Robust HPLC-MS/MS method for levofloxacin and ciprofloxacin determination in human prostate tissue.

Fluoroquinolones are the drugs of choice in the prevention of bacterial infections after transrectal ultrasound guided prostate biopsy. In order to improve assessment of antibacterial efficacy in the target tissue a simple, selective, rapid and robust HPLC-ESI-MS/MS method for the determination of levofloxacin and ciprofloxacin concentrations in human prostate bioptates was developed and validated. Preparation procedure for prostate samples (10mg) was carried out using homogenization and filtration steps. Analyses were performed within 3.5min using RP C18 column in the isocratic elution mode with mobile phase composed of a mixture of 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution (v/v; 79:21). The method was linear between 0.3µg/g and 15µg/g for levofloxacin and ciprofloxacin with coefficient of correlation (r) ≥0.999. The limit of detection and the limit of quantification for levofloxacin were 0.06µg/g and 0.2µg/g and for ciprofloxacin were 0.04µg/g and 0.13µg/g, respectively. Average concentrations (±SD) of levofloxacin and ciprofloxacin obtained from patients tissue were 5.4±2.2µg/g and 3.9±1.5µg/g, respectively. Additionally, during validation procedure a novel, experimental design approach was applied for the robustness study. For evaluation of analytical method robustness, Plackett-Burman design was employed and for sample preparation method robustness Fractional Factorial design was used. The developed and validated method was successfully applied to examine prostate tissue samples obtained from patients enrolled into a clinical study. Up to now, there has been no other HPLC-ESI-MS/MS method reported for the simultaneous determination of levofloxacin and ciprofloxacin in human prostatic tissue.

Ultra-high performance liquid chromatographic determination of levofloxacin in human plasma and prostate tissue with use of experimental design optimization procedures.

Fluoroquinolones are considered as gold standard for the prevention of bacterial infections after transrectal ultrasound guided prostate biopsy. However, recent studies reported that fluoroquinolone- resistant bacterial strains are responsible for gradually increasing number of infections after transrectal prostate biopsy. In daily clinical practice, antibacterial efficacy is evaluated only in vitro, by measuring the reaction of bacteria with an antimicrobial agent in culture media (i.e. calculation of minimal inhibitory concentration). Such approach, however, has no relation to the treated tissue characteristics and might be highly misleading. Thus, the objective of this study was to develop, with the use of Design of Experiments approach, a reliable, specific and sensitive ultra-high performance liquid chromatography- diode array detection method for the quantitative analysis of levofloxacin in plasma and prostate tissue samples obtained from patients undergoing prostate biopsy. Moreover, correlation study between concentrations observed in plasma samples vs prostatic tissue samples was performed, resulting in better understanding, evaluation and optimization of the fluoroquinolone-based antimicrobial prophylaxis during transrectal ultrasound guided prostate biopsy. Box-Behnken design was employed to optimize chromatographic conditions of the isocratic elution program in order to obtain desirable retention time, peak symmetry and resolution of levofloxacine and ciprofloxacine (internal standard) peaks. Fractional Factorial design 2(4-1) with four center points was used for screening of significant factors affecting levofloxacin extraction from the prostatic tissue. Due to the limited number of tissue samples the prostatic sample preparation procedure was further optimized using Central Composite design. Design of Experiments approach was also utilized for evaluation of parameter robustness. The method was found linear over the range of 0.030-10µg/mL for human plasma and 0.300-30µg/g for human prostate tissue samples. The intra-day and inter-day variability for levofloxacine from both plasma and prostate samples were less than 10%, with accuracies between 93 and 108% of the nominal values. The limit of detection and the limit of quantification for human plasma were 0.01µg/mL and 0.03µg/mL, respectively. For the prostate tissue, the limit of detection and the limit of quantification were 0.1µg/g and 0.3µg/g, respectively. The average recoveries of levofloxacin were in the range from 99 to 106%. Also, the method fulfills requirements of robustness what was determined and proved by Design of Experiments. The developed method was successfully applied to examine prostate tissue and plasma samples from 140 hospitalized patients enrolled into the clinical study, 12h after oral administration of LVF at a dose of 500mg. The mean (±SD) LVF concentration in prostate was 6.22±3.52µg/g and in plasma 2.54±1.14µg/mL. Due to simplicity of the method and relative small amount of sample needed for the assay, the method can be applied in clinical practice for monitoring of LVF concentrations in plasma and prostate gland.

Fluoroquinolone-based antimicrobial prophylaxis in patients undergoing transrectal ultrasound-guided prostate biopsy.

