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Background: Nonsterile handling of propofol for anesthesia has been linked with severe sepsis and death. Placing a single check valve in the IV tubing does not prevent retrograde ascension of pathogens into propofol-filled syringes, so we designed an IV tubing set with multiple check valves. To estimate the efficacy of this design, we measured the concentration of pathogens detected upstream in the IV tubing in relation to the pathogen concentration in a model of a contaminated patient., Methods: A glass container with a rubber sealed port was filled with a suspension of either bacteria or phagocytes and kept at 37°C ("contaminated patient" model). A bag of normal saline was connected to an IV cannula, punctured through the rubber sealed port of the patient model. Two additional sidestream infusion lines were connected to syringes in 2 standard infusion pumps. One of the syringes contained propofol and the other contained normal saline as a substitute for an opioid preparation. After 5 hours of infusion, we obtained samples from different parts of the infusion lines and syringes. The samples were streaked out on blood agar plates and incubated at 37°C for 24 hours. We repeated this experiment with 6 different pathogens., Results: We incubated 825 agar plates. Whereas the concentration of bacteria and phagocytes in the "patient" had significantly increased during the 5-hour experiments (positive control), no bacterial growth could be detected in any of the incubated plates., Conclusion: The data from this experimental setting suggest that the design with multiple check valves in paired configuration prevents retrograde contamination. Of note, this does not permit the reuse of propofol syringes because reusing is against the manufacturer's recommendations.

Background: The recently described yeast species Candida dubliniensis is closely related to C. albicans and has been recovered predominantly from the oral cavities of HIV-infected individuals and AIDS patients who are often receiving fluconazole as prophylactic or therapeutic treatment for oropharyngeal candidiasis. Like C. albicans, C. dubliniensis secretes aspartic proteinases which in C. albicans have been shown to be involved in adherence., Objective: To explain the increasing prevalence of C. dubliniensis in AIDS patients and to investigate the virulence factors of this yeast., Methods: An in vitro assay was developed to compare the adherence to epithelial cells of C. dubliniensis from HIV-patients with that of C. albicans., Results: All C. albicans isolates adhered better than the 22 C. dubliniensis isolates. In the presence of fluconazole, the C. dubliniensis isolates tested showed increased adherence as compared with controls without fluconazole. In contrast, all C. albicans isolates decreased in adherence to epithelial cells in the presence of fluconazole independently of their in vitro susceptibility to this drug. Proteinase antigens are present on the surface of C. dubliniensis cells adherent to epithelial target cells. In the presence of fluconazole this proteinase antigen was more strongly expressed., Conclusion: Increased adherence of C. dubliniensis strains in the presence of fluconazole could explain its high recovery rate from HIV-positive patients in recent years. The induction of proteinase secretion in the presence of fluconazole found for most of the C. dubliniensis isolates could be one of the factors involved in adherence.

N-Chlorotaurine, an endogenous long-lived oxidant, demonstrated fungicidal activity against Candida spp. and a postantifungal effect. Secreted aspartyl proteinases, important fungal virulence factors, proved to be a first target of impact. These results provide support for the topical application of N-chlorotaurine as an antimicrobial agent in yeast infections.

Medically important yeasts of the genus Candida secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like Candida albicans, Candida tropicalis secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene SAPT1 as a probe and under low-stringency hybridization conditions, three new closely related gene sequences, SAPT2 to SAPT4, encoding secreted proteinases were cloned from a C. tropicalis lambdaEMBL3 genomic library. All bands identified by Southern blotting of EcoRI-digested C. tropicalis genomic DNA with SAPT1 could be assigned to a specific SAP gene. Therefore, the SAPT gene family of C. tropicalis is likely to contain only four members. Interestingly, the SAPT2 and SAPT3 gene products, Sapt2p and Sapt3p, which have not yet been detected in C. tropicalis cultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast Pichia pastoris as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong SAPT1 signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all other SAPT genes under several conditions tested, showing that these SAPT genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection.

Since the introduction of new anti-retroviral agents such as human immunodeficiency virus (HIV) protease inhibitors, oropharyngeal candidiasis is less often observed in acquired immune deficiency syndrome patients. Secretory aspartic proteases of Candida albicans, which have similarities to the HIV aspartic proteases, are pathogenicity factors that have been intensively investigated in recent years. The inhibitory effect of four different HIV aspartic protease inhibitors (ritonavir, saquinavir, indinavir, and nelfinavir), on the activity of different Candida albicans secretory aspartic proteases was demonstrated. These anti-retroviral agents were able to inhibit Candida albicans secretory aspartic proteases 1, 2, and 3 which are involved in Candida adherence. As a consequence of these results we used selected HIV protease inhibitors in an adherence assay of Candida cells to epithelial cells. Ritonavir and saquinavir inhibited adherence of Candida albicans under the chosen experimental conditions similarly to the in vitro results, whereas indinavir had no effect. This inhibition was shown to be concentration dependent. The specificity of these effects with respect to the secretory aspartic proteases was demonstrated by competitive binding experiments using purified recombinant secretory aspartic proteases. On the basis of these studies we conclude that lower rates of oropharyngeal candidiasis in individuals receiving potent anti-retroviral therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida secretory aspartic proteases by HIV protease inhibitors.

Isoforms of aspartyl proteinase (Sap), which are encoded by at least nine related SAP genes, have been implicated to be a major virulence factor of the opportunistic yeast Candida albicans in experimental infections. Although it is generally assumed that proteinases are important for infections, detailed information on the pathogenetic role of Saps is still lacking. The same applies to the question whether the genes and corresponding isoforms of the enzyme are expressed during oral infection. For in vivo investigations, parts of the lesional oral epithelium were collected from three HIV-infected patients with oropharyngeal candidiasis. Immunoelectron microscopy was performed (pre- and post-embedding gold labeling with silver enhancement) using an anti-Sap murine monoclonal antibody directed against the gene products Sap1-3. It was possible to demonstrate expression of Sap antigens in each of the three samples of human oral candidiasis. This suggests that at least one of the genes SAP1-3 was expressed at the time of sample collection. Furthermore, a possible role of the enzymes during the interaction of yeast cells and mucosal cells is suggested: the majority of Sap antigens is secreted by those C. albicans cells that adhere directly to the epithelial surface. Sap immunoreactivity can be detected in particular at the site of close contact between C. albicans and epithelial cells, suggesting a pathogenetic role of the Saps in host-fungal interaction. Thus, inhibition of the enzyme might prove to be an important alternative in the prevention and treatment of candidiasis.

Previously, it has been shown that human immunodeficiency virus (HIV)-1 envelope proteins gp160 and gp41 bind to Candida albicans. Whether this interaction affects candidal virulence in vitro was investigated. HIV-1 gp160 or gp120 treatment of C. albicans significantly altered neither growth nor phospholipase activity of the fungus. However, treatment of C. albicans with gp160, but not with gp120, led to an elevation of free and cell-bound aspartate proteinase. In addition, culture supernatants obtained from C. albicans treated with gp160 or gp41, but not with gp120, showed a strong increase in proteinase activity. Finally, C. albicans viable yeast cells treated with gp160 or gp41 and serum were phagocytosed by polymorphonuclear leukocytes to a lesser extent than was C. albicans treated with gp120 and serum or serum alone. These findings suggest that the interaction between HIV-1 gp160 and C. albicans may promote the virulence of C. albicans in HIV-1-positive patients.