Polymyxin B Etest® compared with gold-standard broth microdilution in carbapenem-resistant Enterobacteriaceae exhibiting a wide range of polymyxin B MICs.

OBJECTIVES: Polymyxins have been revitalized to combat carbapenem-resistant Enterobacteriaceae (CRE). However, evaluating the activity of these agents by traditional broth dilution methods is not practical for busy clinical laboratories. We compared polymyxin B activity using two quantitative susceptibility testing methods, Etest® and broth microdilution (BMD), against CRE isolates from patients at an academic medical centre. METHODS: Polymyxin B activity against 70 CRE clinical isolates was determined by Etest® according to the manufacturer and by BMD according to CLSI guidelines. Pseudomonas aeruginosa ATCC® 27853 and Escherichia coli NCTC 13846 served as quality control strains. The EUCAST colistin susceptibility breakpoint of Enterobacteriaceae (≤2 mg/L) was used. Essential agreement was isolates with an MIC within 1 log2 dilution over total isolates. Categorical agreement was number of isolates in the same susceptibility category (susceptible or resistant) over total isolates. Major and very major error rates were calculated using number of susceptible and number of resistant isolates, respectively, as the denominator. McNemar's test was used for determining a difference in susceptibility between methods. RESULTS: The CRE isolates were primarily Klebsiella spp. (49%) and Enterobacter spp. (36%). Polymyxin B susceptibility was significantly higher by Etest® compared with BMD (97% versus 77%; p 0.0001). Categorical agreement was 80%, but essential agreement was low (10%). False non-susceptibility was never observed by Etest® (BMD reference), but the very major errors were high (88%). CONCLUSIONS: Etest® reporting of false susceptibility may result in inappropriate antibiotic use and treatment failure clinically. We do not recommend using Etest® for polymyxin B susceptibility testing for routine patient care.

Assessing the optimal liquid volume to be sprayed on isolated olive trees according to their canopy volumes.

The application of pesticides to traditional and intensive olive orchards in Southern Spain has led to environmental problems. More specifically, the lack of an accurate, useful criterion to regulate the spray volume in relation to canopy characteristics has led to spray drift and runoff, which are threats to local ecosystems. The aim of this study was to determine the optimal relationship between canopy volume and the spray application volume, called specific spray volume, CV, through laboratory and field trials. In the laboratory trial, 6 specific spray volumes (0.05, 0.08, 0.10, 0.12, 0.15, and 0.20Lm(-3)) were tested in a specially designed structure containing small, live olive trees in order to simulate an intensive plantation system. The model aimed to evaluate the coverage of pesticide application on water sensitive paper (WSP) collectors. In the field trial, the three laboratory specific spray volumes that gave the best coverage values were tested on live, intensively managed trees, whose crown volume was manually measured. Food dye E-102 was used to determine the spray deposition on artificial targets (10×10cm absorbent paper pieces), and WSP was used to evaluate spray coverage. The spray penetration and deposit homogeneity inside the canopy were also evaluated. Weather conditions during the field trial were monitored with a weather station. The results of the laboratory trial showed that the three best specific spray volumes were 0.08, 0.10, and 0.12Lm(-3), resulting in mean coverage values of approximately 30%. The ANOVA of the field trial results showed that the 0.12Lm(-3) was the optimal specific spray volume for isolated olive trees. This specific spray volume gave the highest mean deposits, the best efficiency (as measured by the greatest normalized deposit), the most favourable penetration and homogeneity, and the highest coverage values.

Comparative performance of herpes simplex virus type 2-specific serologic assays from Meridian Diagnostics and MRL diagnostics.

MRL Diagnostics and Meridian Diagnostics have recently designed herpes simplex virus type 2 (HSV-2)-specific enzyme immunoassays for HSV-2 antibody detection. Blood donor sera were assayed for HSV-2 antibodies by both methods. The sensitivity, specificity, and efficiency were 97.9, 95.4, and 95.9% for the MRL assay and 83.2, 98.2, and 95.5% for the Meridian assay, respectively.

Six-year study of the incidence of herpes in genital and nongenital cultures in a central Kentucky medical center patient population.