The aim of this study was to establish the prevalence of resistance to fluoroquinolones in Escherichia coli strains isolated from patients undergoing transrectal ultrasound-guided prostate biopsy (TRUS-Bx) and to evaluate the incidence of possible infectious complications associated with this procedure. One hundred and four patients undergoing a TRUS-Bx in a single medical centre were prospectively enrolled in this study. In all patients, pre-biopsy rectal swabs were obtained. The analysis determined the antimicrobial susceptibility of E. coli strains to levofloxacin, ciprofloxacin and a panel of other antibiotics. Before biopsy, each of the men received a levofloxacin-based prophylaxis. Telephone follow-up was used to identify patients who had complications after TRUS-Bx. Fluoroquinolone-resistant strains were isolated from 9.62 % of the patients. In all cases, there were related to E. coli and all those strains were resistant to both levofloxacin and ciprofloxacin. Fluoroquinolones showed greater antimicrobial activity against E. coli (p < 0.05) than ampicillin, amoxicillin/clavulanate and cephalothin. Minor infectious complications occurred in three patients (2.91 %). The relation between the resistance of E. coli to fluoroquinolones and the risk of readmission, as well as infectious complications, was statistically significant (p < 0.05). Despite recent reports of increasing prevalence of fluoroquinolone-resistant E. coli and the associated increase of severe infectious complications, the presented results have not confirmed this phenomenon. Resistance to fluoroquinolones of E. coli strains isolated from rectal swab cultures prior to TRUS-Bx is the risk factor for readmission and infectious complications after this procedure.

A multicentre study of the usefulness of liver biopsy in hepatitis C.

The role of liver biopsy in the assessment of chronic hepatitis C is generally accepted yet there is no prospective data available to quantify its contribution. A previous single centre pilot study suggested that the clinician could predict the amount of fibrosis and to a lesser extent, inflammation with moderate accuracy. The 2002 National Institute of Health Hepatitis C Consensus Conference recommended further study of the role of liver biopsy. Our objective was to compare a prediction of biopsy findings by expert clinicians using usually available clinical and laboratory data to actual biopsy results in order to determine whether biopsy is required routinely. This was a prospective observational study conducted at seven university centres in which the accuracy of clinician's predictions of the degree of inflammation and fibrosis were compared with the actual liver biopsy using an adaptation of a standard histological scoring system. We studied 81 adults with previously untreated chronic hepatitis C, raised serum transaminases and positive HCV-RNA in serum. Clinicians predicted the inflammatory grade in 44 of 80 cases (55%) and the fibrosis stage in 46 of 81 cases (57%). Nine of 17 cirrhotic cases were predicted (sensitivity 53%, specificity 56%). No unexpected additional diagnoses were made on the biopsies. Thus despite knowledge of the clinical and laboratory investigations of patients with hepatitis C, clinicians are unable to accurately predict the hepatic inflammatory grade and fibrotic stage. Liver biopsy is an essential investigation to accurately evaluate the grade and stage of liver disease patients with hepatitis C.

Use of interferon in the treatment of chronic viral hepatitis.

Interferon-alpha therapy has been approved for treatment of chronic hepatitis C and B. Candidates for treatment are patients with well-compensated liver disease and histological evidence of chronic hepatitis who have demonstrated abnormal aminotransferase levels for more than 6 months. In patients with chronic hepatitis B, candidates for treatment need to have evidence of viral replication, either hepatitis B e antigen or hepatitis B virus DNA. Successful response in patients with chronic hepatitis C (i.e., normalization or near-normalization of aminotransferase levels) can be expected to occur in approximately 50% of treated individuals. After a successful course of treatment, the relapse rate is at least 50%. With chronic hepatitis B, successful response (i.e., elimination of viral replicative markers) occurs in approximately 40% of treated patients. In this group, relapse is rare, and successfully treated patients may have no detectable hepatitis B surface antigen with long-term follow-up. Variables that may predict a response in patients with chronic hepatitis C include lower pretherapy alanine aminotransferase levels, female sex, and younger age. In individuals with chronic hepatitis B, response is more likely if pretreatment hepatitis B virus DNA is low, pretherapy alanine aminotransferase levels are high, and the duration of infection is short. Current data available in treating chronic delta hepatitis are limited, but suggest that although transient responses can occur, sustained response is unlikely with this disease.

Prevalence and incidence of viral hepatitis in health workers in the prehepatitis B vaccination era.

To assess the impact of hepatitis B virus on health workers, the authors studied baseline prevalences of hepatitis B serologic markers and undertook prospective surveillance to assess hepatitis B attack rates in 837 health workers and 994 blood-donor controls between 1977 and 1982, before the introduction of hepatitis B vaccine. The baseline prevalence of all hepatitis B markers was 14% in health workers and 6% in controls (p < 0.001); exposure to hepatitis B virus was related to the intensity of blood exposure and its duration. In contrast, the frequency of exposure to hepatitis A virus, a nonblood-borne agent, was lower in health workers (11%) than in controls (16%) (p < 0.01) and increased as a function of age. Multivariate logistic regression analysis identified occupational categories with frequent blood contact, rather than duration of exposure, as being the dominant variable for exposure to hepatitis B virus; for hepatitis A virus exposure, age was the most significant variable. Among health workers susceptible to hepatitis B, the incidence of new definite hepatitis B infections was 1.0% per year in 362 health workers (804 person-years of follow-up observation) with frequent blood contact versus 0% per year in 258 health workers (534 person-years of observation) with limited blood contact (p = 0.017). For definite plus probable cases combined, the incidence of new hepatitis B infections was 1.5% per year versus 0.2% per year for the groups with frequent and limited blood exposures, respectively (p = 0.0013). There were no new cases of hepatitis A or B or seroconversions in controls and only one case of hepatitis A acquired outside the hospital by a health worker. These data confirm the high prevalence of hepatitis B exposure and document in a prospective study the high incidence over time of new hepatitis B virus infections in health workers unprotected by vaccination. Such findings reiterate the need for aggressive vaccination programs in health workers exposed to blood.

