External quality assessment programmes for detection of HCV RNA, HIV RNA and HBV DNA in plasma: improved proficiency of the participants observed over a 2-year period.

BACKGROUND AND OBJECTIVES: Two External Quality Assessment Programmes (EQAPs) were run in 2008 and 2009 to evaluate the proficiency of blood centres in detecting, by nucleic acid amplification techniques (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV). MATERIALS AND METHODS: In the EQAP-2008, three customized panels were designed; each containing positive samples with a viral nominal concentration for the three viruses of about three times the 95% DL of the respective commercial NAT assay. In the EQAP-2009, the proficiency of the participants was evaluated with a single panel, independently on the NAT method used. RESULTS: While 84% (102/122) of the participants in the EQAP-2008 correctly identified the positive and negative samples of the panels, in the EQAP-2009 the percentage of proficient laboratories increased to 97% (118/122). Most importantly, in this 2-year experience, we observed a decrease in the number of pre-/postanalytical errors, from 14 in 2008 to two in 2009. CONCLUSIONS: The design of these two EQAPs allowed participants to assess the performance of the NAT methods applied in their routine screening of blood donations, not only with respect to analytical errors but also to human errors that, despite the high level of automation reached by NAT methods, can still occur.

External quality assessment for the detection of HCV RNA, HIV RNA and HBV DNA in plasma by nucleic acid amplification technology: a novel approach.

BACKGROUND AND OBJECTIVES: In this EQA study a novel approach was used to assess the performance of blood centres and blood product manufacturers in detecting the possible contamination of plasma with HCV, HIV and HBV by NAT. MATERIALS AND METHODS: A panel of 12 samples, three negative and three positive for each virus, was distributed to the EQA participants. The positive samples were prepared, using the respective WHO standards, in order to obtain a viral concentration of about three times the 95% DL of the methods most commonly used by laboratories involved in blood screening by NAT. Participants were requested to test each sample of the panel on different days, possibly by different operators using their routine NAT assay. RESULTS: Overall, the participants' performance was satisfactory. In particular, 49 of the 59 participants (83%) were able to correctly identify all samples. Regarding the remaining 10 laboratories, in three cases a deviation from the laboratory's procedure that could be attributed to an operator's mistake was observed, in two cases a possible cross-contamination occurred while in the remaining five cases the failure to detect the positive samples couldn't be ascribed to any relevant deviation in the laboratory's procedure. CONCLUSIONS: The novel design of this EQA study allowed participants to verify their day by day activity as the study was carried out in the context of their routine testing. Under these conditions, it was demonstrated that, despite the high level of automation reached by NAT assays, human errors can still occur.

A study on the receptor for a mycobacteriophage : phage phlei.

From Mycobacterium phlei, glycolipid fractions have been isolated which inactivate phage Phlei. On the basis of the characteristics of the inactivation (specificity, kinetics, requirement for Ca++) typical of the phage-host cell system, it was concluded that these fractions contain the receptor sites for phage Phlei ; this conclusion was supported by electron microscopic studies. All the active fractions contain four kinds of components : fatty acids, glycerol, sugars (D-lyxose, 6-0-methyl-D-glucose, and low amounts of glucose and mannose), and water-soluble acids. These acids are isolated by degradation of the receptor fractions as oxalic and pyruvic acids. Variations of the ratio oxalic acid/pyruvic acid according to the mode of degradation and the absence of the peak characteristic of the protons of a pyruvic acid residue in the NMR spectrum, suggest that these acids might arise from the splitting of oxaloacetic acid. A tentative structure of the receptor is proposed, in many monoglycerides are linked through keto-acid to a polysaccharide core.

Effect of gastric acid suppression on 13C-urea breath test: comparison of ranitidine with omeprazole.