Herpes infections are among the most common sexually transmitted diseases and are the most common cause of genital ulcer disease in the United States. This study addresses the changing distribution of herpes simplex virus type 1 (HSV-1) and HSV-2 in patients presenting for evaluation of herpetic infections. Viral culture results from the University of Kentucky Clinical Microbiology Laboratory were reviewed for a 6-year period (1994 through 1999). Data were collected on patient sex, site of culture, and culture result. These data were analyzed statistically to identify yearly trends. Of the 4,498 cultures analyzed, nearly equal proportions of HSV-1 (13.3%) and HSV-2 (12.0%) were detected for an overall culture positivity rate of 25.3%. Approximately two-thirds of all positive cultures were from women. Although HSV-2 remained the predominant type of genital herpes, over the 6-year span of this study, there was a trend toward increasing proportions of HSV-1 genitalis, with 31.8% of male patients and 44.8% of female patients demonstrating HSV-1 genitalis by 1999. The majority of patients with HSV in nongenital sites grew HSV-1. Although there was significant yearly variation, HSV-2 was isolated from only 9.4% of patients with nongenital HSV for the entire 6-year period. This study therefore concludes that HSV-2 remains primarily a genital pathogen, while HSV-1 is taking on an increasingly important role in causing genital ulcer disease in addition to being the primary nongenital HSV.

Zygomycetes in human disease.

The Zygomycetes represent relatively uncommon isolates in the clinical laboratory, reflecting either environmental contaminants or, less commonly, a clinical disease called zygomycosis. There are two orders of Zygomycetes containing organisms that cause human disease, the Mucorales and the Entomophthorales. The majority of human illness is caused by the Mucorales. While disease is most commonly linked to Rhizopus spp., other organisms are also associated with human infection, including Mucor, Rhizomucor, Absidia, Apophysomyces, Saksenaea, Cunninghamella, Cokeromyces, and Syncephalastrum spp. Although Mortierella spp. do cause disease in animals, there is no longer sufficient evidence to suggest that they are true human pathogens. The spores from these molds are transmitted by inhalation, via a variety of percutaneous routes, or by ingestion of spores. Human zygomycosis caused by the Mucorales generally occurs in immunocompromised hosts as opportunistic infections. Host risk factors include diabetes mellitus, neutropenia, sustained immunosuppressive therapy, chronic prednisone use, iron chelation therapy, broad-spectrum antibiotic use, severe malnutrition, and primary breakdown in the integrity of the cutaneous barrier such as trauma, surgical wounds, needle sticks, or burns. Zygomycosis occurs only rarely in immunocompetent hosts. The disease manifestations reflect the mode of transmission, with rhinocerebral and pulmonary diseases being the most common manifestations. Cutaneous, gastrointestinal, and allergic diseases are also seen. The Mucorales are associated with angioinvasive disease, often leading to thrombosis, infarction of involved tissues, and tissue destruction mediated by a number of fungal proteases, lipases, and mycotoxins. If the diagnosis is not made early, dissemination often occurs. Therapy, if it is to be effective, must be started early and requires combinations of antifungal drugs, surgical intervention, and reversal of the underlying risk factors. The Entomophthorales are closely related to the Mucorales on the basis of sexual growth by production of zygospores and by the production of coenocytic hyphae. Despite these similarities, the Entomophthorales and Mucorales have dramatically different gross morphologies, asexual reproductive characteristics, and disease manifestations. In comparison to the floccose aerial mycelium of the Mucorales, the Entomophthorales produce a compact, glabrous mycelium. The asexually produced spores of the Entomophthorales may be passively released or actively expelled into the environment. Human disease with these organisms occurs predominantly in tropical regions, with transmission occurring by implantation of spores via minor trauma such as insect bites or by inhalation of spores into the sinuses. Conidiobolus typically infects mucocutaneous sites to produce sinusitis disease, while Basidiobolus infections occur as subcutaneous mycosis of the trunk and extremities. The Entomophthorales are true pathogens, infecting primarily immunocompetent hosts. They generally do not invade blood vessels and rarely disseminate. Occasional cases of disseminated and angioinvasive disease have recently been described, primarily in immunocompromised patients, suggesting a possible emerging role for this organism as an opportunist.

PCR detection of Pneumocystis carinii in bronchoalveolar lavage specimens: analysis of sensitivity and specificity.

