Bacteriome and Mycobiome Interactions Underscore Microbial Dysbiosis in Familial Crohn's Disease.

UNLABELLED: Crohn's disease (CD) results from a complex interplay between host genetic factors and endogenous microbial communities. In the current study, we used Ion Torrent sequencing to characterize the gut bacterial microbiota (bacteriome) and fungal community (mycobiome) in patients with CD and their nondiseased first-degree relatives (NCDR) in 9 familial clusters living in northern France-Belgium and in healthy individuals from 4 families living in the same area (non-CD unrelated [NCDU]). Principal component, diversity, and abundance analyses were conducted, and CD-associated inter- and intrakingdom microbial correlations were determined. Significant microbial interactions were identified and validated using single- and mixed-species biofilms. CD and NCDR groups clustered together in the mycobiome but not in the bacteriome. Microbiotas of familial (CD and NCDR) samples were distinct from those of nonfamilial (NCDU) samples. The abundance of Serratia marcescens and Escherichia coli was elevated in CD patients, while that of beneficial bacteria was decreased. The abundance of the fungus Candida tropicalis was significantly higher in CD than in NCDR (P = 0.003) samples and positively correlated with levels of anti-Saccharomyces cerevisiae antibodies (ASCA). The abundance of C. tropicalis was positively correlated with S. marcescens and E. coli, suggesting that these organisms interact in the gut. The mass and thickness of triple-species (C. tropicalis plus S. marcescens plus E. coli) biofilm were significantly greater than those of single- and double-species biofilms. C. tropicalis biofilms comprised blastospores, while double- and triple-species biofilms were enriched in hyphae. S. marcescens used fimbriae to coaggregate or attach with C. tropicalis/E. coli, while E. coli was closely apposed with C. tropicalis Specific interkingdom microbial interactions may be key determinants in CD. IMPORTANCE: Here, we characterized the gut bacterial microbiota (bacteriome) and fungal community (mycobiome) in multiplex families with CD and healthy relatives and defined the microbial interactions leading to dysbiosis in CD. We identified fungal (Candida tropicalis) and bacterial (Serratia marcescens and Escherichia coli) species that are associated with CD dysbiosis. Additionally, we found that the level of anti-Saccharomyces cerevisiae antibodies (ASCA; a known CD biomarker) was associated with the abundance of C. tropicalis We also identified positive interkingdom correlations between C. tropicalis, E. coli, and S. marcescens in CD patients and validated these correlations using in vitro biofilms. These results provide insight into the roles of bacteria and fungi in CD and may lead to the development of novel treatment approaches and diagnostic assays.

The Oral HIV/AIDS Research Alliance Program: lessons learned and future directions.

The Oral HIV/AIDS Research Alliance (OHARA) was established in 2006 to provide the capacity to investigate the oral complications associated with HIV/AIDS within the ACTG infrastructure. Its goals were to explore the effects of potent antiretroviral therapy (ART) on the development of opportunistic infections, and variation and resistance of opportunistic pathogens in the context of immune suppression and long-term ART. The objectives of this talk, presented as part of a plenary session at the 7th World Workshop on Oral Health and Disease in AIDS, were to (i) provide an overview of OHARA's most recent research agenda, and how it evolved since OHARA's inception; (ii) describe OHARA's main accomplishments, including examples of research protocols completed and their key findings; and (iii) describe spin-off projects derived from OHARA, lessons learned, and future directions. OHARA has met its central goal and made key contributions to the field in several ways: (i) by developing/updating diagnostic criteria for oral disease endpoints commonly measured in OHARA protocols and in HIV/AIDS research in general and has creating standardized training modules, both for measuring these oral disease endpoints across clinical specialties, and for collecting oral fluid specimens; (ii) by implementing a total of nine protocols, six of which are completed. Three protocols involved domestic research sites, while three involved international research sites (in Africa, India, and South America); (iii) and by developing and validating a number of laboratory assays used in its protocols and in the field of oral HIV/AIDS research.

Extracorporeal treatment for acetaminophen poisoning: recommendations from the EXTRIP workgroup.

