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N Siafakas

Publications and source records attributed to N Siafakas.

At least 19 recordsLinked to original sources
PubMed2005

Membrane adsorption with direct cell culture combined with reverse transcription-PCR as a fast method for identifying enteroviruses from sewage.

We present a new approach for the detection and identification of enteroviruses concentrated and isolated from sewage. Samples were collected from two study sites located at Nicosia and Limassol sewage treatment plants in Cyprus. Viruses were adsorbed to cellulose nitrate membrane filters, cultured directly from the membrane filters by using the VIRADEN method, and identified by reverse transcription-PCR, followed by 5' untranslated region (5'-UTR) restriction fragment length polymorphism (RFLP) analysis and partial sequencing of the VP1 protein coding region. Initial subgrouping based on the HpaII restriction profile showed that all of the isolates except one belonged to the same genetic subcluster. Partial VP1 sequencing revealed that most isolates belonged to serotypes coxsackie B4 (42.5%) and coxsackie Alpha9 (30%), whereas coxsackie B2 (17.5%) and coxsackie B1 (3%) isolates were less frequently observed. One poliovirus type 2 isolate (2.5%) of vaccine origin was also found. The HpaII digests predicted the genetic subcluster for all isolates. They also accurately differentiated the isolates as nonpolio or polio isolates. This approach seems to be very promising for environmental surveillance of enterovirus circulation and epidemiology, with all of the significant effects that this entails for public health. Partial VP1 sequencing is efficient for molecular serotyping of enteroviruses, while 5'-UTR RFLP analysis with HpaII can also be considered an asset for the initial subclassification of enterovirus isolates.

5' Untranslated Regions↗
PubMed2003

Multiplex PCR: rapid DNA cycling in a conventional thermal cycler.

Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more target sequences are simultaneously amplified in the same reaction. In the present study we investigated the limits to which the duration of multiplex PCR steps can be shortened using the thermal cycler Gene Amp PCR system 9600 (Perkin Elmer, Oak Brook, IL). The present multiplex PCR assay simultaneously detects five different herpes viruses (HSV-1, HSV-2, VZV, CMV, and EBV) and assesses sample suitability in a single amplification round of 40 cycles. It appears that when six target sequences are simultaneously amplified in multiplex PCR, extension time is a critical parameter. Using a PCR protocol of 0 sec at 95 degrees C, 0 sec at 60 degrees C, and 0 sec at 74 degrees C with Platinum Taq DNA polymerase (Life Technologies, Gaithersburg, MD), we were able to reduce the total cycling time of the multiplex PCR assay to as little as 55 min, without affecting the yield of PCR products or the specificity of the assay. It may be necessary to optimize each specific apparatus and template, but any such optimization would be trivial.

DNA↗
PubMed2002

Multiplex polymerase chain reaction: a practical approach.

Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features.

Humans↗
PubMed2002

A reliable RT-PCR/RFLP assay for the molecular classification of enterovirus reference and wild type strains to either of the two genetic clusters on the basis of 5'-UTR.

The available sequence data from a large segment of the 5'-UTR of all enterovirus reference and wild type strains were analysed in an attempt to discover possible restriction sites for reliable, serotypic identification of wild type isolates. No combination of restriction endonucleases, though, was found to produce serotype-, or group-specific haplotypes. Thirteen restriction enzymes were predicted to differentiate between representatives of the two enterovirus genetic clusters on the basis of 5'-UTR. One of these enzymes, BstOI, was tested in practice for the differentiation of 61 enterovirus reference strains from 56 different serotypes and 82 wild type strains which belong to the two genetic clusters on the basis of 5'-UTR. All the representatives of the two clusters were successfully differentiated with the specific restriction enzyme. Consequently, the simple RFLP-based assay presented here could be used as a very rapid and reliable means for the initial determination of whether a clinical isolate of unknown identity belongs to either of the 'poliovirus-like', or the 'CBV-like' genetic cluster on the basis of 5'-UTR classification of human enteroviruses.

5' Untranslated Regions↗
PubMed2001

The role of video-assisted thoracoscopic surgery in the treatment of parapneumonic empyema after the failure of fibrinolytics.

