated the effects of rapamycin on porcine neonatal pancreas cell clusters (NPCCs), which primarily comprised pancreatic precursors, and attempted to find an intracellular mechanism about the harmful effects of rapamycin. METHODS: Porcine NPCCs were treated with rapamycin in a monolayer, and the apoptosis and proliferation were determined via caspase-3 assay and H-thymidine uptake analysis. The expression of transcription factors was assessed via reverse-transcriptase polymerase chain reaction and Western blotting. For the in vivo study, the porcine NPCCs were transplanted into the kidney subcapsules of normal nude mice and treated with rapamycin. RESULTS: Rapamycin treatment significantly reduced the number of ß cells, glucose-stimulated insulin secretion, and the insulin contents in the monolayer-cultured porcine NPCCs. Furthermore, rapamycin treatment increased the apoptosis and inhibited the proliferation of ß cells in the culture dishes. The expressions of the insulin, pancreatic and duodenal homeobox-1, and NeuroD/Beta2 genes were down-regulated via rapamycin treatment. The expression of insulin-like growth factor-II was significantly down-regulated, but the expression of Foxo1 was simultaneously inversely increased, and the translocation of Foxo1 from the cytoplasm to the nucleus was induced by rapamycin treatment. Moreover, rapamycin treatment induced a marked reduction in the relative volume and absolute mass of ß cells in the porcine NPCCs grafts at 8 weeks after transplantation in the normal nude mice. CONCLUSIONS: Here, we demonstrate that rapamycin treatment suppresses the expansion and differentiation of porcine NPCCs, and the alteration of Foxo1 and insulin-like growth factor-II gene expression might be the crucial factors.

Aspergillus PCR testing: results from a prospective PCR study within the AmBiLoad trial.

OBJECTIVES: Invasive fungal infection (IFI) is a major cause of morbidity and mortality in severely immunocompromised patients and is difficult to diagnose. The significance of molecular methods for diagnosis of IFI is still controversial. In a subset of patients treated within the AmBiLoad Trial, samples were investigated prospectively by a nested Aspergillus PCR assay to re-evaluate the significance of PCR in this setting. PATIENTS AND METHODS: In the randomized, prospective multicenter AmBiLoad trial, patients with proven or probable IFI were randomized to receive liposomal amphotericin B (L-AMB) 3 or 10 mg/kg QD for 14 d followed by L-AMB 3 mg/kg QD. From 91 patients, 459 serial samples (98% blood samples) were investigated by a nested PCR assay for Aspergillus DNA. All samples were investigated in our laboratory with a previously described nested and a quantitative PCR assay. As required by the study protocol, serial Aspergillus antigen galactomannan was performed. IFI was defined according to modified EORTC/MSG 2002 criteria as applied in the AmBiLoad trial. RESULTS: Seven and 52 patients had proven and probable IFI according to modified EORTC/MSG criteria, respectively. The median number of samples investigated per patient was 4. Seventy percent of samples were obtained during treatment with antifungal study medication. Forty-three samples gave positive PCR results. Patients with an unfavorable outcome had a significantly higher rate of positive PCR results (48% versus 21%). CONCLUSIONS: The sensitivity of Aspergillus PCR testing is limited during antifungal therapy. The tendency for persistently positive PCR results to indicate a poor prognosis has to be confirmed in further studies.

Impact of valganciclovir on Epstein-Barr Virus polymerase chain reaction in pediatric liver transplantation: preliminary report.

UNLABELLED: Preemptive therapy with ganciclovir has been recommended in the pediatric liver transplant strategy to avoid the development of posttransplant lymphoproliferative disorder (PTLD) from an high Epstein-Barr virus (EBV) is detected. We sought viral load to analyze the response to preemptive therapy with valganciclovir (VGC) in children with liver transplantations and an high quantitative EBV-PCR. METHODS: From June 2005 to December 2007, we tested 979 EBV-PCR among 80 pediatric liver transplant recipients, from those 21/80 PCR were tested from the date of transplantation and 59/80 belonged to the historical cohort (7/59 had a prior history of PTLD). Patients were divided into 2 groups depending upon whether they did (n = 22) or did not (n = 19) receive VGC treatment. The response to VGC was considered complete, if the PCR was negative at 30 and 60 days of treatment; and partial, when the PCR decreased at least 50%. Ganciclovir blood levels tested in 109 cases instances and correlated with the EBV-PCR. RESULTS: A total of 369 (33%) positive PCR were detected in 36/80 patients (mean, 75,000 copies; range = 5000-4,200,000). Among the 22 episodes treated for 30 days, 34% showed complete responses, 41%, partial, and 23%, no response. Among the non-treated group the rates were 6%, 25%, and 68%, respectively (P = .01). However, no differences were observed among those episodes treated for 60 days. At the administered doses, hardly any patient reached the recommended ganciclovir therapeutic level at 2 hours (6 micro/mL). However, the mean PCR was lower when the ganciclovir levels were greater than 4 mg/L when compared with lower levels (P = .03). CONCLUSION: After 30 days of treatment there was a response to VGC in the EBV viral load. There was high interpatient variability of ganciclovir serum concentrations, suggesting the need for pharmacokinetic monitoring to optimize treatment. There was a relationship between the concentration of ganciclovir and the EBV viral load.

Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental water samples.

Ethidium monoazide bromide (EMA) treatment of pure culture and environmental waters at low concentrations (1.0-7.5 microg/ml) indicated effective enumeration of viable and viable but nonculturable Escherichia coli in pure cultures, creek waters, and secondary activated sludge effluent samples by quantitative polymerase chain reaction (qPCR) amplification of the uidA and fliC gene targets at turbidity values < 10 NTU. However, EMA treatment was not effective in primary clarifier and secondary trickling filter effluents where turbidities were > or = 10 NTU. In viable pure cultures, rapidly dividing and senescent cells were most affected by increasing EMA concentrations. Amplification of heat-killed pure bacterial cultures decreased 4 to 6 logs depending on EMA concentration and culture age. The greatest difference was observed in 5-h cultures using 7.5 microg/ml EMA. Turbidity (> or = 100 NTU) in environmental samples inhibited EMA effectiveness on viability discrimination. Enumeration of E. coli in certain wastewaters using EMA-qPCR was similar to culture suggesting that EMA treatment could be incorporated into qPCR assays for the quantification of viable bacteria increasing assay time no more than 30 min. Our results indicate that EMA can be used in routine qPCR assays, but optimum conditions for exposure must be identified for each sample type due to sample matrix effects such as turbidity.

A simple and effective method to overcome the inhibition of Fe to PCR.

Fe inhibition to PCR can be overcome by adding greatly excessive EDTA first, and then the same amount Mg(2+) as EDTA to PCR mix. By this method impure DNA was amplified successfully. DNA samples containing metals whose complexation constant with EDTA was much higher than Mg(2+) can also be amplified.

Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification.

In this study, we found that deoxyinosine triphosphate (dITP) could inhibit polymerase chain reaction (PCR) amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from deoxyadenosine triphosphate during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not deoxyuridine triphosphate. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.

Do fluoroquinolones predispose patients to Clostridium difficile associated disease? A review of the evidence.

BACKGROUND: Clostridium difficile associated diarrhea (CDAD) is an important cause of hospital-acquired diarrhea, and increasingly of community-acquired diarrhea. The occurrence of CDAD in the hospitalized patient is associated with increased length of stay, morbidity, mortality, and healthcare costs. Exposure to antimicrobials is the single most important predisposing factor for acquiring CDAD. The data suggesting that fluoroquinolones are an important risk factor for CDAD is becoming stronger. Also, different fluoroquinolones may pose different risks for CDAD development. OBJECTIVES: The aim of this commentary is to summarize the literature as it relates to the role that fluoroquinolones may have in CDAD. METHODS: PubMed and Ovid MEDLINE were searched using the terms fluoroquinolones, ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin in combination with C. difficile, CDAD, pseudomembranous colitis and antibiotic associated diarrhea. RESULTS: The evidence for an association between fluoroquinolone use and CDAD, especially CDAD due to the hypervirulent NAP1 strain or the polymerase chain reaction ribotype 027, is becoming stronger. CONCLUSIONS: Fluoroquinolones appear to predispose patients to CDAD. The data are suggestive but not conclusive. More studies are needed to define the role that fluoroquinolones play in the development of CDAD. Meticulous and enhanced infection control practices at all times and the judicious use of antimicrobials will help contain the epidemic of CDAD.

Bacterial DNA persists for extended periods after cell death.

The fate of DNA from bacteria that infect the root canal but cannot survive is currently unknown, yet such information is essential in establishing the validity of polymerase chain reaction (PCR)-based identification methods for root canal samples. This in vitro study tested the hypothesis that PCR-detectable DNA from dead bacteria might persist after cell death and investigated the efficiency of sodium hypochlorite (NaOCl) as a field decontamination agent. Using heat-killed Enterococcus faecalis, the persistence of DNA encoding the 16S rRNA gene was monitored by PCR. While most probable number analysis showed an approximate 1000-fold decay in amplifiable template, E. faecalis DNA was still PCR-detectable 1 year after cell death. NaOCl (1%) eliminated amplifiable DNA within 60 seconds of exposure. Our findings also disclosed a previously overlooked problem of concentration-dependent inhibition of the PCR reaction by thiosulfate-inactivated NaOCl. These results highlight the challenges of reliably identifying the authentic living root canal flora with PCR techniques.