Although PCR detection of Pneumocystis carinii DNA has been described, little is known about the sensitivity or specificity of the assay in routine laboratory practice. We had the unique opportunity to use a bronchoalveolar lavage (BAL) specimen bank with samples for which the direct examination results for P. carinii were known. DNA purified from 129 selected specimens was amplified by using the primers described previously (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moton, and J. M. Hopkin, Mol. Biochem. Parasitol. 43:69-76, 1990). Of the 129 specimens, 37 were positive for P. carinii by direct examination. All 37 specimens were positive for P. carinii by PCR, yielding a 100% sensitivity and 100% negative predictive value for the assay. An additional 23 specimens were repeatedly positive for P. carinii by PCR but were not positive by direct examination. Review of the patient charts for these specimens with discordant results demonstrated that five of the patients were actually positive for P. carinii, as determined by either biopsy or examination of repeat or prior BAL specimens. A response to empiric therapy for P. carinii pneumonia was seen in an additional two patients. Of the remaining specimens, 8 produced no significant isolates other than P. carinii, while 12 contained culture-confirmed significant respiratory pathogens in addition to P. carinii (two fungal, nine bacterial, and one viral pathogen). Cytomegalovirus, which was of unknown significance, was isolated from 16 additional specimens. Overall, the specificity of the PCR assay was 79.3% compared to the results of direct examination. We hypothesized that the apparently poor specificity of the PCR assay was due to the increased sensitivity of the assay compared to that of direct examination. The sensitivity of the PCR assay was therefore assessed with BAL specimens containing P. carinii cysts. Serial dilutions of this preparation were evaluated by direct examination and PCR. PCR was found to be 100-fold more sensitive than direct examination, which detected one to two cysts per amplification. No false-positive results were detected in controls containing no DNA or by using target DNA from various fungal, viral, or bacterial respiratory pathogens. We conclude that PCR detection of P. carinii in BAL specimens is very sensitive and should be considered for patients whose specimens do not yield a diagnosis. The increased sensitivity of the PCR assay may help to identify those patients with low-titer infections who might benefit from directed antibiotic therapy for P. carinii and would otherwise be missed by direct examination alone.

Effects of ionic and nonionic radiographic contrast agents on endothelial cells in vitro.

Intravenous administration of radiographic contrast agents results in the occasional occurrence of thrombotic complications, which are more common after the use of ionic agents than nonionic agents. To investigate the pathophysiologic basis of this thrombotic tendency, we compared the effects of ionic and nonionic contrast agents on endothelial cells in vitro. Exposure of cultured human umbilical vein endothelial cells to ionic contrast medium for 10 minutes resulted in lifting of 76% +/- 8% of cells, significantly greater than that after exposure to nonionic medium (6% +/- 4%; p less than 0.005). A modified Baumgartner chamber was used to evaluate the effects of contrast agents on adhesion of platelets in anticoagulated whole blood to everted segments of fresh or stored deendothelialized rabbit aorta segments. Exposure of fresh vessels to ionic contrast medium led to a significant increase in platelet adhesion (31% +/- 7%; p less than .01), whereas the increase was smaller after exposure to nonionic contrast medium (25% +/- 3%). Platelet adhesion to stored vessels (41% +/- 4%) was significantly greater than adhesion to fresh aorta segments (15% +/- 2%; p less than 0.001), and contrast agents did not further increase adhesion. Microscopic examination of perfused aorta segments exposed to ionic contrast medium showed platelet adherence to intact endothelial cells, a phenomenon that did not occur without prior exposure of fresh aorta segments to ionic contrast medium. These findings demonstrate that exposure of endothelial cells to ionic contrast medium results in marked changes in cell viability and adhesive properties that may contribute to their thrombotic potential.

Multimer size dependence of von Willebrand factor binding to crosslinked or noncrosslinked fibrin.

von Willebrand factor (vWF) is synthesized in endothelial cells (EC) and may be either secreted constitutively or stored in Weibel-Palade bodies (WPB) for regulated release. Because fibrin stimulates rapid vWF release from EC, we examined the binding of EC synthesized vWF to fibrin. Culture medium containing constitutively secreted vWF was removed from metabolically labeled primary cultures of human umbilical vein EC, and vWF released from WPB was obtained after stimulation by A23187. vWF-deficient fibrinogen with or without factor XIII was added to releasate or media and clotted with thrombin to form crosslinked or noncrosslinked fibrin. vWF was immunopurified from releasate or media before and after clotting, and the amount and multimeric pattern of vWF bound was determined after sodium dodecyl sulfate agarose gel electrophoresis. High molecular weight multimers of vWF, whether secreted constitutively or released from WPB, bound preferentially to fibrin. Multimers of greater than 20 subunits represented 60% +/- 4% (SEM) of A23187 released vWF and 11% +/- 5% of media vWF, but binding to fibrin was similar, 96% +/- 1% and 94% +/- 2%, respectively. A progressively smaller proportion of vWF bound as multimer size decreased, and dimeric vWF binding was least, with 34% +/- 5% binding from A23187 releasate and 51% +/- 4% from media. The amount of vWF binding to crosslinked or noncrosslinked fibrin was similar, and preferential binding of high molecular weight multimers occurred with both. As measured by enzyme-linked immunosorbent assay, 45% +/- 2% of constitutively secreted vWF bound to crosslinked fibrin and 50% +/- 2% to noncrosslinked fibrin. The propolypeptide of vWF did not bind to fibrin. These findings indicate that binding of EC secreted vWF binding to fibrin depends on multimeric size but not on factor XIII crosslinking. This suggests that vWF released from EC in the presence of fibrin will bind locally, thereby facilitating platelet adhesion to the hemostatic plug or thrombus.

