Osteoarticular Mycoses.

Osteoarticular mycoses are chronic debilitating infections that require extended courses of antifungal therapy and may warrant expert surgical intervention. As there has been no comprehensive review of these diseases, the International Consortium for Osteoarticular Mycoses prepared a definitive treatise for this important class of infections. Among the etiologies of osteoarticular mycoses are Candida spp., Aspergillus spp., Mucorales, dematiaceous fungi, non-Aspergillus hyaline molds, and endemic mycoses, including those caused by Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides species. This review analyzes the history, epidemiology, pathogenesis, clinical manifestations, diagnostic approaches, inflammatory biomarkers, diagnostic imaging modalities, treatments, and outcomes of osteomyelitis and septic arthritis caused by these organisms. Candida osteomyelitis and Candida arthritis are associated with greater events of hematogenous dissemination than those of most other osteoarticular mycoses. Traumatic inoculation is more commonly associated with osteoarticular mycoses caused by Aspergillus and non-Aspergillus molds. Synovial fluid cultures are highly sensitive in the detection of Candida and Aspergillus arthritis. Relapsed infection, particularly in Candida arthritis, may develop in relation to an inadequate duration of therapy. Overall mortality reflects survival from disseminated infection and underlying host factors.

Pharmacodynamic and immunological interactions of amphotericin B formulations and voriconazole with human neutrophils against mature Scedosporium apiospermum and Fusarium spp. biofilms.

BACKGROUND: Mould infections caused by Scedosporium apiospermum and Fusarium solani species complex (FSSC) biofilms are rising among immunocompromised and immunocompetent patients. Little is known about the immunomodulatory effects of antifungal agents against these moulds. We examined the effects of deoxycholate and liposomal amphotericin B (DAmB, LAmB) and voriconazole on antifungal activities and immune responses of neutrophils (PMNs) against mature biofilms compared with their planktonic counterparts. METHODS: Antifungal activity of human PMNs exposed to mature biofilms and planktonic cells for 24 h was determined at effector-to-target ratios of 2:1 and 5:1, alone or combined with DAmB, LAmB and voriconazole, assessed as fungal damage by XTT assay. Cytokine production was evaluated by multiplex ELISA, following PMN stimulation with biofilms in the presence/absence of each drug. RESULTS: All drugs showed additive or synergistic effects with PMNs against S. apiospermum at 0.03-32 mg/L. They showed antagonism primarily against FSSC at 0.06-64 mg/L. Increased IL-8 was produced by PMNs exposed to S. apiospermum biofilms plus DAmB or voriconazole compared with PMNs exposed to biofilms alone (P < 0.01). During combined exposure, IL-1ß was increased, an effect only counteracted by increased levels of IL-10 caused by DAmB (P < 0.01). LAmB and voriconazole caused similar IL-10 levels with those released by biofilm-exposed PMNs. CONCLUSIONS: The synergistic, additive or antagonistic effects of DAmB, LAmB or voriconazole on biofilm-exposed PMNs are organism-specific, with FSSC exhibiting greater resilience than S. apiospermum to antifungals. Biofilms of both moulds caused dampened immune responses. The drug-mediated immunomodulating effect on PMNs, evidenced by IL-1ß, enhanced host protective functions.

Activity of Amphotericin B Formulations and Voriconazole, Alone or in Combination, against Biofilms of Scedosporium and Fusarium spp.