A randomized, controlled trial of interferon alfa-2b alone and after prednisone withdrawal for the treatment of chronic hepatitis B. The Hepatitis Interventional Therapy Group.

BACKGROUND AND METHODS: Chronic hepatitis B is a common and often progressive liver disorder for which there is no accepted therapy. To assess the efficacy of treatment with interferon, we randomly assigned patients with chronic hepatitis B to one of the following regimens: prednisone for 6 weeks followed by 5 million units of recombinant interferon alfa-2b daily for 16 weeks; placebo followed by 5 million units of interferon daily for 16 weeks; placebo followed by 1 million units of interferon daily for 16 weeks; or observation with no treatment. RESULTS: Hepatitis B e antigen and hepatitis B viral DNA disappeared from serum significantly more often in the patients given prednisone plus interferon (16 of 44 patients, or 36 percent) or 5 million units of interferon alone (15 of 41; 37 percent) than in the untreated controls (3 of 43; 7 percent; P less than 0.001); the difference between those given 1 million units of interferon (7 of 41; 17 percent) and the controls was not significant. The strongest independent predictor of a response to treatment was the amount of hepatitis B viral DNA in serum at entry (P less than 0.0001). Of the 38 patients who responded to interferon, 33 (87 percent) had normal serum aminotransferase levels after therapy; 11 patients who responded (29 percent), but no controls, lost the hepatitis B surface antigen. Blinded histologic assessment revealed a significant improvement in periportal necrosis in the treated patients (P = 0.03). CONCLUSIONS: In chronic hepatitis B, treatment with interferon alfa-2b (5 million units per day for 16 weeks) was effective in inducing a sustained loss of viral replication and achieving remission, assessed biochemically and histologically, in over a third of patients. Moreover, in about 10 percent of the patients treated with interferon, hepatitis B surface antigen disappeared from serum.

Cure of hemophilia A by orthotopic liver transplantation.

A patient with hemophilia A and transfusion-associated end-stage chronic liver disease underwent orthotopic liver transplantation. He had no requirement for exogenous factor VIII replacement during the 27 mo he survived. Although his hemophilia was cured, he had antibodies to the human immunodeficiency virus; ultimately he died of complications arising from acquired immunodeficiency syndrome. Liver transplantation for cirrhotic hemophiliacs can free them of the need for antihemophilic-factor therapy; however, application of this approach may be limited by the high prevalence of human immunodeficiency virus infection in multitransfused hemophiliacs.

Outcome of toxic dilatation in ulcerative and Crohn's colitis.

A review of 1,236 patients admitted to The Mount Sinai Hospital with inflammatory bowel disease between 1960 and 1979 yielded 75 cases (6%) with toxic dilatation of the colon. There were 61 cases among 613 patients (10%) with ulcerative colitis (UC), and 14 of 623 (2.3%) with Crohn's disease (CD). Fifty-nine of the 75 patients (79%) underwent surgery during their hospitalization with toxic dilatation. Twelve of the 75 patients (16%) died. Both UC and CD groups had similar mean ages at onset of colitis (32 years and 31 years, respectively) and at development of toxic dilatation (37 years); similar durations of overall disease (4.8 and 5.9 years) and of toxic dilatation prior to surgery (11 days and 13 days); and similar anatomic distributions of disease. Both UC and CD also had similar mortality rates (16% and 14%). Mean duration of presenting attack up to onset of toxic megacolon was longer in CD than in UC (62 days versus 31 days) and in unoperated versus operated cases (64 days versus 37 days), but was not significantly different between survivors and mortalities (43 days versus 39 days). Mortality rates were also unaffected by total duration of inflammatory bowel disease, first attack versus relapse (14% versus 18%), or medical versus surgical therapy (13% versus 17%). Factors which affected mortality included age (30% for patients over 40 years old, versus 5% for those younger than 40), sex (21% in women versus 13% in men), and especially the occurrence of colonic perforation (44% for cases with perforation versus only 2% in those without). Of the 12 patients who died, 11 had suffered colonic perforation.(ABSTRACT TRUNCATED AT 250 WORDS)