BACKGROUND: The Extracorporeal Treatments in Poisoning (EXTRIP) workgroup was created to provide evidence-based recommendations on the use of extracorporeal treatments (ECTR) in poisoning and the results are presented here for acetaminophen (APAP). METHODS: After a systematic review of the literature, a subgroup selected and reviewed the articles and summarized clinical and toxicokinetic data in order to propose structured voting statements following a pre-determined format. A two-round modified Delphi method was chosen to reach a consensus on voting statements, and the RAND/UCLA Appropriateness Method was used to quantify disagreement. Following discussion, a second vote determined the final recommendations. RESULTS: Twenty-four articles (1 randomized controlled trial, 1 observational study, 2 pharmacokinetic studies, and 20 case reports or case series) were identified, yielding an overall very low quality of evidence for all recommendations. Clinical data on 135 patients and toxicokinetic data on 54 patients were analyzed. Twenty-three fatalities were reviewed. The workgroup agreed that N-acetylcysteine (NAC) is the mainstay of treatment, and that ECTR is not warranted in most cases of APAP poisoning. However, given that APAP is dialyzable, the workgroup agreed that ECTR is suggested in patients with excessively large overdoses who display features of mitochondrial dysfunction. This is reflected by early development of altered mental status and severe metabolic acidosis prior to the onset of hepatic failure. Specific recommendations for ECTR include an APAP concentration over 1000 mg/L if NAC is not administered (1D), signs of mitochondrial dysfunction and an APAP concentration over 700 mg/L (4630 mmol/L) if NAC is not administered (1D) and signs of mitochondrial dysfunction and an APAP concentration over 900 mg/L (5960 mmol/L) if NAC is administered (1D). Intermittent hemodialysis (HD) is the preferred ECTR modality in APAP poisoning (1D). CONCLUSION: APAP is amenable to extracorporeal removal. Due to the efficacy of NAC, ECTR is reserved for rare situations when the efficacy of NAC has not been definitively demonstrated.

A second look at efficacy criteria for onychomycosis: clinical and mycological cure.

BACKGROUND: Approval of topical onychomycosis drugs by regulatory agencies may be negatively impacted by an overly stringent definition of complete cure, which includes nail clearing plus mycological cure. OBJECTIVES: In this position paper, we discuss interpretation of mycological outcome and clinical trial length. METHODS: We reviewed data from seven international onychomycosis trials that enrolled subjects with positive KOH and dermatophyte-positive culture at screening followed by 48 weeks of treatment. Further, we examined 94 KOH-positive/culture-negative week 52 follow-up samples for morphological hyphal damage. RESULTS: From 3054 samples collected at week 52 follow-up visits, 2360 were culture-negative. However, a significant percentage (78.7%) of these subungual samples (n = 1857) remained KOH-positive. From the subset of follow-up samples examined for morphological changes, we identified hyphal breakage or distortion in 56 direct smears (60%), which may indicate nonviability. CONCLUSIONS: Reassessment of the definition of onychomycosis cure is critical. For clinical trials of topical agents, length of treatment should be re-examined. Further, in our experience, a high rate of subungual debris samples remained direct smear-positive while converting to negative culture. Evidence of morphological hyphal damage suggests that late-visit microscopic results may be false-positives. Therefore, the absence of clinical signs following an adequate washout period, coupled with a negative culture, with or without negative microscopy, should be considered the definition of onychomycosis cure.

Successful prevention of respiratory syncytial virus nosocomial transmission following an enhanced seasonal infection control program.

Respiratory syncytial virus (RSV) infections can be serious in severely immunocompromised patients. Use of a targeted infection control program (TICP) has been shown to reduce RSV nosocomial transmission. We evaluated the impact of an enhanced seasonal infection control program (ESICP) vs standard TICP in a hematology-oncology ward. TICP was applied from 1999 to 2001 and ESICP applied from 2001 to 2003. ESICP consisted of strict isolation for all patients admitted on the ward during the RSV season. We prospectively evaluated the incidence, related morbidity and mortality of nosocomial RSV in both field interventions. A total of 40 hospitalized RSV infections were documented. The cumulative incidence of nosocomial RSV during TICP and ESICP was respectively of 42.8 and 3.9 cases/1000 admissions (relative risk = 0.09). ESICP needed to be implemented on 26 admitted patients on our ward to prevent one RSV nosocomial case. Furthermore, implementation of ESICP prevented four pneumonias and two deaths per RSV season. We conclude that ESICP is significantly more efficient than TICP to reduce the occurrence of nosocomial RSV infections and its related morbidity and mortality in patients with hematological malignancy and recipients of hematopoietic SCT.