Scedosporium and Fusarium species are emerging opportunistic pathogens, causing invasive fungal diseases in humans, particularly in immunocompromised patients. Biofilm-related infections are associated with increased morbidity and mortality. Here, we assessed the ability of Scedosporium apiospermum and Fusarium solani species complex (FSSC) isolates to form biofilms and evaluated the efficacy of deoxycholate amphotericin B (D-AMB), liposomal amphotericin B (L-AMB), and voriconazole (VRC), alone or in combination, against mature biofilms. Biofilm formation was assessed by safranin staining and spectrophotometric measurement of optical density. Planktonic and biofilm damage was assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] reduction assay. Planktonic cell and biofilm MIC50s were determined as the minimum concentrations that caused ≥50% fungal damage compared to untreated controls. The combined activity of L-AMB (0.5 to 32 mg/liter) and VRC (0.125 to 64 mg/liter) against biofilms was determined by the checkerboard microdilution method and analyzed by the Bliss independence model. Biofilm MIC50s of D-AMB and L-AMB against S. apiospermum isolates were 1 and 2 mg/liter and against FSSC isolates were 0.5 and 1 mg/liter, respectively. Biofilm MIC50s of VRC against S. apiospermum and FSSC were 32 mg/liter and >256 mg/liter, respectively. Synergistic effects were observed at 2 to 4 mg/liter of L-AMB combined with 4 to 16 mg/liter of VRC against S. apiospermum biofilms (mean ΔE ± standard error, 17% ± 3.7%). Antagonistic interactions were found at 0.5 to 4 mg/liter of L-AMB combined with 0.125 to 16 mg/liter of VRC against FSSC isolates, at -28% ± 2%. D-AMB and L-AMB were more efficacious against S. apiospermum and FSSC biofilms than VRC.

Dose-Dependent Synergistic Interactions of Colistin with Rifampin, Meropenem, and Tigecycline against Carbapenem-Resistant Klebsiella pneumoniae Biofilms.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) can cause biofilm-related bloodstream infections associated with significant morbidity and mortality worldwide. We investigated the bactericidal activities of colistin (CST), rifampin (RIF), meropenem (MEM), gentamicin (GEN), and tigecycline (TGC) alone and that of CST in combination with RIF, MEM, GEN, or TGC against CR-Kp mature biofilms. Twenty CR-Kp blood isolates were derived from an equal number of bloodstream infections in adult patients. Biofilm formation was assessed by staining with 0.4% crystal violet and measuring the optical density spectrophotometrically at 545 nm. Biofilm damage was measured as the percent reduction of metabolic activity by an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] assay. The MIC50 for biofilms was determined as the minimum concentration that caused ≥50% bacterial damage compared to that for untreated controls. Antibacterial drug interactions were analyzed by the Bliss independence model. Four of the 20 CR-Kp isolates were biofilm producers. Biofilm MIC50s of CST, RIF, MEM, GEN, and TGC for these isolates were 64, 8, >256, 128, and 8 mg/liter, respectively. Synergistic interactions were observed at 32 to 64 mg/liter of CST combined with 0.25 to 4 mg/liter of RIF, at 32 mg/liter of CST combined with 0.007 to 0.25 mg/liter of MEM, and at 16 to 32 mg/liter of CST combined with 16 to 64 mg/liter of TGC. The synergy was highest for CST plus RIF, with a mean ΔE ± standard error (SE) of 49.87% ± 9.22%, compared to 29.52% ± 4.97% for CST plus MEM (P < 0.001) and 32.44% ± 6.49% for CST plus TGC (P < 0.001). Indifferent results were exhibited by CST plus GEN. None of the combinations exhibited antagonism. These drug interaction findings, especially those for CST with RIF, may be of importance in the treatment of biofilm-related CR-Kp infections.

Pharmacodynamic and Immunomodulatory Effects of Micafungin on Host Responses against Biofilms of Candida parapsilosis in Comparison to Those of Candida albicans.