The influence of antimicrobial therapy on the sensitivity of Legionella PCR.

The aim of our study was to establish the sensitivity of Legionella DNA detection in lower respiratory tract samples in 3 cases of Legionnaires' disease after initiation of specific antibiotic therapy. The results showed that Legionella amplicon intensity was highest in the sputum or bronchial aspirates collected at or before the start of appropriate therapy and decreased markedly within 3 days of therapy. PCR testing was negative within 4 to 6 days of therapy. These data suggest that within a few days specific antimicrobial therapy induces a significant drop of bacterial concentration in respiratory secretions reaching the detection limit of PCR assay. Respiratory samples for Legionella PCR should be obtained before or early after initiating antimicrobial therapy.

The effect of antibiotics and endodontic antimicrobials on the polymerase chain reaction.

The effectiveness of endodontic antimicrobial treatment could be determined using sensitive molecular methods. The purpose of this study was to determine if antibiotics or endodontic reagents interfere with the ability of PCR to detect Enterococcus faecalis in vitro. Amoxicillin (25 mg/ml), clindamycin (15 mg/ml), tetracycline (25 mg/ml), doxycycline (10 mg/ml), calcium hydroxide, 1% buffered sodium hypochlorite (NaOCl1), 3% and 6% unbuffered NaOCl (NaOCl3 and NaOCl6), 2% chlorhexidine (CHX), 5% tincture iodine (TI), 2% iodine potassium iodide (IKI), chloroform (CF), 70% ethyl alcohol, 5% sodium thiosulphate, 5% citric acid or saline were added to 10 or 10 cells/ml E. faecalis ATCC 19433 for 1 h (1 wk for Ca(OH)2). Using PCR, all specimens were positive except for NaOCl3 and NaOCl6. PCR with Ca(OH)2 was positive with 10 cells/ml but negative with 10 cells/ml. The following reagents yielded negative culturing results: all antibiotics, Ca(OH)2, CHX, IKI, TI, NaOCl3, NaOCl6, and CF. BacLight nuclear staining revealed the presence of viable cells in all PCR positive, culture negative combinations, except for those with CF. Therefore, in the presence of threshold values of bacterial concentrations, all reagents tested except for NaOCl3 and NaOCl6 do not interfere with the detection of E. faecalis using PCR.

Evaluation of three methods for effective extraction of DNA from human hair.

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.

Effects of cidofovir on cell death and replication of feline herpesvirus-1 in cultured feline corneal epithelial cells.

OBJECTIVE: To assess the effect of cidofovir on viability of feline corneal epithelial (FCE) cells, replication of feline herpesvirus (FHV)-1, and virus-induced cytopathic changes. SAMPLE POPULATION: Healthy eyes from 14 recently euthanatized cats. PROCEDURE: Cidofovir at concentrations ranging from 0.05 to 0.000005 mg/mL was added to primary cultures of FCE cells, and cytopathic changes and effects on cell proliferation and cell viability were determined during the subsequent 48 hours. Efficacy of cidofovir (0.02 and 0.05 mg/mL) to prevent in vitro infection of FCE cells with FHV-1 was determined during 72 hours of culture by assessing viral cytopathic effects and viral titers. RESULTS: Cidofovir at concentrations of 0.05, 0.005, and 0.0005 mg/mL significantly reduced mean viable cell counts, and cidofovir at a concentration of 0.05 mg/mL significantly reduced the percentage viability of cultured FCE cells. Minimal cytopathic changes were observed at concentrations of 0.02 and 0.05 mg of cidofovir/mL. Cidofovir at concentrations of 0.05 and 0.02 mg/mL abrogated the cytopathic effects attributable to FHV-1 infection and reduced viral titers from > or =10(14) TCID(50)/mL to < or =10(3.5) TCID50/mL. CONCLUSIONS AND CLINICAL RELEVANCE: Cidofovir in vitro was highly efficacious against FHV-1 infection of a primary culture of FCE cells but had cytostatic effects on cultured cells.

Kinetics and regulation of cytochrome P450-mediated etoposide metabolism.