The Oral HIV/AIDS Research Alliance: updated case definitions of oral disease endpoints.

The Oral HIV/AIDS Research Alliance (OHARA) is part of the AIDS Clinical Trials Group (ACTG), the largest HIV clinical trials organization in the world. Its main objective is to investigate oral complications associated with HIV/AIDS as the epidemic is evolving, in particular, the effects of antiretrovirals on oral mucosal lesion development and associated fungal and viral pathogens. The OHARA infrastructure comprises: the Epidemiologic Research Unit (at the University of California San Francisco), the Medical Mycology Unit (at Case Western Reserve University) and the Virology/Specimen Banking Unit (at the University of North Carolina). The team includes dentists, physicians, virologists, mycologists, immunologists, epidemiologists and statisticians. Observational studies and clinical trials are being implemented at ACTG-affiliated sites in the US and resource-poor countries. Many studies have shared end-points, which include oral diseases known to be associated with HIV/AIDS measured by trained and calibrated ACTG study nurses. In preparation for future protocols, we have updated existing diagnostic criteria of the oral manifestations of HIV published in 1992 and 1993. The proposed case definitions are designed to be used in large-scale epidemiologic studies and clinical trials, in both US and resource-poor settings, where diagnoses may be made by non-dental healthcare providers. The objective of this article is to present updated case definitions for HIV-related oral diseases that will be used to measure standardized clinical end-points in OHARA studies, and that can be used by any investigator outside of OHARA/ACTG conducting clinical research that pertains to these end-points.

Zygomycosis: the re-emerging fungal infection.

Invasive fungal infections are major medical complications in immunocompromised patients. The recent rise in the incidence of cancer and the increased use of newer medical treatment modalities, including organ transplantations, have resulted in growing numbers of highly immunosuppressed individuals. Although aspergillosis and candidiasis are among the most common invasive mycoses in such patients, there is evidence that the incidence of infectious diseases caused by Zygomycetes has risen significantly over the past decade. Patients with diabetes, malignancies, solid organ or bone marrow transplants, or iron overload and those receiving immunosuppressive agents, deferoxamine therapy, or broad-spectrum antimicrobial drugs are at highest risk for zygomycosis. This review details the emergence and importance of zygomycosis in current clinical practice and its manifestations and management. The etiologic species, pathogenesis and risk factors for zygomycosis are reviewed and updated. The clinical spectrum of zygomycosis is now broader, and it can be difficult to distinguish between mucormycosis and enthomophthoramycosis, both of which can manifest as disease ranging from a superficial infection to an angioinvasive infection with high mortality. Finally, the three-part treatment strategy (antifungal drugs, surgery, control of underlying diseases) is reviewed. Lipid formulations of amphotericin B are the antifungal agents of choice for treatment of zygomycosis. A novel antifungal triazole, posaconazole, has been developed and may become approved for treatment of zygomycosis. The clinical experience with adjunctive treatments like colony-stimulating factors, interferon-gamma, and hyperbaric oxygen therapy is still limited.

Human beta-defensins: differential activity against candidal species and regulation by Candida albicans.

Oral epithelial cell-derived human beta-defensins-1, -2, and -3 participate in innate immune responses against Candida. We hypothesized that these peptides utilize several mechanisms for protection. Recombinant hBD-1 and -2 were produced with the use of an insect cell/baculovirus expression system, while rhBD-3 was expressed as a fusion protein in E. coli. RhBD-2 and -3 were more effective at killing the candidal species at low micromolar concentrations than was rhBD-1, except for C. glabrata. While this species was relatively resistant to rhBD fungicidal activity, its adherence to oral epithelial cells was strain-specifically inhibited by the rhBDs. C. albicans hyphae were important in regulating hBD2 and -3 mRNA expression in primary human oral epithelial cells. Confocal microscopy of rhBD-2-challenged C. albicans suggests disruption of the fungal membrane. Results support the hypothesis that hBDs control fungal colonization through hyphal induction, direct fungicidal activity, and inhibition of candidal adherence.

Choices aplenty: antifungal prophylaxis in hematopoietic stem cell transplant recipients.