BACKGROUND: The assessment of the effect of H2 antagonists on the results of the urea breath test has produced controversial results. AIM: To assess whether standard doses of both omeprazole and H2 blockers can adversely influence the accuracy of the urea breath test. METHODS: Sixty dyspeptic patients with ascertained Helicobacter pylori infection were recruited for this prospective, open study. They were randomized to receive either omeprazole 20 mg at 08:00 hours (n = 30) or ranitidine 300 mg at 22:00 hours (n = 30) for 14 days. The urea breath test was performed at baseline, on day 14, while patients were still taking the antisecretory drugs, and on day 21, 1 week after their cessation. Duplicate breath samples were collected after ingestion of 75 mg 13C-urea + citric acid. A delta value > 5 per thousand was considered positive. RESULTS: On day 14 the median delta values had declined, but not significantly (P = 0. 07) compared to baseline (13.79 vs. 22.39) with omeprazole, while they had increased (P = 0.27) with ranitidine (27.21 vs. 19.46). On the same day there were five out of 30 (17%) and five out of 28 (18%) false-negative results in the omeprazole and ranitidine groups, respectively. All these cases became positive again on day 21. However, in eight cases treated with omeprazole and 13 treated with ranitidine, there was an increase of 14-day delta values compared to baseline. CONCLUSIONS: Our study shows that both omeprazole and ranitidine at standard doses are able to negatively affect the results of the urea breath test. Their adverse effect resolves within 7 days of drug cessation and therefore the withdrawal of these drugs 7 days before testing seems to be sufficient to avoid false-negative results. The surprising finding that both antisecretory drugs reduce delta values in one group and increase them in another group of patients deserves further study.

Head-to-head comparison of 1-week triple regimens combining ranitidine or omeprazole with two antibiotics to eradicate Helicobacter pylori.

BACKGROUND: Triple therapies containing omeprazole and ranitidine have been shown to be equivalent in eradicating H. pylori infection, but have been assessed either separately or head-to-head, only in small trials. AIM: To carry out a large randomized controlled study comparing omeprazole and ranitidine combined with two antibiotic combinations for 1 week. METHODS: Three hundred and twenty H. pylori-positive patients were randomly subdivided into four equal-sized groups and received one of the following treatments: OAM = omeprazole 20 mg b.d. + amoxycillin 1 g b.d. + metronidazole 500 mg b.d.; RAM = ranitidine 300 mg b.d. + amoxycillin 1 g b.d. + metronidazole 500 mg b.d.; OAC = omeprazole 20 mg b.d. + amoxycillin 1 g b.d. + clarithromycin 250 mg t.d.s.; RAC = ranitidine 300 mg b.d. + amoxycillin 1 g b.d. + clarithromycin 250 mg t.d.s. The assessment of H. pylori status was performed before and 4 weeks after the end of therapy by means of CLO-test and histology. H. pylori infection was considered to be eradicated when both tests were negative. RESULTS: OAM and RAM eradicated H. pylori in 89% and 85% of cases on per protocol (P = 0.48) and in 77% and 75% of cases on intention-to-treat analyses (P = 0.71). OAC and RAC eradicated H. pylori in 67% and 70% of cases on per protocol (P = 0.68) and in 57% and 64% of cases on intention-to-treat analyses (P = 0.41). In contrast, there was significant difference between OAM and OAC (P<0.01) and between RAM and RAC (P<0.05). Side-effects occurred in 15%, 10%, 17% and 16% of patients with respect to the above four subgroups. CONCLUSIONS: Omeprazole and ranitidine combined with two antibiotics for 1 week are equally effective in the eradication of H. pylori infection, and these results question the role of profound acid suppression in the eradication of the bacterium.

Optimal duration of therapy combining ranitidine bismuth citrate with clarithromycin and metronidazole in the eradication of Helicobacter pylori infection.

