Assessing cross-resistance within the pyrethroids in terms of their interactions with key cytochrome P450 enzymes and resistance in vector populations.

BACKGROUND: It is important to understand whether the potential impact of pyrethroid resistance on malaria control can be mitigated by switching between different pyrethroids or whether cross-resistance within this insecticide class precludes this approach. METHODS: Here we assess the relationships among pyrethroids in terms of their binding affinity to, and depletion by, key cytochrome P450 enzymes (hereafter P450s) that are known to confer metabolic pyrethroid resistance in Anopheles gambiae (s.l.) and An. funestus, in order to identify which pyrethroids may diverge from the others in their vulnerability to resistance. We then investigate whether these same pyrethroids also diverge from the others in terms of resistance in vector populations. RESULTS: We found that the type I and II pyrethroids permethrin and deltamethrin, respectively, are closely related in terms of binding affinity to key P450s, depletion by P450s and resistance within vector populations. Bifenthrin, which lacks the common structural moiety of most pyrethroids, diverged from the other pyrethroids tested in terms of both binding affinity to key P450s and depletion by P450s, but resistance to bifenthrin has rarely been tested in vector populations and was not analysed here. Etofenprox, which also lacks the common structural moiety of most pyrethroids, diverged from the more commonly deployed pyrethroids in terms of binding affinity to key P450s and resistance in vector populations, but did not diverge from these pyrethroids in terms of depletion by the P450s. The analysis of depletion by the P450s indicated that etofenprox may be more vulnerable to metabolic resistance mechanisms in vector populations. In addition, greater resistance to etofenprox was found across Aedes aegypti populations, but greater resistance to this compound was not found in any of the malaria vector species analysed. The results for pyrethroid depletion by anopheline P450s in the laboratory were largely not repeated in the findings for resistance in malaria vector populations. CONCLUSION: Importantly, the prevalence of resistance to the pyrethroids α-cypermethrin, cyfluthrin, deltamethrin, λ-cyhalothrin and permethrin was correlated across malaria vector populations, and switching between these compounds as a tool to mitigate against pyrethroid resistance is not advised without strong evidence supporting a true difference in resistance.

Hybrid macroporous gelatin/bioactive-glass/nanosilver scaffolds with controlled degradation behavior and antimicrobial activity for bone tissue engineering.

A new composition of gelatin/bioactive-glass/silver nanoparticle was synthesized and employed to prepare antibacterial macroporous scaffolds with potential applications in bone tissue engineering. A set of macroporous nanocomposite scaffolds were developed from an aqueous solution of gelatin by freeze-drying and crosslinking using genipin at ambient temperature. Silver nanoparticles were successfully synthesized in situ in gelatin solution by heat treatment reduction as a simple and "green" method in which gelatin acted as a natural reducing and stabilizing agent. The effect of the incorporation of the bioactive-glass and the silver nanoparticle concentration on the physicochemical properties of the scaffolds, such as the gel fraction, porosity, in vitro enzyme degradation, morphology, and swelling behavior was studied. Furthermore, the in vitro viability of human mesenchymal stem cells (hMSC) and the antibacterial activity against gram-negative Escherichia coli and gram-positive Staphylococcus aureus were tested on the scaffolds. It was found that upon the addition of silver nanoparticles the porosity, pore size, swelling, and antibacterial properties were enhanced. The silver nanoparticles increased the in vitro enzyme degradation in samples without bioactive-glass; however, the degradation was remarkably reduced by addition of bioactive-glass. In addition, formation of apatite particles, the main inorganic constituent of the bone, on the surface of the bioactive-glass containing scaffolds were confirmed after immersion in simulated body fluid (SBF). The viability of hMSC on the scaffold suggested that gelatin/bioactive-glass/nanosilver scaffolds can be used as an antibacterial scaffolds.

Surface modification of stainless steel orthopedic implants by sol-gel ZrTiO4 and ZrTiO4-PMMA coatings.