The incidence of invasive fungal infection (IFIs) in hematopoietic stem cell transplantation (HSCT) recipients ranges from 10 to 25% with an overall case fatality rate of up to 70-90%. Candida and Aspergillus genera remain the two most common pathogens. Although fluconazole prophylaxis in this population has been moderately effective in reducing mortality due to invasive candidiasis, this agent does not have activity against invasive aspergillosis (IA) and other mould. Several new agents such as voriconazole and caspofungin have enhanced potency and broad-spectrum antifungal activity and show promising results against yeasts and filamentous fungi when given as therapy and as chemoprophylaxis. Further, new diagnostic tools to detect circulating fungal antigens in biological fluids and PCR-based methods to detect species or genus-specific DNA or RNA have been developed. Incorporating these techniques along with clinical criteria appear to improve the accuracy of preclinical diagnosis of IFIs. Such approaches may alter the current treatment strategy from prophylaxis to pre-emptive therapy, thereby potentially decreasing cost and toxicity in high-risk patients.

Indoor mold, toxigenic fungi, and Stachybotrys chartarum: infectious disease perspective.

Damp buildings often have a moldy smell or obvious mold growth; some molds are human pathogens. This has caused concern regarding health effects of moldy indoor environments and has resulted in many studies of moisture- and mold-damaged buildings. Recently, there have been reports of severe illness as a result of indoor mold exposure, particularly due to Stachybotrys chartarum. While many authors describe a direct relationship between fungal contamination and illness, close examination of the literature reveals a confusing picture. Here, we review the evidence regarding indoor mold exposure and mycotoxicosis, with an emphasis on S. chartarum. We also examine possible end-organ effects, including pulmonary, immunologic, neurologic, and oncologic disorders. We discuss the Cleveland infant idiopathic pulmonary hemorrhage reports in detail, since they provided important impetus for concerns about Stachybotrys. Some valid concerns exist regarding the relationship between indoor mold exposure and human disease. Review of the literature reveals certain fungus-disease associations in humans, including ergotism (Claviceps species), alimentary toxic aleukia (Fusarium), and liver disease (Aspergillys). While many papers suggest a similar relationship between Stachybotrys and human disease, the studies nearly uniformly suffer from significant methodological flaws, making their findings inconclusive. As a result, we have not found well-substantiated supportive evidence of serious illness due to Stachybotrys exposure in the contemporary environment. To address issues of indoor mold-related illness, there is an urgent need for studies using objective markers of illness, relevant animal models, proper epidemiologic techniques, and examination of confounding factors.

Effects of fluconazole singly and in combination with 5-fluorocytosine or amphotericin B in the treatment of cryptococcal meningoencephalitis in an intracranial murine model.

In this study we developed a highly reproducible intracranial murine model of cryptococcosis. Mice (Balb/c, 5-7 weeks old) were challenged intracranially and treated with intermediate (30 mg/kg) or high (90 mg/kg) dose fluconazole, and amphotericin B (0.75 mg/kg), administered singly or in combination with flucytosine (100 mg/kg). Survival and brain CFU analyses were performed. Effect of fluconazole prophylaxis was also determined. Our data show that the developed model mimics clinical signs of cryptococcal meningitis. In single treatment, fluconazole (30 mg/kg) was more efficacious than amphotericin B or flucytosine (P < 0.0001). Combination treatment led to significantly increased anticryptococcal activity, which was highest for high dose fluconazole + flucytosine (P < 0.0001). However, no significant difference was observed between high dose fluconazole treatment with and without flucytosine (P >0.05). Fluconazole prophylaxis led to a significant decrease in brain CFU. In conclusion, high dose fluconazole administered post-infection, or as prophylaxis, may be highly efficacious in the treatment and prevention of meningoencephalitis.

Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins.