Micafungin (MFG) demonstrates potent activity against biofilms of Candida albicans and Candida parapsilosis, the most frequent opportunistic fungal pathogens. Little is known about its immunopharmacologic effect on antibiofilm activity of phagocytic cells following exposure to Candida biofilms. In this study, we investigated the effects of MFG on human neutrophil-mediated damage of C. albicans and C. parapsilosis biofilms by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] and the potential mechanisms underlying the immunomodulatory MFG activities on cultured monocyte-derived THP-1 cells in response to these biofilms by reverse transcription-PCR and sandwich and multiplex enzyme-linked immunosorbent assay. Preexposure of C. albicans to subinhibitory MFG concentrations significantly enhanced neutrophil-mediated biofilm damage, an effect that appears to be species specific since a comparable effect was not observed with drug-pretreated C. parapsilosis biofilms. Human THP-1 cells responded to both Candida biofilms through Toll-like receptor 2 (TLR2) and TLR4 upregulation, modest TLR6 involvement, and enhanced NLRP3 activation, whereas the signal was relayed to the nucleus via NF-κB p65 activation. MFG caused 2- to 3-fold lower TLR2 and TLR4 mRNA levels than those caused by either organism. C. albicans biofilms induced a robust proinflammatory response, whereas C. parapsilosis biofilms either alone or in the presence of MFG caused increased interleukin-1ß (IL-1ß) production, but small amounts of IL-8, IL-23, and tumor necrosis factor alpha. In conclusion, MFG may condition THP-1 cells toward an inflammatory response through TLR2/TLR4 recruitment. Inflammatory signals observed with C. albicans biofilms are considerably reduced upon exposure of THP-1 cells to C. parapsilosis biofilms, possibly enhancing fungal survival and increasing biofilm pathogenicity.

Differential effects of antifungal agents on expression of genes related to formation of Candida albicans biofilms.

The purpose of this study was to analyse specific molecular mechanisms involved in the intrinsic resistance of C. albicans biofilms to antifungals. We investigated the transcriptional profile of three genes (BGL2, SUN41, ECE1) involved in Candida cell wall formation in response to voriconazole or anidulafungin after the production of intermediate and mature biofilms. C. albicans M61, a well-documented biofilm producer strain, was used for the development of intermediate (12 h and 18 h) and completely mature biofilms (48 h). After exposure of cells from each biofilm growth mode to voriconazole (128 and 512 mg l(-1)) or anidulafungin (0.25 and 1 mg l(-1)) for 12-24 h, total RNA samples extracted from biofilm cells were analysed by RT-PCR. The voriconazole and anidulafungin biofilm MIC was 512 and 0.5 mg l(-1) respectively. Anidulafungin caused significant up-regulation of SUN41 (3.7-9.3-fold) and BGL2 (2.2-2.8 fold) in intermediately mature biofilms; whereas, voriconazole increased gene expression in completely mature biofilms (SUN41 2.3-fold, BGL2 2.1-fold). Gene expression was primarily down-regulated by voriconazole in intermediately, but not completely mature biofilms. Both antifungals caused down-regulation of ECE1 in intermediately mature biofilms.

Antipseudomonal agents exhibit differential pharmacodynamic interactions with human polymorphonuclear leukocytes against established biofilms of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the most common pathogen infecting the lower respiratory tract of cystic fibrosis (CF) patients, where it forms tracheobronchial biofilms. Pseudomonas biofilms are refractory to antibacterials and to phagocytic cells with innate immunity, leading to refractory infection. Little is known about the interaction between antipseudomonal agents and phagocytic cells in eradication of P. aeruginosa biofilms. Herein, we investigated the capacity of three antipseudomonal agents, amikacin (AMK), ceftazidime (CAZ), and ciprofloxacin (CIP), to interact with human polymorphonuclear leukocytes (PMNs) against biofilms and planktonic cells of P. aeruginosa isolates recovered from sputa of CF patients. Three of the isolates were resistant and three were susceptible to each of these antibiotics. The concentrations studied (2, 8, and 32 mg/liter) were subinhibitory for biofilms of resistant isolates, whereas for biofilms of susceptible isolates, they ranged between sub-MIC and 2 × MIC values. The activity of each antibiotic alone or in combination with human PMNs against 48-h mature biofilms or planktonic cells was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. All combinations of AMK with PMNs resulted in synergistic or additive effects against planktonic cells and biofilms of P. aeruginosa isolates compared to each component alone. More than 75% of CAZ combinations exhibited additive interactions against biofilms of P. aeruginosa isolates, whereas CIP had mostly antagonistic interaction or no interaction with PMNs against biofilms of P. aeruginosa. Our findings demonstrate a greater positive interaction between AMK with PMNs than that observed for CAZ and especially CIP against isolates of P. aeruginosa from the respiratory tract of CF patients.