In this paper, the biocompatibility of a medical-grade stainless steel coated with sol-gel derived, nanostructured inorganic ZrTiO4 and hybrid ZrTiO4-PMMA thin films is correlated with surface characteristics. The surfaces of the samples are characterized by atomic force microscopy, the sessile drop technique, and electrochemical corrosion experiments. The viability of adult human mesenchymal stem cells on the surfaces after one day of culture is also assessed quantitatively and morphologically. According to the results, both of the coatings improve the hydrophilicity, corrosion resistance, and thereby cytocompatibility of the substrate. Despite the higher corrosion protection by the hybrid coating, the sample coated with the inorganic thin film exhibits a better cell response, suggesting the domination of wettability. In summary, the ZrTiO4-based sol-gel films can be considered to improve the biocompatibility of metallic implants.

The performance and safety of a pleural drainage unit under hyperbaric conditions.

The performance of a proprietary dry suction pleural drainage unit was measured under hyperbaric oxygenation conditions. The test pleural drainage unit was connected to pressure gauges that allowed the pressures created in the suction and collection chambers to be measured as well as the pleural drainage catheter pressures under varied suction regulator settings during compression, hyperbaric steady states and decompression. The maximum flow capacity of the unit was also measured under varying hyperbaric conditions. TheAtrium Oasis Dry Suction 3600 Chest Drain brand was dramatically affected by pressure change. Nevertheless, based upon our testing, we believe it can be used safely in a hyperbaric environment provided that the following precautions are taken. Suction should not be applied during pressurization. Pressurization needs to be slow, 10 kpa/min or less. Suction is needed for air leaks of 4/min or more at pressure. At stable hyperbaric pressure, the level of suction delivered can be set by adjusting the suction regulator with reference to the conversion table we have determined. Suction must be applied during depressurization if there is an air leak of 5/min or greater coming from the patient, otherwise suction is not essential. As the features of many brands and models of proprietary drains are similar, we would expect other types could be hyperbaric compatible, but individual testing should be performed before acceptance.

The concentration-independent effect of monoexponential and biexponential decay in vancomycin concentrations on the killing of Staphylococcus aureus under aerobic and anaerobic conditions.

An in-vitro pharmacodynamic system was used to generate time-kill curves to demonstrate the concentration-independent pharmacodynamics of vancomycin against Staphylococcus aureus ATCC 29213. Initial vancomycin concentrations of 5, 10, 20 and 40 mg/L were studied monoexponentially while simulating a 6 h half-life. One parallel experiment was performed in duplicate using an initial peak concentration of 40 mg/L where both a distribution alpha-phase half-life of 0.66 h for 1 h and an elimination beta-phase half-life of 6 h for 11 h were simulated to determine if the transient distribution phase concentrations of vancomycin have any impact on bacterial killing beyond that provided by the elimination phase concentrations. Additionally, two monoexponential experiments with peak concentrations of 40 and 20 mg/L and a half-life of 6 h were repeated in an anaerobic chamber to determine if killing of S. aureus was affected. The time to achieve a 3 log10 kill was calculated from the linear portion of the regression line and averaged (mean +/- S.D.) 9.0 +/- 1.4 h for all aerobic monoexponential experiments and was 8.4 and 8.6 h for the aerobic biexponential experiments (P > 0.05). For the anaerobic studies, the times to reach 3 log10 kill were significantly greater averaging 18.9 +/- 1.7 h. The slopes of the bacterial kill curves were virtually identical for both monoexponential and biexponential aerobic experiments averaging -0.34 +/- 0.04, yet significantly different from the anaerobic bacterial kill curve slopes of -0.16 +/- 0.015 (P < 0.05). Time-kill curve analyses suggest that varying the concentration of vancomycin does not affect the rate or extent of bacterial killing aerobically or anaerobically against S. aureus and more efficient killing was achieved under aerobic conditions. The simulated distribution phase concentrations did not contribute to more effective killing of this strain of S. aureus.

Effect of aerobic and anaerobic environments on antistaphylococcal activities of five fluoroquinolones.