BACKGROUND: Approximately 10% of patients treated with intrapleural instillation of fibrinolytics fail to respond and therefore need more invasive techniques, such as video-assisted thoracoscopic surgery (VATS). METHODS: During the period 1994-99, we treated 20 consecutive patients with complicated parapneumonic effusion (CPE) and pleural empyema (PE) that did not resolve with urokinase instillation given through the chest tube in a dose of 100,000 IU diluted in 100 ml of normal saline/daily for 3-5 days. The patients' ages ranged from 21 to 68 years (median, 46); 14 were male and six female. All patients had pleural fluid pH <7.1, LACTATE DEHYDRAGENASE (LDH) >1000, glucose <40 mg/dl and were submitted to VATS. RESULTS: Complete drainage was observed in 17 patients (85%), in the other three (15%), the procedure had to be converted to open thoracotomy due to a thickened visceral pleural peel. The mean operative time was 80.3 min (range, 55-140), and the mean duration of postoperative hospital stay was 7.5 days (range, 4-19). CONCLUSION: We found that VATS is a safe, effective, and well-tolerated surgical procedure in CPE and PE patients who have failed to resolve with initial treatment with fibrinolytics.

Empyema, Pleural↗
PubMed2001

Molecular detection and identification of an enterovirus during an outbreak of aseptic meningitis.

Stool samples from sixteen cases of children with meningitis originating from four different and geographically isolated parts of Greece were investigated for enteroviruses. The conventional method of cell culture in four different cell lines was initially used for the isolation of enteroviruses. The results showed a cytopathic effect (CPE) in all cases after two, or even more successive passages in only one cell line (RD), although a less-than-satisfactory CPE was obtained in many cases. Seroneutralization with RIVM mixed hyperimmune antisera followed and the isolates were typed as Coxsackie B viruses. The method of RT-PCR with enterovirus-specific primers targeted to the highly conserved 5'-UTR of the genome was initially used for the detection of enteroviruses from the inoculated cell cultures. A positive RT-PCR result was obtained for all of the clinical samples rapidly and accurately and the isolates were further characterized with the aid of Restriction Fragment Length Polymorphism (RFLP) analysis and Single Strand Conformation Polymorphism analysis (SSCP) of the amplicons. The RFLP analysis showed first of all that the isolates had an identical restriction pattern with Coxsackie B5 Faulkner reference strain with 4 out of 5 restriction enzymes and secondly, both RFLP and SSCP analysis indicated the epidemiological association of the isolates. The speed of the molecular methodology that was used in comparison with the conventional methods and its possible significance for the description of virus evolution and circulation in the populations is discussed.

Animals↗
PubMed2001

Laboratory diagnosis of common herpesvirus infections of the central nervous system by a multiplex PCR assay.

A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of alpha-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.

Central Nervous System Viral Diseases↗
PubMed2000

Isolation of polioviruses and other enteroviruses in south Greece between 1994 and 1998.

During the five-year period between 1994 and 1998, a total of 217 clinical samples were assessed for the isolation of enteroviruses at the Enterovirus Reference Centre for South Greece. Fourteen enterovirus strains belonging to different serotypes were isolated. These field strains were detected by cell culture in appropriate cell lines. They were subsequently identified by neutralizing antibodies with the LBM (Lim-Benyesh Melnick) mixed antisera pools up to 1995 and RIVM (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) pools from 1996 onwards. The isolated viruses included two strains of poliovirus type 2 Sabin-like, three strains of poliovirus type 1 non-Sabin-like, one Coxsackie B2 (CBV2) strain, one Coxsackie B5 (CBV5) strain, one Echo 5 (ECV5) strain, one Echo 7 (ECV7) strain, three Coxsackie A16 (CAV16) strains, and two currently enteroviral strains unidentified by RIVM pools. Reverse transcription-polymerase chain reaction (RT-PCR) using poliovirus-specific primers or poliovirus type-specific primers and enterovirus specific primers from the highly conserved 5'-UTR, the latter followed by RFLP, was also applied in 6 clinical isolates (3 strains of poliovirus type 1 non-Sabin-like, 1 polio type 2 Sabin-like, and 2 non-identified by RIVM pools enteroviruses). The advantages and the drawbacks of these assays against the conventional ones are discussed here. The isolations and the subsequent identification of the strains were carried out from fecal samples of clinical cases that included hand-foot-and-mouth disease, meningitis, and acute flaccid paralysis. The reappearance of non-Sabin-like poliovirus strains in Greece in 1996 after 14 years is considered to have an important medical and clinical value.