Biofilms, likely the predominant mode of device-related microbial infection, exhibit resistance to antimicrobial agents. Evidence suggests that Candida biofilms have dramatically reduced susceptibility to antifungal drugs. We examined antifungal susceptibilities of Candida albicans and Candida parapsilosis biofilms grown on a bioprosthetic model. In addition to conventional agents, we determined if new antifungal agents (triazoles, amphotericin B lipid formulations, and echinocandins) have activities against Candida biofilms. We also explored effects of preincubation of C. albicans cells with subinhibitory concentrations (sub-MICs) of drugs to see if they could modify subsequent biofilm formation. Finally, we used confocal scanning laser microscopy (CSLM) to image planktonic- and biofilm-exposed blastospores to examine drug effects on cell structure. Candida biofilms were formed on silicone elastomer and quantified by tetrazolium and dry weight (DW) assays. Susceptibility testing of fluconazole, nystatin, chlorhexidine, terbenafine, amphotericin B (AMB), and the triazoles voriconazole (VRC) and ravuconazole revealed resistance in all Candida isolates examined when grown as biofilms, compared to planktonic forms. In contrast, lipid formulations of AMB (liposomal AMB and AMB lipid complex [ABLC]) and echinocandins (caspofungin [Casp] and micafungin) showed activity against Candida biofilms. Preincubation of C. albicans cells with sub-MIC levels of antifungals decreased the ability of cells to subsequently form biofilm (measured by DW; P < 0.0005). CSLM analysis of planktonic and biofilm-associated blastospores showed treatment with VRC, Casp, and ABLC resulted in morphological alterations, which differed with each agent. In conclusion, our data show that Candida biofilms show unique susceptibilities to echinocandins and AMB lipid formulations.

Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans.

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase-encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild-type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37 degrees C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild-type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage-like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.

Candida albicans and Candida krusei differentially induce human blood mononuclear cell interleukin-12 and gamma interferon production.

Protection against Candida infection involves both innate and acquired immune responses, and cytokines produced by monocytes during the innate response may modify the acquired immune response by T cells. We hypothesized that Candida species which differ in pathogenicity can differentially induce production of immunoregulatory cytokines by human monocytes, which in turn modify T cells for immune responses to Candida. To test this hypothesis, we examined the effects of Candida albicans and Candida krusei on immunoregulatory cytokine production by human monocytes and gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells (PBMC). Purified monocytes were incubated with live or heat-killed strains of C. albicans and C. krusei at the optimal Candida/monocyte ratio of 0.5. Cytokines in the supernatants were measured by enzyme-linked immunosorbent assay. Our data demonstrated that live C. albicans and C. krusei significantly induced interleukin-10 (IL-10), monocyte chemotactic factor 1, IL-1beta, and tumor necrosis factor alpha production by monocytes relative to unstimulated monocytes. In contrast, unlike C. krusei, pathogenic live strains of C. albicans induced no or only a minimal level of IL-12. The expression of IL-12 p40 mRNA levels by reverse transcription-PCR corroborated the IL-12 protein (p70) findings. In human PBMC, human blood monocytes were the major source of both IL-10 and IL-12 production in response to C. albicans and C. krusei. Upon activation of T cells in the presence of Candida-modified monocytes and antigen-presenting cells, IL-12 production by PBMC treated with Candida organisms correlated strongly with the level of IFN-gamma production by T cells. These results indicate that the virulence of C. albicans may be related to its ability to induce the monocytic type II cytokine IL-10, with a selective inhibition of IL-12 production, which may be responsible for the observed lack of T-cell IFN-gamma and may restrain an effective type I immune response to Candida.

Endothelial cell injury caused by Candida albicans is dependent on iron.

Although it is known that Candida albicans causes endothelial cell injury, in vitro and in vivo, the mechanism by which this process occurs remains unknown. Iron is critical for the induction of injury in many types of host cells. Therefore, we investigated the role of iron in Candida-induced endothelial cell injury. We found that pretreatment of endothelial cells with the iron chelators phenanthroline and deferoxamine protected them from candidal injury, even though the organisms germinated and grew normally. Loading endothelial cells with iron reversed the cytoprotective effects of iron chelation. Moreover, chelation of endothelial cell iron significantly reduced phagocytosis of C. albicans by these cells, while candidal adherence to chelator-treated endothelial cells was slightly enhanced. Since endothelial cell phagocytosis of C. albicans is required for endothelial cell injury to occur, inhibition of phagocytosis is likely the principal mechanism of the cytoprotective effects of iron chelation. The production of toxic reactive oxygen intermediates by host cells is known to be inhibited by iron chelation. Therefore, we investigated whether treating endothelial cells with antioxidants could mimic the cytoprotective effects of iron chelation. Neither extracellular nor membrane-permeative antioxidants reduced candidal injury of endothelial cells. Furthermore, depleting endothelial cells of the endogenous antioxidant glutathione did not render them more susceptible to damage by C. albicans. These results suggest that candidal injury of endothelial cells is independent of the production of reactive oxygen intermediates and that the cytoprotective effects of iron chelation are not due to inhibition of the synthesis of these toxic intermediates.