BACKGROUND: Ranitidine bismuth citrate (RBC) co-prescribed with clarithromycin and metronidazole for 1 week has been shown to be an effective eradicating regimen for Helicobacter pylori. AIM: To determine the optimal duration of this regimen. METHODS: A series of 165 dyspeptic patients were recruited for this randomized, open, parallel-group study. They were subdivided into three groups receiving RBC 400 mg b.d. plus clarithromycin 250 mg b.d. and metronidazole 500 mg b.d. for three different periods (4, 7 and 10 days). H. pylori infection was assessed by the concomitant positivity of CLO-test and histology performed at the pre-entry endoscopy. The bacterium was considered eradicated on the basis of a negative 13C-urea breath test performed at least 28 days after the completion of treatment. RESULTS: The three subgroups were well matched and 16 patients dropped out of the study for many reasons (six in the 4-day, five in the 7-day and five in the 10-day treatment regimens). Intention-to-treat cure rates were 60%, 84% and 85%, and the per-protocol rates 67%, 92% and 94% in the 4-day, 7-day and 10-day treatment regimens, respectively. There was a significant difference, P = 0.003-0.006 on intention-to-treat and P = 0.001-0. 002 on per protocol analysis between the 4-day and the 7-day and the 4-day and the 10-day periods, respectively. The 7-day and 10-day periods did not differ from each other. Side-effects were reported in 9%, 14% and 20% of the 4-, 7- and 10-day regimens. They led to stopping treatment in four cases (one in the 7-day and three in the 10-day period). There was no statistical difference among them. CONCLUSIONS: Reducing the duration of RBC-based triple therapy to 4 days provides a low and unacceptable rate of H. pylori eradication. As there is no difference between 7 and 10 days of treatment, 1 week represents the optimal time period for this kind of treatment, based on RBC plus two antibiotics.

Comparison of 24-h control of gastric acidity by three different dosages of pantoprazole in patients with duodenal ulcer.

BACKGROUND: It is now clear that the extent to which gastric acid secretion must be suppressed varies with the clinical condition being treated. AIM: To assess the 24-h control of gastric acidity and the individual response variability of three different doses of pantoprazole. METHODS: Sixty-four duodenal ulcer patients were recruited for this prospective, randomized, multicentre, double-blind, parallel-group study. They were subdivided into three well-matched groups treated with 20 mg o.m., 40 mg o.m. and 40 mg b.d. of pantoprazole, respectively. Endoscopy and intragastric pH monitoring were performed in each patient before and after 14 days of treatment. RESULTS: Fifty-five patients were eligible for final analysis (17 treated with 20 mg o.m., 18 with 40 mg o.m. and 20 with 40 mg b.d. pantoprazole). The ulcer crater healed in 94, 88 and 95% of cases, respectively. The three dosages of pantoprazole produced significant increases in gastric pH compared to basal levels (P < 0.0001). There was also a clear dose-dependent pharmacodynamic effect, which augmented on moving from the lowest dosage of 20 mg o.m. pantoprazole to the highest dosage of 40 mg b.d. (P < 0.01-0.001). The inter-individual response variability within the three treatment groups was more marked with the dose of 20 mg than with the two higher doses of pantoprazole. CONCLUSIONS: All three doses of pantoprazole we tested are highly effective in decreasing gastric acidity and there is a clear dose-dependent pharmacodynamic effect on moving from the lowest to the highest dosage. The greatest inter individual variation in the degree of acid inhibition was seen with pantoprazole 20 mg o.m., while the majority of patients responded adequately to the two higher doses of the drug.

The impact of antibiotic resistance on the efficacy of three 7-day regimens against Helicobacter pylori.

BACKGROUND: Antibiotic resistance affects the success of anti-Helicobacter pylori therapies and varies greatly from country to country. AIM: To compare the efficacy of three short-term triple regimens in relation to H. pylori primary resistance in our region. METHODS: We enrolled 210 H. pylori-positive dyspeptic patients for this randomized, open, parallel-group study. Three arms of 70 patients each received the following 1-week regimens: (1) ranitidine bismuth citrate 400 mg b.d. + clarithromycin 250 mg b.d. + metronidazole 500 mg b.d. (RCM); (2) bismuth subcitrate 240 mg b.d. + amoxycillin 1000 mg b.d. + metronidazole 500 mg b.d. (BAM); (3) omeprazole 20 mg o.d. + clarithromycin 250 mg b.d. + metronidazole 500 mg b.d. (OCM). H. pylori was assessed by CLO-test and histology before and 4 weeks after therapy. Antibiotic resistance was assessed by E-test. RESULTS: On intention-to-treat analysis RCM was more effective than OCM (84% vs. 69%; P < 0.03) and BAM (84% vs. 63%; P < 0.004). MIC determination was successful in 117 out of 210 patients (55%); metronidazole resistance was present in 52 out of 117 patients (44%) and clarithromycin resistance was present in 17 out of 117 patients (14%). Excellent cure rates were achieved when strains were sensitive to both antibiotics (100% with RCM and BAM and 90% with OCM), whereas RCM was superior to OCM (P=0.009) and BAM (P=0.001) with respect to overall resistant strains (94% vs. 57% and 38%, respectively). CONCLUSIONS: One-week RCM is the best regimen to eradicate H. pylori in our geographical area. This seems to be linked to the better ability of RCM compared to OCM and BAM in overcoming the high prevalence of H. pylori resistance to both metronidazole and clarithromycin in our region.