A previously established in vitro pharmacodynamic system was used to evaluate the antistaphylococcal activities of five fluoroquinolones under both aerobic and anaerobic conditions. Staphylococcus aureus ATCC 29213 was exposed to a 5-micrograms/ml concentration of each of the following fluoroquinolones: ciprofloxacin, ofloxacin, temafloxacin, sparfloxacin, and clinafloxacin. Terminal elimination half-lives of 4, 6, 8, 8, and 13 h were simulated for the respective drugs. Each fluoroquinolone was bactericidal under both aerobic and anaerobic conditions. However, the bactericidal activity of each fluoroquinolone was delayed by anaerobiosis. This difference in fluoroquinolone activity under aerobic and anaerobic conditions could not be attributed to any particular parameter or physiochemical property but was most likely caused by a combination of factors (e.g., variations in hydrophobicity, intracellular pH, antibiotic concentration, and structure-activity relationships). Fluoroquinolone uptake studies were also performed to investigate the possibility of active, energy-dependent transport mechanisms in S. aureus ATCC 29213. Uptake studies indicated that active efflux does occur in S. aureus ATCC 29213.

Evaluation of antimicrobial activities of clarithromycin and 14-hydroxyclarithromycin against three strains of Haemophilus influenzae by using an in vitro pharmacodynamic model.

An in vitro pharmacodynamic model was used to simulate the in vivo pharmacokinetics of clarithromycin and 14-hydroxyclarithromycin in order to generate time-kill curves for three clinical isolates of Haemophilus influenzae (isolates 2019, 91-183, and 1746). Representative concentrations in serum or lung tissue and the pharmacokinetic parameters of clarithromycin and the 14-hydroxy metabolite, separately and in combination, were simulated for the time-kill studies. Amoxicillin-clavulanic acid was used as a control drug. The simulation of typical concentrations of the macrolides in serum in time-kill studies resulted in magnitudes of bacterial killing that were less than (for strains 2019 and 91-183, MICs = 4 mg/liter for clarithromycin and 14-hydroxy-clarithromycin) or equal to (for strain 1746, MIC = 1 mg/liter for clarithromycin and 14-hydroxyclarithromycin) those observed in amoxicillin-clavulanic acid studies. When typical concentrations in lung tissue were simulated, total log decreases in bacterial counts were greater than those achieved with typical concentrations in serum and, in the case of strain 1746, exceeded the magnitude observed with the control drug. In each case, the time to 3-log-unit killing was longer for the macrolides than for amoxicillin-clavulanic acid. Time-kill curve analyses demonstrated the presence of synergy (defined as a 2-log-unit decrease in the CFU per milliliter between the combination and the most active constituent at any time point) for the combination of clarithromycin and 14-hydroxyclarithromycin at simulated concentrations in serum for one strain of H. influenzae (isolate 91-183). Synergism is likely bacterial strain specific, and the presence of synergy may be dependent on the antibiotic concentrations that are tested. Evaluation of the kill curve kinetics in terms of bactericidal rate for the various starting concentrations of clarithromycin did not result in a clear demonstration of either concentration-dependent or concentration-independent bactericidal activity.

Evaluation of activity of temafloxacin against Bacteroides fragilis by an in vitro pharmacodynamic system.

An in vitro pharmacodynamic system has been successfully adapted to simulate in vivo antimicrobial pharmacokinetics under anaerobic conditions. This system was used to perform time-kill kinetic studies which were designed to compare the activity of temafloxacin to ciprofloxacin and cefotetan against two strains of Bacteroides fragilis (ATCC 25285 and ATCC 23745). All experiments were performed as single-dose, 24-h, duplicate runs. Starting bacterial inocula of 10(7) CFU/ml were exposed to starting antimicrobial concentrations of 5 micrograms of temafloxacin per ml, 5 micrograms of ciprofloxacin per ml, and 100 micrograms of cefotetan per ml. Terminal half-lives of 8, 4, and 4 h were simulated for each antimicrobial agent. Temafloxacin was rapidly bactericidal against B. fragilis. Ciprofloxacin was not bactericidal (< 3 log10 unit decline in bacterial numbers) to either strain of B. fragilis. Cefotetan was bactericidal (> or = 3 log10 unit decline in bacterial numbers) to each strain but killed at a slower rate than temafloxacin. Times to 3 log10 unit declines of strain ATCC 25285 were 2, 4, and > 24 h, whereas those of strain ATCC 23745 were 4, 4, and > 24 h for temafloxacin, cefotetan, and ciprofloxacin, respectively. Total logarithmic declines of strain ATCC 25285 were > 4.5, > 4.5, and 2.9 log10 CFU/ml, whereas those of strain ATCC 23745 were 4.1, > 4.5, and 1.2 log10 CFU/ml for each drug, respectively. These and other studies demonstrated that temafloxacin showed potential as an agent that could have been further developed for use in the treatment of anaerobic infections. However, the drug was removed from the market by its manufacturer because of toxicity issues. Although the release of newer fluoroquinolones that possess significant activity against anaerobic bacteria does not appear imminent, the time-kill studies performed in this study demonstrate that further research is warranted in the development of fluoroquinolones which possess significant antianaerobic activity.

