Candida colonization index and subsequent infection in critically ill surgical patients: 20 years later.

INTRODUCTION: For decades, clinicians dealing with immunocompromised and critically ill patients have perceived a link between Candida colonization and subsequent infection. However, the pathophysiological progression from colonization to infection was clearly established only through the formal description of the colonization index (CI) in critically ill patients. Unfortunately, the literature reflects intense confusion about the pathophysiology of invasive candidiasis and specific associated risk factors. METHODS: We review the contribution of the CI in the field of Candida infection and its development in the 20 years following its original description in 1994. The development of the CI enabled an improved understanding of the pathogenesis of invasive candidiasis and the use of targeted empirical antifungal therapy in subgroups of patients at increased risk for infection. RESULTS: The recognition of specific characteristics among underlying conditions, such as neutropenia, solid organ transplantation, and surgical and nonsurgical critical illness, has enabled the description of distinct epidemiological patterns in the development of invasive candidiasis. CONCLUSIONS: Despite its limited bedside practicality and before confirmation of potentially more accurate predictors, such as specific biomarkers, the CI remains an important way to characterize the dynamics of colonization, which increases early in patients who develop invasive candidiasis.

Treatment and timing in invasive mould disease.

Invasive mould disease is a growing threat for immunocompromised patients. The optimum time to use mould-active antifungal agents is much debated. Current approaches to antifungal prophylaxis, early treatment (empirical and pre-emptive therapy) and treatment of documented mould infections in onco-haematology patients are discussed.

Contribution of CgPDR1-Regulated Genes in Enhanced Virulence of Azole-Resistant Candida glabrata.

In Candida glabrata, the transcription factor CgPdr1 is involved in resistance to azole antifungals via upregulation of ATP binding cassette (ABC)-transporter genes including at least CgCDR1, CgCDR2 and CgSNQ2. A high diversity of GOF (gain-of-function) mutations in CgPDR1 exists for the upregulation of ABC-transporters. These mutations enhance C. glabrata virulence in animal models, thus indicating that CgPDR1 might regulate the expression of yet unidentified virulence factors. We hypothesized that CgPdr1-dependent virulence factor(s) should be commonly regulated by all GOF mutations in CgPDR1. As deduced from transcript profiling with microarrays, a high number of genes (up to 385) were differentially regulated by a selected number (7) of GOF mutations expressed in the same genetic background. Surprisingly, the transcriptional profiles resulting from expression of GOF mutations showed minimal overlap in co-regulated genes. Only two genes, CgCDR1 and PUP1 (for PDR1upregulated and encoding a mitochondrial protein), were commonly upregulated by all tested GOFs. While both genes mediated azole resistance, although to different extents, their deletions in an azole-resistant isolate led to a reduction of virulence and decreased tissue burden as compared to clinical parents. As expected from their role in C. glabrata virulence, the two genes were expressed as well in vitro and in vivo. The individual overexpression of these two genes in a CgPDR1-independent manner could partially restore phenotypes obtained in clinical isolates. These data therefore demonstrate that at least these two CgPDR1-dependent and -upregulated genes contribute to the enhanced virulence of C. glabrata that acquired azole resistance.

Invasive Candida infections in the ICU

Invasive candidiasis, including candidemia and deep-seated Candida infections, is a severe opportunistic infection with an overall mortality in ICU patients comparable to that of severe sepsis/septic shock. With an incidence ranging from 5 to 10 cases per 1000 ICU admissions, invasive candidiasis represents 510% of all ICU-acquired infections. Although a high proportion of critically ill patients is colonised with Candida spp., only 540% develop an invasive infection. The occurrence of this complication is difficult to predict and an early diagnosis remains a major challenge. Indeed, blood cultures are positive in a minority of cases and often late in the course of infection. New non-culture based laboratory techniques may contribute to early diagnosis and management of invasive candidiasis. Recent data suggest that prediction rules based on risk factors, clinical and microbiological parameters or monitoring of Candida colonisation may efficiently identify critically ill patients at high risk of invasive candidiasis who may benefit of preventive or pre-emptive antifungal therapy. In many cancer centres, exposure to azoles antifungals has been associated with an epidemiological shift from Candida albicans to non-albicans Candida species with reduced antifungal susceptibility or intrinsic resistance. This trend has not been observed in recent surveys on candidemia in non-immunocompromised ICU patients. Prophylaxis, pre-emptive or empirical antifungal treatment are possible approaches for prevention or early management of invasive candidiasis. However, the selection of high-risk patients remains critical for an efficient management aimed at reducing the number needed to treat and thus avoiding unnecessary treatments associated with the emergence of resistance, drug toxicity and costs.

Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHAO.

In Pseudomonasfluorescens strain CHAO, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.

Diagnosis of invasive candidiasis in the ICU.

Invasive candidiasis ranges from 5 to 10 cases per 1,000 ICU admissions and represents 5% to 10% of all ICU-acquired infections, with an overall mortality comparable to that of severe sepsis/septic shock. A large majority of them are due to Candida albicans, but the proportion of strains with decreased sensitivity or resistance to fluconazole is increasingly reported. A high proportion of ICU patients become colonized, but only 5% to 30% of them develop an invasive infection. Progressive colonization and major abdominal surgery are common risk factors, but invasive candidiasis is difficult to predict and early diagnosis remains a major challenge. Indeed, blood cultures are positive in a minority of cases and often late in the course of infection. New nonculture-based laboratory techniques may contribute to early diagnosis and management of invasive candidiasis. Both serologic (mannan, antimannan, and betaglucan) and molecular (Candida-specific PCR in blood and serum) have been applied as serial screening procedures in high-risk patients. However, although reasonably sensitive and specific, these techniques are largely investigational and their clinical usefulness remains to be established. Identification of patients susceptible to benefit from empirical antifungal treatment remains challenging, but it is mandatory to avoid antifungal overuse in critically ill patients. Growing evidence suggests that monitoring the dynamic of Candida colonization in surgical patients and prediction rules based on combined risk factors may be used to identify ICU patients at high risk of invasive candidiasis susceptible to benefit from prophylaxis or preemptive antifungal treatment.

Polymorphisms in tumor necrosis factor-α increase susceptibility to intra-abdominal Candida infection in high-risk surgical ICU patients*.

OBJECTIVES: To evaluate the influence of genetic polymorphisms on the susceptibility to Candida colonization and intra-abdominal candidiasis, a blood culture-negative life-threatening infection in high-risk surgical ICU patients. DESIGN: Prospective observational cohort study. SETTING: Surgical ICUs from two University hospitals of the Fungal Infection Network of Switzerland. PATIENTS: Eighty-nine patients at high risk for intra-abdominal candidiasis (68 with recurrent gastrointestinal perforation and 21 with acute necrotizing pancreatitis). MEASUREMENTS AND MAIN RESULTS: Eighteen single-nucleotide polymorphisms in 16 genes previously associated with development of fungal infections were analyzed from patient's DNA by using an Illumina Veracode genotyping platform. Candida colonization was defined by recovery of Candida species from at least one nonsterile site by twice weekly monitoring of cultures from oropharynx, stools, urine, skin, and/or respiratory tract. A corrected colonization index greater than or equal to 0.4 defined "heavy" colonization. Intra-abdominal candidiasis was defined by the presence of clinical symptoms and signs of peritonitis or intra-abdominal abscess and isolation of Candida species either in pure or mixed culture from intraoperatively collected abdominal samples. Single-nucleotide polymorphisms in three innate immune genes were associated with development of a Candida corrected colonization index greater than or equal to 0.4 (Toll-like receptor rs4986790, hazard ratio = 3.39; 95% CI, 1.45-7.93; p = 0.005) or occurrence of intra-abdominal candidiasis (tumor necrosis factor-α rs1800629, hazard ratio = 4.31; 95% CI, 1.85-10.1; p= 0.0007; β-defensin 1 rs1800972, hazard ratio = 3.21; 95% CI, 1.36-7.59; p = 0.008). CONCLUSION: We report a strong association between the promoter rs1800629 single-nucleotide polymorphism in tumor necrosis factor-α and an increased susceptibility to intra-abdominal candidiasis in a homogenous prospective cohort of high-risk surgical ICU patients. This finding highlights the relevance of the tumor necrosis factor-α functional polymorphism in immune response to fungal pathogens. Immunogenetic profiling in patients at clinical high risk followed by targeted antifungal interventions may improve the prevention or preemptive management of this life-threatening infection.