A new 1-week therapy for Helicobacter pylori eradication: ranitidine bismuth citrate plus two antibiotics.

BACKGROUND: One-week triple regimens are currently the most recommended therapy for the eradication of Helicobacter pylori. No previous study has evaluated the efficacy of a short-term regimen combining ranitidine bismuth citrate with two antibiotics. METHODS: Seventy-two consecutive H. pylori-positive dyspeptic patients were recruited for this randomized, three-centre, open, parallel-group study. They were subdivided into two groups receiving either ranitidine bismuth citrate 400 mg b.d. + clarithromycin 250 mg b.d. and metronidazole 500 mg b.d. (group A) or ranitidine bismuth citrate 400 mg b.d. + clarithromycin 250 mg b.d. and metronidazole 250 mg q.d.s (group B) for 1 week. H. pylori infection was assessed by CLO-test and histology on both antral and corpus biopsies before and at least 4 weeks after the end of therapy. The bacterium was considered eradicated when both tests were negative. Eradication rates and the number of side-effects were evaluated in each group. The Chi-squared test was used for statistical analysis. RESULTS: One patient with only CLO-test positivity was erroneously randomized to group B and four patients dropped out of the study (two in group A and two in group B), mainly because they refused the second endoscopy. In group A, H. pylori was eradicated in 31 of 36 patients (intention-to-treat = 86%; 95% CI = 71-95% and per protocol 31/34 = 91%; 95% CI = 76-98%). Side-effects occurred in 10 patients (27%) and they were generally mild. In group B, H. pylori was eradicated in 29 of 35 patients (intention-to-treat = 83%; 95% CI = 66-93%; and per protocol 29/33 = 88%; 95% CI = 72-97%). Seven patients (20%) complained of modest side-effects. There was no significant difference between the two treatment arms (P = N.S.): no severe adverse events occurred and none of the patients was withdrawn from the study because of them. CONCLUSIONS: The co-administration of ranitidine bismuth citrate plus clarithromycin at low dosage and metronidazole in twice daily doses for 1 week is a short, effective and well-tolerated regimen for the eradication of H. pylori. These findings should provide the impetus for large-scale investigations.

Differential reactivation by HI-6 in vivo of paraoxon-inhibited rat brain acetylcholinesterase molecular forms.

The effects of the cholinesterase reactivator HI-6, [1-(((4-(aminocarbonyl)-piridinio)methoxy)methyl-2-(hydroxy- imino)methyl pyridinium dichloride], on paraoxon-inhibited brain acetylcholinesterase (AChE) and its molecular forms were studied in rats. Treatment with paraoxon (0.25 mg/kg s.c.) caused approx. 60% inhibition of total AChE from frontal cerebral cortex, while that including HI-6 (140 mg/kg i.m.) and atropine (50 mg/kg i.m.) reduced such inhibition to only 25%. Two molecular forms of the enzyme, 10S and 4S, corresponding to globular tetrameric (G4) and monomeric (G1), were detected by sucrose gradient sedimentation. In paraoxon treated rats the G4 form was inhibited by approx. 65% while G1 only by 35%. The G4 form was considerably and selectively reactivated by HI-6 while the G1 form was not reactivated at all. The data show that HI-6 penetrates the blood-brain barrier and reactivates the molecular forms preferentially inhibited by paraoxon and involved in synaptic neurotransmission.

Transient global brain hypoxia-ischemia in adult rats: neuronal damage, glial proliferation, and alterations in inositol phospholipid hydrolysis.