Mediation of fibrin-induced release of von Willebrand factor from cultured endothelial cells by the fibrin beta chain.

The exposure of endothelial cells (EC) to fibrin has been shown to stimulate the rapid release of von Willebrand factor (vWf) from storage sites in Weibel-Palade bodies. We have now investigated the fibrin structural features required for stimulation of release. The role of fibrinopeptide cleavage was examined by preparing fibrin with thrombin to remove both fibrinopeptide A (FPA) and fibrinopeptide B (FPB) and with reptilase or Agkistrodon contortrix procoagulant to selectively remove FPA or FPB, respectively. vWf release was found to require FPB cleavage, whereas removal of FPA and Factor XIIIa cross-linking of fibrin were without effect. The dependence of release on FPB cleavage suggested that a site involving the NH2 terminus of the beta chain could mediate vWf secretion. To test this hypothesis, B beta chain derivatives were prepared and examined for their capacity to induce release. Purified B beta chain had no effect on release at a concentration of 20 nM but stimulated release from 26 +/- 6% of cells at 200 nM, the maximum solubility. However, after thrombin cleavage of FPB, release occurred from 36 +/- 9% of cells at 20 nM and from 60 +/- 7% at 200 nM, both significantly greater than before cleavage. FPB and B beta 1-42 showed no activity, whereas beta 15-42, representing the NH2 terminus of the thrombin cleaved beta chain, stimulated significant release at concentrations of 0.1 and 1 mM. We conclude that FPB cleavage from fibrin is required for stimulation of vWf release from EC and that this is mediated by a site that includes the NH2 terminus of the beta chain.

Fibrin induces release of von Willebrand factor from endothelial cells.

Addition of fibrinogen to human umbilical vein endothelial cells in culture resulted in release of von Willebrand factor (vWf) from Weibel-Palade bodies that was temporally related to formation of fibrin in the medium. Whereas no release occurred before gelation, the formation of fibrin was associated with disappearance of Weibel-Palade bodies and development of extracellular patches of immunofluorescence typical of vWf release. Release also occurred within 10 min of exposure to preformed fibrin but did not occur after exposure to washed red cells, clot liquor, or structurally different fibrin prepared with reptilase. Metabolically labeled vWf was immunopurified from the medium after release by fibrin and shown to consist of highly processed protein lacking pro-vWf subunits. The contribution of residual thrombin to release stimulated by fibrin was minimized by preparing fibrin clots with nonstimulatory concentrations of thrombin and by inhibiting residual thrombin with hirudin or heating. We conclude that fibrin formed at sites of vessel injury may function as a physiologic secretagogue for endothelial cells causing rapid release of stored vWf.

Effects of streptozotocin-induced diabetes and insulin on phospholipid content of R3230AC mammary tumor cells.

The influence of diabetes and insulin treatment on the phospholipid content of R3230AC mammary tumors, a hormonally responsive neoplasm, was studied. Diabetes was induced by administration of streptozotocin 3 days prior to tumor implantation. Protamine zinc insulin, 3 IU/rat twice daily, was administered to tumor-bearing rats for 3 days. Enzymatically dissociated tumor cells from diabetic animals showed significant increases in phosphatidyl choline, lysophosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, and phosphatidic acid, compared to controls. Diabetic animals treated with insulin displayed reductions in phosphatidyl choline, lysophosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, and phosphatidic acid to levels approximating those found in intact (control) animals. However, neither diabetes nor insulin treatment altered sphingomyelin levels. Mammary tumor cells from diabetic animals showed a 21% increase in DNA content compared to that in intact controls and treatment of diabetic animals with insulin lowered DNA level significantly. The responsiveness of both phospholipids and DNA content to changes in the insulin milieu of the host suggest that phospholipids may play an important role in mediating the effects of insulin on growth of R3230AC tumors.