Role of gluconic acid production in the regulation of biocontrol traits of Pseudomonas fluorescens CHA0.

The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Deltagcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.

ESCMID* guideline for the diagnosis and management of Candida diseases 2012: non-neutropenic adult patients.

This part of the EFISG guidelines focuses on non-neutropenic adult patients. Only a few of the numerous recommendations can be summarized in the abstract. Prophylactic usage of fluconazole is supported in patients with recent abdominal surgery and recurrent gastrointestinal perforations or anastomotic leakages. Candida isolation from respiratory secretions alone should never prompt treatment. For the targeted initial treatment of candidaemia, echinocandins are strongly recommended while liposomal amphotericin B and voriconazole are supported with moderate, and fluconazole with marginal strength. Treatment duration for candidaemia should be a minimum of 14 days after the end of candidaemia, which can be determined by one blood culture per day until negativity. Switching to oral treatment after 10 days of intravenous therapy has been safe in stable patients with susceptible Candida species. In candidaemia, removal of indwelling catheters is strongly recommended. If catheters cannot be removed, lipid-based amphotericin B or echinocandins should be preferred over azoles. Transoesophageal echocardiography and fundoscopy should be performed to detect organ involvement. Native valve endocarditis requires surgery within a week, while in prosthetic valve endocarditis, earlier surgery may be beneficial. The antifungal regimen of choice is liposomal amphotericin B +/- flucytosine. In ocular candidiasis, liposomal amphotericin B +/- flucytosine is recommended when the susceptibility of the isolate is unknown, and in susceptible isolates, fluconazole and voriconazole are alternatives. Amphotericin B deoxycholate is not recommended for any indication due to severe side effects.

Invasive candidiasis: comparison of management choices by infectious disease and critical care specialists.

OBJECTIVE: To compare the management of invasive candidiasis between infectious disease and critical care specialists. DESIGN AND SETTING: Clinical case scenarios of invasive candidiasis were presented during interactive sessions at national specialty meetings. Participants responded to questions using an anonymous electronic voting system. PATIENTS AND PARTICIPANTS: Sixty-five infectious disease and 51 critical care physicians in Switzerland. RESULTS: Critical care specialists were more likely to ask advice from a colleague with expertise in the field of fungal infections to treat Candida glabrata (19.5% vs. 3.5%) and C. krusei (36.4% vs. 3.3%) candidemia. Most participants reported that they would change or remove a central venous catheter in the presence of candidemia, but 77.1% of critical care specialists would start concomitant antifungal treatment, compared to only 50% of infectious disease specialists. Similarly, more critical care specialists would start antifungal prophylaxis when Candida spp. are isolated from the peritoneal fluid at time of surgery for peritonitis resulting from bowel perforation (22.2% vs. 7.2%). The two groups equally considered Candida spp. as pathogens in tertiary peritonitis, but critical care specialists would more frequently use amphotericin B than fluconazole, caspofungin, or voriconazole. In mechanically ventilated patients the isolation of 10(4) Candida spp. from a bronchoalveolar lavage was considered a colonizing organism by 94.9% of infectious disease, compared to 46.8% of critical care specialists, with a marked difference in the use of antifungal agents (5.1% vs. 51%). CONCLUSIONS: These data highlight differences between management approaches for candidiasis in two groups of specialists, particularly in the reported use of antifungals.

Milbemycins: more than efflux inhibitors for fungal pathogens.