Clostridium difficile colonization in residents of long-term care facilities: prevalence and risk factors.

OBJECTIVE: To determine the period prevalence of Clostridium difficile disease and asymptomatic carriage in the residents of long-term care facilities (LTCF) and to characterize the risk factors for colonization or associated disease. DESIGN: Period prevalence survey. SETTING: Two long-term care facilities in St. Paul, MN. PARTICIPANTS: Specimens were collected from 225 LTCF residents. MEASUREMENTS: The dependent variable was the culture result for C. difficile, which was isolated and identified using selective culture media and a commercial anaerobe identification kit. Tissue culture assay was used to detect the ability of each C. difficile isolate to produce toxin. Independent variables (including gender, age, race, current medical diagnoses, severity of underlying disease, case mix, current clinical symptoms, current medications, antibiotic use within 4 weeks prior to specimen procurement, and other pertinent history) were obtained from the current medical record of each participant. RESULTS: Of 225 stool cultures that were obtained, 16 (7.1%) were positive for C. difficile. None of the residents with a positive culture was symptomatic. History of nosocomial infection and the use of antibiotics in general, cephalosporins, trimethoprim/sulfamethoxazole (TMP/SMX), and histamine-2 blockers were significantly associated with positive C. difficile culture (P < or = 0.05) by univariate analyses. Trends towards significance (0.05 < 0.10) were noted for narcotic use, previous hospitalization, LTCF, and non-insulin-dependent diabetes mellitus. Logistic regression analysis revealed significant, independent predictors of positive culture: antibiotic use in general (P = 0.028; relative risk = 3.31), histamine-2 antagonist use (P = 0.038; relative risk = 3.27), cephalosporin use (P = 0.038; relative risk = 4.66), and TMP/SMX use (P = 0.007; relative risk = 8.45). CONCLUSIONS: The use of antibiotics, particularly cephalosporins and TMP/SMX, is a significant risk factor for asymptomatic carriage of C. difficile in long-term care facilities. The use of H-2 blockers was also a significant risk factor for carriage; however, this finding has not been reported previously and should be confirmed by independent studies. These medications should be used judiciously in the LTCF population. When diarrheal diseases are encountered in LTCF residents, a high index of suspicion for C. difficile infection should be maintained and the appropriate diagnostic and therapeutic measures taken.

Elimination of quinolone antibiotic carryover through use of antibiotic-removal beads.

To prove the utility of antibiotic-removal beads in separating antibiotics from bacterial samples, Escherichia coli ATCC 25922 was exposed to five separate quinolones before and after each was exposed to antibiotic-removal beads. Plates treated with antibiotic solutions that were exposed to beads demonstrated antibiotic removal, and plates treated with antibiotic solutions that were not exposed to beads demonstrated antibiotic carryover. After exposure to beads, fluoroquinolone concentrations decreased from 5 micrograms/ml to 0.14 micrograms/ml (ciprofloxacin), 0.04 micrograms/ml (temafloxacin), < 0.01 microgram/ml (ofloxacin), < 0.01 microgram/ml (sparfloxacin), and 0.02 micrograms/ml (clinafloxacin). These data indicate that antibiotic carryover can be successfully circumvented through the use of antibiotic-removal beads.

Letters.

JOURNAL POLICY DEBATE COMPARING DRESSINGS.

Pharmacotherapy and pharmacodynamics in the management of bacterial infection.

Minimum inhibitory concentration testing is the most common standard used to evaluate antibacterial activity of antimicrobials against specific pathogens. The consideration of pharmacodynamic factors in conjunction with these tests can improve the management of bacterial infections. Further, the incorporation of MIC values into pharmacodynamic ratios may provide clinically useful tools for selecting optimal antibiotic selection, determining proper dosing strategies, and predicting therapeutic outcomes. Physiologic consequences of infection and antibiotic treatment, such as endotoxin release and initiation of the septic cascade, also must be considered when choosing appropriate anti-infective therapy. The introduction of adjuvant immunotherapy, along with improvement, validation, and implementation of pharmacodynamic predictors of antibiotic efficacy, undoubtedly will provide the medical community with an effective arsenal to further reduce the morbidity and mortality rates associated with bacterial infections.