Molecular epidemiology and antifungal susceptibility of Cryptococcus neoformans isolates from Ugandan AIDS patients.

Little is known of the antifungal susceptibility patterns and molecular epidemiology of Cryptococcus neoformans from tropical regions. We studied 164 clinical isolates of C. neofomans from 120 Ugandan AIDS patients with cryptococcal meningitis by analyzing their electrophoretic karyotypes and antifungal susceptibility profiles. Computer-assisted analysis of karyotype patterns was performed to generate dendrograms. MICs of fluconazole and flucytosine were determined by reference methods. A total of 43 distinguishable DNA types were identified among the 164 isolates. Only 30 patients (25%) were infected with their own unique strain of c. neoformans, whereas 75% of the patients shared their infecting strain with at least one other patient. Among 17 patients with more than one CSF isolate of C. neoformans, sequential isolates were identical or highly related in 12 (71%) and were different in five patients (29%). The isolates were susceptible to both fluconazole and flucytosine and there were no instances in which a stepwise increase in either fluconazole or flucytosine MICs was observed among serial isolates. These findings suggest that the epidemiology of cryptococcal disease in AIDS patients from tropical regions may be somewhat different from that observed in more temperate climates.

PIP: Even though Cryptococcus neoformans var. neoformans is a leading cause of life-threatening mycotic infection among AIDS patients worldwide, little is known about its antifungal susceptibility patterns and molecular epidemiology in tropical regions. The authors studied 164 clinical isolates of C. neoformans from 120 Ugandan AIDS patients with cryptococcal meningitis by analyzing their electrophoretic karyotypes and antifungal susceptibility profiles. Computer-assisted analysis of karyotype patterns was performed to generate dendrograms, while the MICs of fluconazole and flucytosine were determined using reference methods. 43 distinguishable C. neoformans DNA types were identified among the 164 isolates. 30 patients (25%) were infected with their own unique strain of C. neoformans, while 75% of the patients shared their infecting strain with at least 1 other patient. Among 17 patients with more than 1 cerebrospinal fluid isolate of C. neoformans, sequential isolates were identical or highly related in 12 (71%) and were different in 5 patients (29%). The isolates were susceptible to both fluconazole and flucytosine, and there was no instance in which a stepwise increase in either fluconazole or flucytosine MICs was observed among serial isolates. These findings suggest that the epidemiology of cryptococcal disease in AIDS patients from tropical regions may be somewhat different from that observed in more temperate climates.

International Conference for the Development of a Consensus on the Management and Prevention of Severe Candidal Infections.

Because of the rapidly increasing incidence of serious candidal infections, a consensus conference of 22 investigators from the United States, Europe, and Japan was held to discuss strategies for the prevention and treatment of deep-organ infections caused by Candida species. Commonly asked questions concerning the management of candidal infections were selected for discussion by the participating investigators. Possible answers to the questions were developed by the investigators, who then voted anonymously for their preferences. In certain instances, unanimity or a strong consensus was the result. In all cases, the full spectrum of responses was recorded and is presented in this report. The forms of candidal infection addressed included candidemia, candiduria, hepatosplenic candidiasis (chronic systemic candidiasis), candidal endophthalmitis, and candidal peritonitis. Prevention and treatment strategies were considered for patients who have undergone surgery, for neutropenic and nonneutropenic patients, and for patients who have undergone bone marrow and solid organ transplantation. The therapeutic roles of amphotericin B (standard and lipid formulations) and the azoles were considered.

Resistance to platelet microbicidal protein results in increased severity of experimental Candida albicans endocarditis.