Existing antifungal agents are still confronted to activities limited to specific fungal species and to the development of resistance. Several improvements are possible either by tackling and overcoming resistance or exacerbating the activity of existing antifungal agents. In Candida glabrata, azole resistance is almost exclusively mediated by ABC transporters (including C. glabrata CDR1 [CgCDR1] and CgCDR2) via gain-of-function mutations in the transcriptional activator CgPDR1 or by mitochondrial dysfunctions. We also observed that azole resistance was correlating with increasing virulence and fitness of C. glabrata in animal models of infection. This observation motivated the re-exploitation of ABC transporter inhibitors as a possible therapeutic intervention to decrease not only the development of azole resistance but also to interfere with the virulence of C. glabrata. Milbemycins are known ABC transporter inhibitors, and here we used commercially available milbemycin A3/A4 oxim derivatives to verify this effect. As expected, the derivatives were inhibiting C. glabrata efflux with the highest activity for A3 oxim below 1 μg/ml. More surprising was that oxim derivatives had intrinsic fungicidal activity above 3.2 μg/ml, thus highlighting effects additional to the efflux inhibition. Similar values were obtained with C. albicans. Our data show that the fungicidal activity could be related to reactive oxygen species formation in these species. Transcriptional analysis performed both in C. glabrata and C. albicans exposed to A3 oxim highlighted a core of commonly regulated genes involved in stress responses, including genes involved in oxidoreductive processes, protein ubiquitination, and vesicle trafficking, as well as mitogen-activated protein kinases. However, the transcript profiles contained also species-specific signatures. Following these observations, experimental treatments of invasive infections were performed in mice treated with the commercial A3/A4 oxim preparation alone or in combination with fluconazole. Tissue burden analysis revealed that oxims on their own were able to decrease fungal burdens in both Candida species. In azole-resistant isolates, oxims acted synergistically in vivo with fluconazole to reduce fungal burden to levels of azole-susceptible isolates. In conclusion, we show here the potential of milbemycins not only as drug efflux inhibitors but also as effective fungal growth inhibitors in C. glabrata and C. albicans.

Evaluation of the susceptibility of pathogenic Candida species to fluconazole. Fluconazole Global Susceptibility Study Group.

A fluconazole 25 microg disk diffusion test was used to test 2230 consecutively isolated Candida strains from 42 different hospital laboratories in 23 countries. Ninety seven percent of 1634 Candida albicans isolates and 83.4% of 596 non-Candida albicans isolates were susceptible to fluconazole, applying the proposed breakpoints (> or = 26 mm for susceptible strains and 18-25 mm for dose-dependent susceptible strains). This is the first hospital laboratory study to evaluate a large number and wide range of sequential Candida isolates from patients with all types of hospital infections. The fluconazole disk diffusion test appears to be a low-cost, reproducible, and accurate means of assessing the in vitro susceptibility of Candida isolates.

Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites.

At mucosal surfaces, we must co-exist with a high density of diverse microorganisms; therefore, protection against these occurs on multiple levels. Leukocyte- and epithelial derived-antimicrobial peptides and proteins (AMPs) comprise an essential component of immune defense. These molecules possess antibacterial, antifungal and signalling properties and probably contribute to defence and maintenance of homeostasis between the host and commensal microorganisms. Among these AMPs is bactericidal/permeability-increasing protein (BPI), an antimicrobial protein with potent endotoxin-neutralising activity, and several homologs. This review explores the roles of BPI and and its homologs at the mucosal interface. Congeners of BPI are under biopharmaceutical development as novel anti-infective agents, highlighting the potential therapeutic relevance of this protein family.

Cryptococcose disseminée: Manifestation inaugurale d'un SIDA [Disseminated cryptococcosis as inaugural manifestation of AIDS]

Disseminated cryptococcal disease is typically seen in patients with HIV infection. We report here the evolution of a patient with disseminated cryptococcosis whose treatment failed after ten weeks of induction therapy with amphotericin B. This case illustrates the importance of careful initial evaluation, and close clinical follow-up of these patients who are at risk of developing other opportunistic infections and drug-related complications.