Pharmacodynamic factors of antibiotic efficacy.

The primary focus of the pharmaceutical industry in past years has been on developing more potent antibiotics rather than on establishing optimum therapy with currently available agents. Concepts that can be used to tailor patient- and pathogen-specific antimicrobial regimens include concentration-dependent killing, concentration-independent killing, and postantibiotic effect. It is possible to administer single daily doses (SDD) of aminoglycosides; however, a fixed SDD regimen cannot meet the goals for therapy in all patients. Instead, it is necessary to consider both pharmacodynamic concepts and pharmacokinetic principles. Even with tailored, patient-specific regimens, however, limitations exist with antibiotic therapy alone. Immunotherapy, used as an adjunct to antimicrobial therapy, may play a role in improving patient outcomes.

Heterogeneity of oestrogen receptor expression in normal and malignant breast tissue.

The heterogeneity of oestrogen receptor (ER) expression has been examined in both normal and malignant breast tissue using an immunohistochemical assay. In both instances the ER status and cellular ER negativity were influenced by the patients' menopausal status, with tissues removed from premenopausal women being more often ER-negative, and when ER-positive, containing a high proportion of apparently ER-negative cells. Since the breast is normally regarded as hormone sensitive and since tumour cell ER negativity is apparently under a degree of hormonal regulation, our results suggest that the proportion of breast cancer cells that are ER-negative should be viewed with a degree of caution.

Influence of the antioestrogen tamoxifen on normal breast tissue.

Immunohistochemical assays have been employed to study the expression of ER, PgR, EGFR and Ki67 immunostaining in normal breast tissue (n = 76). The expression of ER and PgR was highly variable in both pre and postmenopausal women and was characterised by large numbers of apparently negative cells. This was most evident for ER-ICA staining in tissues removed from premenopausal women. PgR levels were highest in the ducts of premenopausal women, while EGFR expression was elevated in both ducts and lobules. Ki67 expression was observed in less than 10% of all normal cells and was suppressed by the menopause in lobular tissue. Tamoxifen therapy (40 mg d-1) did not influence the expression of PgR, EGFR or Ki67 immunostaining in cancer associated normal tissue (n = 17). A significant increase, however, was observed in the mean percentage ER positivity in ductal tissue. No effect of duration of tamoxifen therapy was observed on the expression of the antigens studied.

Hormone sensitivity in breast cancer: influence of heterogeneity of oestrogen receptor expression and cell proliferation.

The percentage of oestrogen receptor (ER) positive cells in a series of 118 breast cancers has been examined by immunohistochemistry in relation to patients' response to endocrine therapy. Positive and negative predictive values have been used to calculate appropriate cut-off points. The rate of response to treatment was significantly higher in women with receptor positive tumours, especially where the tumours contained more than 70% positive cells. Tumours that were apparently negative for ER expression rarely responded to endocrine therapy. The hormone sensitivity of ER positive breast cancer was also influenced by the rate of tumour cell proliferation, with tumours expressing high levels of Ki67 immunostaining rarely responding to therapy.

Zoladex plus tamoxifen versus Zoladex alone in pre- and peri-menopausal metastatic breast cancer.

Phase II studies examining the endocrinological and clinical efficacy of Zoladex and Zoladex plus tamoxifen have been examined in pre- and peri-menopausal women with advanced breast cancer. No adverse endocrinological interaction between the drugs have been observed. Although a higher proportion of static disease was observed with the combination of the drugs, which possibly occurred at the expense of partial remissions, the time to disease progression was extended in women who received Zoladex plus tamoxifen. Remissions were primarily restricted to patients whose tumours were ER positive. Only occasional responses were seen in ER negative disease. This was especially evident where the ER negative tumours were EGF-R positive and showed high rates of cell proliferation.

Goserelin (Zoladex) in premenopausal advanced breast cancer: duration of response and survival.