Adult↗
PubMed1999

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection.

The development of a quadriplex PCR method with amplification of HCMV in a single-step procedure using primers taken from four different regions of the viral genome is described. Different concentrations of dNTPs and MgCl2 were assayed in order to optimize the constitution of the buffer for the multiplex PCR. The specificity of the PCR was tested with 100 ng, 10 ng, and 1 ng of genomic MRC-5 cell DNA infected with CMV in the presence of 10 microg of uninfected MRC-5 cell DNA. The sensitivity of the PCR was evaluated by the amplification of various amounts (100 ng, 10 ng, 1 ng, and 0.1 ng) of genomic MRC-5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10 ng of genomic MRC-5 cell DNA with each primer pair. When all four sets of primers were combined in a single reaction tube, the sensitivity of the assay was equivalent to 10 ng of genomic MRC-5 cell DNA, whereas amplification from 1 ng genomic MRC-5 cell DNA produced only a subset of the amplimers. By amplifying four target-sequences of HCMV simultaneously with minimum incubation time at each temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allowed the detection of variants and achieved maximal sensitivity and specificity which are essential for a diagnostic utilization.

Cell Line↗
PubMed1999

Effects of chemical feedback on respiratory motor and ventilatory output during different modes of assisted mechanical ventilation.

The purpose of the study was to examine the effects of chemical feedback on respiratory motor and ventilatory output in conscious subjects ventilated on various modes of assisted mechanical ventilation. Seven subjects were connected to a ventilator and randomly ventilated on assist-volume control (AVC), pressure support (PS) or proportional assist ventilation (PAV). On each mode, the assist level was set to the highest comfortable level. Airway and oesophageal (Poes) pressures, tidal volume, respiratory frequency (fR) and end-tidal carbon dioxide tension (PET,CO2) were measured breath-by-breath. When the subjects were stable on each mode, the fraction of inspired carbon dioxide (FI,CO2) was increased stepwise, and changes in minute ventilation (V'E) and respiratory motor output, estimated by the pressure-time product of all the respiratory muscles per breath (PTPrm) and per minute (PTPminute), were observed. At zero FI,CO2, PTPminute/PET,CO2 did not differ between modes, while V'E/ PTPminute was significantly lower with PAV than that with PS and AVC. As a result V'E/PET,CO2 was significantly lower with PAV, preventing, unlike AVC and PS, a significant drop in PET,CO2. With PAV, independent of CO2, V'E/PTPminute remained constant, while it decreased significantly with increasing CO2 stimulus with PS and AVC. At high PET,CO2 respiratory effort was significantly lower with PAV than that with PS and AVC. In conclusion, the mode of mechanical ventilation modifies the effects of chemical feedback on respiratory motor and ventilatory output. At all carbon dioxide stimulus levels neuroventilatory coupling was better preserved with proportional assist ventilation than with pressure support and assist-volume control ventilation.

Adult↗
PubMed1999

Response of respiratory motor output to varying pressure in mechanically ventilated patients.

It has been shown in mechanically ventilated patients that pressure support (PS) unloads the respiratory muscles in a graded fashion depending on the PS level. The downregulation of respiratory muscles could be mediated through chemical or load-related reflex feedback. To test this hypothesis, 8 patients with acute lung injury mechanically ventilated on PS mode (baseline PS) were studied. In Protocol A, PS was randomly decreased or increased by at least 5 cmH2O for two breaths. During this time, which is shorter than circulation delay, only changes in load-related reflex feedback were operating. Sixty trials where PS increased (high PS) for two breaths and 62 trials where PS decreased (low PS), also for two breaths were analysed. Thereafter, the patients were assigned randomly to baseline, low or high PS and ventilated in each level for 30 min (Protocol B). The last 2 min of each period were analysed. Respiratory motor output was assessed by total pressure generated by the respiratory muscles (Pmus), computed from oesophageal pressure (Poes). In Protocol A, alteration in PS caused significant changes in tidal volume (VT) without any effect on Pmus waveform except for neural expiratory time (ntE). ntE increased significantly with increasing PS. In Protocol B, Pmus was significantly down-regulated with increasing PS. Carbon dioxide tension in arterial blood (Pa,CO2) measured at the end of each period increased with decreasing PS. There was not any further alteration in ntE beyond that observed in Protocol A. These results indicate that the effect of load-related reflex on respiratory motor output is limited to timing. The downregulation of pressure generated by the respiratory muscles with steady-state increase in pressure support is due to a slow feedback system, which is probably chemical in nature.