A model of ischemic-hypoxic brain injury which combines bilateral occlusion of common carotid arteries for 10 min and mild hypoxia (15% O2 for 10 min before and during occlusion) was developed. Global ischemia was assessed by a simplified EEG recording indicating isoelectric line, i.e. full arrest of cortical electrical activity. Histological examination of brain 7 days after ischemic insult showed from moderate to severe damage, mainly in the cerebral cortex (layers III, V and VI) and hippocampus (mainly CA1 subfield). The injury consisted of neuronal degeneration and necrosis with nuclear pyknosis and karyorrhexis. Immunohistochemical staining for gliofibrillar acidic protein showed a marked glial proliferation in the cerebral cortex and hippocampus. In the cortical slices, inositol phosphates accumulation stimulated by excitatory amino acid agonists (ACPD, ibotenate and quisqualate), as well as by norepinephrine and carbachol, was enhanced significantly (p < 0.01) with respect to sham-operated rats 7 days, but not 24 h, after the ischemic insult. The overall data show that the relatively simple transient brain hypoxia/ischemia rat model produces full arrest of cortical EEG, histopathological alterations and those relative to post-receptor neurochemical mechanisms characteristic of four-vessel occlusion model.

Change in the distribution of acetylcholinesterase molecular forms in frontoparietal cortex of the rat following nucleus basalis lesions with kainic acid.

The unilateral injection of kainic acid into the nucleus basalis magnocellularis (NBM) resulted in an alteration of the distribution of acetylcholinesterase (AChE) molecular forms in frontoparietal cortex ipsilaterally to the lesion. The G4/G1 ratio fell from 5.4 +/- 0.8 in contralateral to 3.0 +/- 0.5 in ipsilateral cortex. The NBM lesion effect thus, mimicks, the loss of tetrameric G4 form reported for various brain cortical areas of Alzheimer's disease (AD) patients. The data support the suggestion that G4 form is enriched in presynaptic nerve terminals.

Brain acetylcholinesterase and its molecular forms in a precocial murid, Acomys cahirinus, and rat during post-natal development.

Brain acetylcholinesterase (AChE) and its molecular forms of a precocial murid, Acomys cahirinus, characterized by a large hippocampus, were measured during post-natal development and compared with rat. The activity of soluble AChE in Acomys increased slightly up to 4 weeks after birth. The total AChE activity increased somewhat more but, in rats, this increase was still greater. Three main molecular forms of AChE were separated by 7.5% polyacrylamide gel electrophoresis. Their close similarity to the rat AChE forms was assessed by gradient polyacrylamide gel electrophoresis and electrofocusing. Maturation of these forms, i.e., conversion of simple into more complex forms in the soluble fraction of AChE was, however, considerably delayed reaching only after 4 weeks the pattern comparable to that of rat.

Molecular forms of cholinesterases in CSF of Alzheimer's disease/senile dementia of Alzheimer type patients and matched neurological controls.

Acetylcholinesterase, pseudocholinesterase and their molecular forms were measured in the CSF of patients affected by Alzheimer's disease and of matched neurological controls. Three different molecular forms of ChE were found in the CSF of both groups of patients, but only two of them belonged to 'true' AChE. No differences were found between Alzheimer's disease patients and neurological controls in all the examined parameters.

Differential inhibition of rat brain acetylcholinesterase molecular forms by 7-methoxytacrine in vitro.

The effects of 7-methoxytacrine (7-MEOTA), a less toxic derivative of tetrahydroaminoacridine, on the activity of acetylcholinesterase (AChE) molecular forms were investigated in vitro. AChE molecular forms were separated by sucrose gradient sedimentation from homogenates of the frontal cerebral cortex prepared with buffer containing Triton X-100 (soluble + membrane-bound enzyme). Two molecular forms, namely 10S and 4S corresponding to globular tetrameric (G4) and monomeric (G1) forms, respectively, were detected; their molecular weights were 220,000 and 54,000 Da. A significantly higher sensitivity to 7-MEOTA of G4 than of G1 forms was observed. The Ki values were 0.21 +/- 0.07 microM for the former and 0.70 +/- 0.15 microM for the latter. The differential inhibition of AChE molecular forms by 7-MEOTA is discussed in relation to its possible clinical application for treatment of disorders such as Alzheimer's disease, in which a reduction of brain cholinergic neurotransmission is believed to play a role.