In premenopausal women with advanced breast cancer the luteinising hormone-releasing hormone agonist goserelin (Zoladex, ICI plc) will produce serum levels of oestradiol equivalent to those following surgical oophorectomy or the menopause. This paper reports our further experience of using this drug in 75 premenopausal patients with advanced breast cancer. In addition to response rates, duration of response is reported. An objective response was seen in 25 patients (33%), the median duration of which was in excess of 15 months. Seven patients (9%) showed a complete response to therapy; median duration greater than 37 months. There was no significant difference in time to disease progression (Lee-Desu statistic 18.26, 1 d.f., P = 0.43) and probability of survival (Lee-Desu statistic 3.41, 1 d.f., P = 0.07) between those patients assessed as having either static disease, or those showing a partial response at six months. Response to therapy correlates significantly with the oestrogen receptor status of the primary tumour (X2 = 20.59, 6 d.f., P less than 0.005). The modest side-effects, ease of administration and reversibility make this approach to therapy very attractive. This is to be remembered in that 53% of patients had disease progression whilst receiving goserelin. These patients thus avoided the unnecessary and irreversible morbidity associated with surgical oophorectomy. With the proven efficacy and minimal morbidity associated with goserelin we believe there is no current role for surgical oophorectomy in the management of premenopausal patients with advanced breast cancer.

Automated quantitation of immunocytochemically localized estrogen receptors in human breast cancer.

Frozen sections of breast tumor tissue have been stained using an immunoperoxidase [estrogen receptor (ER)-immunocytochemistry] kit incorporating a monoclonal antiserum [H222] to visualize nuclear human ERs. Quantitation of specific staining has been performed by manual procedures using optical microscopy and by a computer-assisted image analysis system (CAS 100). Initial investigations with a test panel of ER-immunocytochemistry-positive tumors revealed a good qualitative agreement between CAS and manual assessments. Reduced variance was, however, observed between quantified ER-immunocytochemistry results from four experienced investigators using the CAS analysis. An extended study confirmed the relationships between CAS and manual methods of assessment. These findings were evident when studies were scored either by assessment of the percentage of positively stained cells (n = 92; r = 0.919; P less than 0.01) or by H-score calculations (n = 92; r = 0.913; P less than 0.01). A good correlation was also found between CAS quantification and the results of an ER enzyme immunoassay of 48 primary breast cancer specimens (r = 0.715; P less than 0.05). In 49 cases it was possible to relate CAS-defined ER status and levels to the subsequent response of patients to endocrine therapy. ER was assessed on specimens obtained prior to commencement of treatments for recurrent breast cancer. Presuming the presence of ER to be a prerequisite for successful therapy, very good correlations between response and both status and levels of positivity were recorded. None of 16 patients with CAS-ER-negative tumors responded to treatment, while 16 of 33 (48.4%) CAS-ER-positive patients achieved an objective response according to International Union Against Cancer criteria. A relationship between response and the degree of CAS-ER positivity was obtained when the CAS score divisions of 0, 1-100, and greater than 100 (response rates, 0, 41, and 64%, respectively) were used. These data demonstrate that automated image analysis offers a reliable, reproducible procedure for quantifying ER in immunocytochemically stained sections. It has potential advantages over manual procedures, providing less opportunity for subjective influences in scoring sections. Future advances in software design should further reduce elements of subjectivity and increase both the speed and reliability of results. We anticipate image analysis becoming a valuable tool in investigations concerning, for example, the influence of heterogeneity of steroid receptor distribution on the rate of recurrence of breast cancer after mastectomy and in the clinical course of the disease.

Estrogen deprivation in breast cancer. Clinical, experimental, and biological aspects.

The endocrinological and clinical effects of an LH-RH agonist, Zoladex, and an antiestrogen, Nolvadex, in patients with advanced breast cancer are outlined and their potential in the therapy of nonmalignant diseases of the breast and high-risk states is briefly discussed. Additional data are presented to indicate that new antiestrogens are now available for experimental studies that, unlike tamoxifen, do not possess partial estrogen-like activity and that show favorable antitumor properties against DMBA-induced mammary tumors and MCF-7 human breast cancer cells in culture. The lack of agonistic effects of this new class of pharmacological agents now allows a state of total estrogen deprivation to be approached, a previously unobtainable clinical goal.