Etoposide is a DNA topoisomerase II inhibitor widely used in the treatment of a variety of malignancies that is also associated with therapy-related leukemia. The cytochrome P450 (P450)-derived catechol and quinone metabolites of etoposide may be important in the damage to the MLL (mixed lineage leukemia) gene and other genes resulting in leukemia-associated chromosomal translocations. Kinetic analysis of catechol formation by recombinant P450s was determined using liquid chromatography/selected reaction monitoring/mass spectrometry. CYP3A4 was found to play a major role in etoposide metabolism (K(m) = 77.7 +/- 27.8 microM; V(max) = 314 +/- 84 pmol of catechol/min/nmol of P450). However, CYP3A5 (K(m) = 13. 9 +/- 3.1 microM; V(max) = 19.4 +/- 0.4 pmol of catechol/min/nmol of P450) may be involved in etoposide metabolism at therapeutic concentrations of free drug. Other P450s do not appear to be involved in etoposide catechol formation. Real-time polymerase chain reaction and Western blot analysis revealed significantly increased CYP3A4 mRNA and protein levels in hepatocytes treated with 10 microM rifampicin compared with untreated cells, but only modest effects of rifampicin on CYP3A5 induction. Etoposide (40, 5, 1, and 0.25 microM) caused a slight increase in CYP3A4 mRNA in three of five batches of hepatocytes but did not result in proportionately increased CYP3A4 protein levels. At high concentrations, etoposide induced only a modest increase in CYP3A5 mRNA and protein levels in four of five batches of hepatocytes. Alternatively, coadministration of other drugs with etoposide may account for the increase in etoposide catechol formation during therapy with etoposide.

Inverse correlation of epidermal growth factor receptor messenger RNA induction and suppression of anchorage-independent growth by OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in glioblastoma multiforme cell lines.

OBJECT: Quantitative and qualitative alterations in the epidermal growth factor receptor (EGFR) commonly occur in many cancers in humans, including malignant gliomas. The aim of the current study was to evaluate molecular and cellular effects of OSI-774, a novel EGFR tyrosine kinase inhibitor, on nine glioblastoma multiforme (GBM) cell lines. METHODS: The effects of OSI-774 on expression of EGFR messenger (m)RNA and protein, proliferation, anchorage-independent growth, and apoptosis were examined using semiquantitative reverse transcription-polymerase chain reaction, immunocytochemical analysis, Coulter counting, soft agar cloning, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling/fluorescence-activated cell sorting, respectively. All p53 genes were completely and bidirectionally sequenced. Suppression of anchorage-independent growth by OSI-774 was inversely correlated to the induction of EGFR mRNA during relative serum starvation (r = -0.74) and was unrelated to p53 status. Overall, suppression of anchorage-independent growth was a considerably stronger effect of OSI-774 than inhibition of proliferation. The extent of OSI-774-induced apoptosis positively correlated with both proliferation and anchorage-independent growth of GBM cell lines (r = 0.75 and 0.79, respectively). In a single cell line derived from a secondary GBM, exposure to concentrations of greater than or equal to 1 micromol/L resulted in a substantial net cell loss during proliferation studies. CONCLUSIONS: The induction of EGFR mRNA may constitute a cellular mechanism to counteract the inhibitory effect of OSI-774 on the anchorage-independent growth of GBM cells. In contrast, no considerable correlation could be established between baseline expression levels of EGFR (both mRNA and protein) in GBM cell lines and their biological response to OSI-774. The OSI-774 induced greater (p53-independent) apoptosis in more malignant GBM phenotypes and may be a promising therapeutic agent against secondary GBM.

Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR.

Contamination of polymerase chain reaction (PCR) reagents continues to be a major problem when consensus primers are used for detection of low concentrations of bacterial DNA. We designed a real-time polymerase chain reaction (PCR) for quantification of bacterial DNA by using consensus primers that bind specifically to the 16S region of bacterial DNA. We have tested four different methods of decontamination of PCR reagents in a project aimed at detecting bacterial DNA at low concentrations: deoxyribonuclease (DNAse) treatment, restriction endonuclease digestion, UV irradiation, and 8-methoxypsoralen in combination with long-wave UV light to intercalate contaminating DNA into double-stranded DNA. All four methods result in inhibition of the PCR reaction, and most of the decontamination procedures failed to eliminate the contaminating bacterial DNA. Only the DNAse decontamination proved to be efficient in eliminating contaminating DNA while conserving PCR efficiency. All four decontamination methods are time consuming and have the possibility of carrying new contamination into the reaction mixture. However, decontamination with DNAse may help, together with the use of highly purified PCR reagents, in detecting small amounts of bacterial DNA in clinical specimens.

A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp.

BACKGROUND: Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. METHODS: The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. RESULTS AND CONCLUSIONS: The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.