Aged↗
PubMed1999

Formoterol and beclomethasone versus higher dose beclomethasone as maintenance therapy in adult asthma.

A total of 132 adult asthmatics who were symptomatic on 500 microg x day(-1) inhaled beclomethasone dipropionate (BDP) were studied in an open-label randomized, parallel group, 12 week, clinical trial. The addition of 12 microg formoterol fumarate solution aerosol (pressurized metered dose inhaler) b.i.d. to BDP at a dose of 500 microg x day(-1) was compared with a higher dose of 1,000 microg x day(-1) BDP. Mean morning premedication peak expiratory flow rate (PEF) during the final week of treatment (primary end-point) increased in both groups compared to baseline. The estimated treatment difference of 20.4 L x min(-1) (95% confidence interval 3.2-37.6) after 12 weeks of treatment was statistically significant (p<0.05) in favour of the formoterol/BDP group. The overall mean morning premedication PEF for the entire treatment period was higher in the formotero/BDP group (p=0.002). The overall number of puffs of rescue medication and asthma symptom scores were less in the formotero/BDP group (p<0.01). Safety and tolerability evaluations were satisfactory in both groups. In conclusion, the results suggest that the addition of formoterol fumarate to the existing dose of an inhaled corticosteroid should be considered as an alternative to increasing the dose of inhaled corticosteroid in the inadequately controlled asthmatic.

Administration, Inhalation↗
PubMed1999

Intrapleural urokinase versus normal saline in the treatment of complicated parapneumonic effusions and empyema. A randomized, double-blind study.

Intrapleural administration of fibrinolytic agents has been shown to be effective and safe in the treatment of loculated parapneumonic pleural effusions. However, controlled studies of the possible role of the activity of urokinase (UK) through the volume effect are lacking. We therefore investigated the hypothesis that UK is effective through the lysis of pleural adhesions and not through the volume effect. Thirty-one consecutive patients with multiloculated pleural effusions were randomly assigned to receive either intrapleural UK (15 patients) or normal saline (NS) (16 patients) for 3 d, in a double-blind manner. All patients had inadequate drainage through a chest tube (< 70 ml/24 h). UK was given daily through the chest tube in a dose of 100.000 IU diluted in 100 ml of NS. Controls were given the same volume of NS intrapleurally. Response was assessed by clinical outcome, fluid drainage, chest radiography, pleural ultrasonography (US) and/or computed tomography (CT). Clinical and radiographic improvement was noted in all but two patients in the UK group but in only four in the control group. The net mean volume drained during the 3-d treatment period was significantly greater in the UK group (970 +/- 75 ml versus 280 +/- 55 ml, p < 0.001). Pleural fluid drainage was complete in 13 (86.5%) patients in the UK group (two patients were treated through video-assisted thoracoscopy) but in only four (25%) in the control group. Twelve patients in the control group were subsequently treated with UK and six of them had complete drainage; the remaining six patients had complete drainage after video-assisted thoracoscopy. Our results suggest that UK is effective in the treatment of loculated pleural effusions through the lysis of pleural adhesions and not through the volume effect.

Adult↗
PubMed1999

Utility of fibrinolytic agents for draining intrapleural infections.