Thrombin-induced platelet microbicidal protein (tPMP) exerts potent in vitro microbicidal activity against pathogens commonly found in the bloodstream, including Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. Localized platelet release of tPMP may be important in defense against infections involving the vascular endothelium caused by tPMP-susceptible organisms. In contrast, pathogens capable of surviving in the presence of tPMP could then exploit the platelet as an adhesive surface for attachment to damaged endothelium. To examine these hypotheses, we derived a tPMP-resistant (tPMP(r)) C. albicans strain from its tPMP-sensitive (tPMP(s)) parental strains were equivalent in vitro as assessed by genotyping (electrophoretic karyotype and restriction endonuclease analysis of genomic DNA), biotyping, germination, platelet aggregation, adherence to vascular endothelial cells, and growth characteristics. In addition, the tPMP(r) phenotype was stable following multiple in vitro and in vivo passages. We then investigated the in vivo relevance of tPMP susceptibility on endovascular infection using a rabbit model of endocarditis and hematogenous dissemination. Rabbits with transaortic catheters (n = 15 in each group) were challenged with either the tPMP(s) or tPMP(r) C. albicans strain. All rabbits developed C. albicans-induced endocarditis, as determined by the presence of infected vegetations. In rabbits challenged with tPMP(s) strain (P < 0.001). These results were seen in the absence of differences in either initial adherence of strains to cardiac valves or vegetation weights. Furthermore, although these C. albicans strains induced equivalent rates and extent of hematogenous renal infection, only the tPMP(r) strain disseminated hematogenously to the spleen (15 of 15 rabbits) versus 0 of 15 [tpmp(s) strain]; P < 0.0001). Thus, tPMP(r) C. albicans caused more-severe endocarditis and produced greater metastatic sequelae than the tPMP(s) counterpart.

Identification of patients with acute AIDS-associated cryptococcal meningitis who can be effectively treated with fluconazole: the role of antifungal susceptibility testing.

No method currently exists to predict which patients with acute AIDS-associated cryptococcal meningitis can be effectively treated with fluconazole. The objective of this study was to determine the relationship of cryptococcal susceptibility to fluconazole, along with clinical variables, to the risk of treatment failure for patients with acute AIDS-associated cryptococcal meningitis. Results of in vitro fluconazole susceptibility testing of cryptococcal isolates and data from two clinical trials were analyzed. Susceptibility to fluconazole was determined by means of both microtiter and macrobroth (M27-P) dilution methods. Treatment was defined as successful if the patient was alive at 10 weeks and if a cerebrospinal fluid culture was sterile at that time. Seventy-six patients receiving fluconazole +/- flucytosine were included; therapy failed for 19. Patients whose therapy failed were more likely to have a positive blood and urine culture and a higher titer in serum and cerebrospinal fluid of cryptococcal antigen, and the MIC of fluconazole against their isolates (as determined by the microtiter method) was more likely to be higher; they were less likely to have received flucytosine. Logistic regression modeling revealed that a negative blood culture, a low MIC of fluconazole (per the microtiter method), and treatment with flucytosine were factors independently associated with successful treatment.

Candida albicans antifungal-resistant strains: studies on adherence and other pathogenicity related characteristics.

In search of adhesion-variant strains of Candida albicans the adherence of a number of polyenes and/or azole-resistant strains of this yeast was studied (C. albicans 6406, 6406/8 and 799-XL, -XS, -YS, -R and YL). For comparison C. albicans KCCC 14172, known for its high adhesion and proteinase production, was also used. All isolates showed significantly lower adhesion (P < 0.001) compared with KCCC 14172. The exception was 6406/8 which showed superior adherability to all strains tested (2.5-4.8 times more adherent). This superiority prompted us to study the possible variation between this strain and the others in parameters that contribute to pathogenicity. Strain 6406/8 had the smallest average cell size (0.5-0.75 the size of cells from other strains). Variation in proteinase production and germ-tube formation existed among strains, with strain 6406/8 producing the lowest levels of inducible proteinase (2-4-fold less than the others), as well as being the least germ-tube former (10 times less than other strains). Ultrastructural comparisons between strain 6406/8 and its parent showed that the mutant strain had a thinner cell wall with a dense floccular layer throughout the cell wall compared to the parent strain. The cytoplasmic membrane of the mutant was more conspicuous than that of the parent strain. Comparison of the pathogenicity of strain 6406/8 and its parent (6406) revealed that although the mutant strain initially showed higher colonization than the parent strain, it was cleared much faster.(ABSTRACT TRUNCATED AT 250 WORDS)