Methylprednisolone impairs conidial phagocytosis but does not attenuate hyphal damage by neutrophils against Exserohilum rostratum.

Exserohilum rostratum caused a multistate fungal meningitis outbreak following iatrogenic inoculation of contaminated methylprednisolone in the United States. To gain insight into the immunopathogenesis of this infection, we studied the innate host responses of human neutrophils against E. rostratum conidia and hyphae with or without methylprednisolone. The neutrophil-induced percentage fungal damage against conidia and hyphae was effector-to-target ratio dependent (≤55%). While methylprednisolone did not affect neutrophil-induced fungal damage by treatment of Exserohilum or neutrophils, it compromised phagocytosis of conidia (P < 0.05). These findings suggest that methylprednisolone-treated neutrophils may have altered phagocytic clearance of Exserohilum conidia, reducing host capacity to contain the invasive process.

Caspofungin at catheter lock concentrations eradicates mature biofilms of Candida lusitaniae and Candida guilliermondii.

The antibiofilm activities of caspofungin, anidulafungin, micafungin, and liposomal amphotericin B were studied against Candida lusitaniae, Candida guilliermondii, and a Candida albicans control strain. While anidulafungin and micafungin (0.007 to 2,048 mg/liter) showed reduced activity against biofilms of both test species, caspofungin displayed concentration-dependent antibiofilm activity, reaching complete and persistent eradication at concentrations achievable during lock therapy (512 to 2,048 mg/liter, P < 0.05). Although liposomal amphotericin B strongly inhibited mature biofilms, it possessed lower antibiofilm activity than caspofungin (P < 0.05).

Pathogenesis and host defence against Mucorales: the role of cytokines and interaction with antifungal drugs.

Innate immune response, including macrophages, neutrophils and dendritic cells and their respective receptors, plays an important role in host defences against Mucorales with differential activity against specific fungal species, while adaptive immunity is not the first line of defence. A number of endogenous and exogenous factors, such as cytokines and growth factors as well as certain antifungal agents have been found that they influence innate immune response to these organisms. Used alone or especially in combination have been shown to exert antifungal effects against Mucorales species. These findings suggest novel ways of adjunctive therapy for patients with invasive mucormycosis.

Species-specific and drug-specific differences in susceptibility of Candida biofilms to echinocandins: characterization of less common bloodstream isolates.

Candida species other than Candida albicans are increasingly recognized as causes of biofilm-associated infections. This is a comprehensive study that compared the in vitro activities of all three echinocandins against biofilms formed by different common and infrequently identified Candida isolates. We determined the activities of anidulafungin (ANID), caspofungin (CAS), and micafungin (MFG) against planktonic cells and biofilms of bloodstream isolates of C. albicans (15 strains), Candida parapsilosis (6 strains), Candida lusitaniae (16 strains), Candida guilliermondii (5 strains), and Candida krusei (12 strains) by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. Planktonic and biofilm MICs were defined as ≥ 50% fungal damage. Planktonic cells of all Candida species were susceptible to the three echinocandins, with MICs of ≤ 1 mg/liter. By comparison, differences in the MIC profiles of biofilms in response to echinocandins existed among the Candida species. Thus, C. lusitaniae and C. guilliermondii biofilms were highly recalcitrant to all echinocandins, with MICs of ≥ 32 mg/liter. In contrast, the MICs of all three echinocandins for C. albicans and C. krusei biofilms were relatively low (MICs ≤ 1 mg/liter). While echinocandins exhibited generally high MICs against C. parapsilosis biofilms, MFG exhibited the lowest MICs against these isolates (4 mg/liter). A paradoxical growth effect was observed with CAS concentrations ranging from 8 to 64 mg/liter against C. albicans and C. parapsilosis biofilms but not against C. krusei, C. lusitaniae, or C. guilliermondii. While non-albicans Candida planktonic cells were susceptible to all echinocandins, there were drug- and species-specific differences in susceptibility among biofilms of the various Candida species, with C. lusitaniae and C. guilliermondii exhibiting profiles of high MICs of the three echinocandins.