Multiple studies have shown that the intrapleural instillation of fibrinolytic agents provides an effective and safe mode of treatment for complicated parapneumonic effusions and empyemas that decrease the need for surgical interventions. Although most investigators use streptokinase and urokinase, the technique of instillation is not standardized. The usual dose of streptokinase is 250,000 IU, but doses range from 50,000 to 220,000 IU for urokinase. Reported success rates range from 38% to 100%, but outcomes depend on the stage of progression of the parapneumonic effusion when fibrinolytics are employed. Fibrinolytics are more effective in complicated parapneumonic effusions than in established empyemas. Although complications of fibrinolytic therapy rarely occur, they result most often from allergic reactions to streptokinase. Urokinase is safer but more expensive. More randomized, comparative, controlled studies are needed to further define the most effective mode of fibrinolytic therapy for subgroups of patients with pleural infection.

Drainage↗
PubMed1999

Fibrinolytics in the treatment of parapneumonic effusions.

Intrapleural instillation of fibrinolytic agents has been shown, in a number of studies, to be an effective and safe mode of treatment in complicated parapneumonic effusions and empyema, minimizing the need for surgical intervention. Streptokinase and urokinase are the fibrinolytics used, but the technique of instillation is not yet standardized. The usual dose of streptokinase is 250,000 IU, 100,000 IU for urokinase. Fibrinolytics are diluted in 30-100 mL normal saline and the success rate ranges 50-100%, depending on the stage of pleural effusion. Generally, fibrinolytics are more successful if used early in the process of pleural infection (in complicated parapneumonic effusions rather than in empyemas). Adverse reactions are rare, of the allergic type and more frequent for streptokinase. Urokinase is safer but more expensive.

Empyema, Pleural↗
PubMed1998

Lung function in patients with inflammatory bowel disease.

Previous studies on baseline pulmonary function testing (PFT) abnormalities in patients with inflammatory bowel disease (IBD) are conflicting because most of them have incorporated patients suffering from both ulcerative colitis (UC) and Crohn's disease (CD). The aim of the study is to investigate whether any PFT abnormalities could be detected in a large group of IBD patients and whether there are differences between the two IBD entities. A total of 132 patients, 47 with CD (mean age 35 years) and 85 with UC (mean age 40 years) were studied. Pulmonary function tests (PFTs), lung transfer factor for carbon monoxide (TLCO) were examined and compared with those of 36 healthy controls. No significant difference of mean values of spirometric indices, TLCO and ABG was found between the two groups of patients and controls, or between patients with CD and UC. However, nine (19%) patients with CD and 15 (17.6%) with UC had a reduction in TLCO, a percentage significantly higher than in controls (P < 0.05). The majority of the patients with TLCO reduction were in an active phase of disease (P < 0.05). Our results suggest that there is no difference in routine PFTs between UC and CD patients, as well as between both these groups and normal controls. However, TLCO abnormalities related to the degree of disease activity are found in patients with both UC and CD.

Adult↗
PubMed1998

Angiogenic growth factor mRNA responses to passive and contraction-induced hyperperfusion in skeletal muscle.

It has been proposed that, in skeletal muscle, the angiogenic response to exercise may be signaled by the increase in muscle blood flow, via biomechanical changes in the microcirculation (increased shear stress and/or wall tension). To examine this hypothesis, we compared the change in abundance of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta1 (TGF-beta1) mRNA in skeletal muscles of the canine leg after 1 h of pump-controlled high blood flow alone (passive hyperperfusion; protocol A) and electrical stimulation of the femoral and sciatic nerves producing muscle contraction (protocol B). The increase in leg blood flow (5.4- and 5. 9-fold change from resting values, respectively) was similar in both groups. Passive hyperperfusion alone did not increase message abundance for VEGF (ratio of mRNA to 18S signals after vs. before hyperperfusion, 0.94 +/- 0.08) or bFGF (1.08 +/- 0.05) but slightly increased that of TGF-beta1 (1.14 +/- 0.07; P < 0.03). In contrast, as previously found in the rat, electrical stimulation provoked more than a threefold increase in VEGF mRNA abundance (3.40 +/- 1.45; P < 0.02). However, electrical stimulation produced no significant changes in either bFGF (1.16 +/- 0.13) or TGF-beta1 (1.31 +/- 0.27). These results suggest that the increased muscle blood flow of exercise does not account for the increased abundance of these angiogenic growth factor mRNA levels in response to acute exercise. We speculate that other factors, such as local hypoxia, metabolite concentration changes, or mechanical effects of contraction per se, may be responsible for the effects of exercise.

Animals↗