Neonatal Bloodstream Infection with Ceftazidime-Avibactam-Resistant blaKPC-2-Producing Klebsiella pneumoniae Carrying blaVEB-25.

BACKGROUND: Although ceftazidime/avibactam (CAZ/AVI) has become an important option for treating adults and children, no data or recommendations exist for neonates. We report a neonatal sepsis case due to CAZ/AVI-resistant blaKPC-2-harboring Klebsiella pneumoniae carrying blaVEB-25 and the use of a customized active surveillance program in conjunction with enhanced infection control measures. METHODS: The index case was an extremely premature neonate hospitalized for 110 days that had been previously treated with multiple antibiotics. Customized molecular surveillance was implemented at hospital level and enhanced infection control measures were taken for early recognition and prevention of outbreak. Detection and identification of blaVEB-25 was performed using next-generation sequencing. RESULTS: This was the first case of a bloodstream infection caused by KPC-producing K. pneumoniae that was resistant to CAZ/AVI without the presence of a metalo-ß-lactamase in the multiplex PCR platform in a neonate. All 36 additional patients tested (12 in the same NICU and 24 from other hospital departments) carried wild-type blaVEB-1 but they did not harbor blaVEB-25. CONCLUSION: The emergence of blaVEB-25 is signal for the horizontal transfer of plasmids at hospital facilities and it is of greatest concern for maintaining a sharp vigilance for the surveillance of novel resistance mechanisms. Molecular diagnostics can guide appropriate antimicrobial therapy and the early implementation of infection control measures against antimicrobial resistance.

Evaluation of Five Host Inflammatory Biomarkers in Early Diagnosis of Ventilator-Associated Pneumonia in Critically Ill Children: A Prospective Single Center Cohort Study.

Background: Early diagnosis of ventilator-associated pneumonia (VAP) remains a challenge due to subjective clinical criteria and the low discriminative power of diagnostic tests. We assessed whether rapid molecular diagnostics in combination with Clinically Pulmonary Index Score (CPIS) scoring, microbiological surveillance and biomarker measurements of PTX-3, SP-D, s-TREM, PTX-3, IL-1ß and IL-8 in the blood or lung could improve the accuracy of VAP diagnosis and follow-up in critically ill children. Methods: A prospective pragmatic study in a Pediatric Intensive Care Unit (PICU) was conducted on ventilated critically ill children divided into two groups: high and low suspicion of VAP according to modified Clinically Pulmonary Index Score (mCPIS). Blood and bronchial samples were collected on days 1, 3, 6 and 12 after event onset. Rapid diagnostics were used for pathogen identification and ELISA for PTX-3, SP-D, s-TREM, IL-1ß and IL-8 measurements. Results: Among 20 enrolled patients, 12 had a high suspicion (mCPIS > 6), and 8 had a low suspicion of VAP (mCPIS < 6); 65% were male; and 35% had chronic disease. IL-1ß levels at day 1 correlated significantly with the number of mechanical ventilation days (rs = 0.67, p < 0.001) and the PICU stay (r = 0.66; p < 0.002). No significant differences were found in the levels of the other biomarkers between the two groups. Mortality was recorded in two patients with high VAP suspicion. Conclusions: PTX-3, SP-D, s-TREM, IL-1ß and IL-8 biomarkers could not discriminate patients with a high or low suspicion of VAP diagnosis.

Daptomycin exerts differential immunomodulatory effects on host responses against methicillin-resistant Staphylococcus aureus biofilms.

Daptomycin (DAP) is indicated for difficult-to-treat Gram-positive infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). Exposure of S. aureus to subinhibitory antimicrobial concentrations (sub-MICs) has been shown to alter cell morphology and biofilm formation. This study aimed to investigate the influence of DAP biofilm sub-MICs on the damage caused by human polymorphonuclear neutrophils (PMNs) against MRSA biofilms and the potential immunomodulatory activity of DAP on human monocytes (MNCs) exposed to MRSA biofilms. DAP activity against biofilms and the impact of DAP on PMN-induced biofilm damage were evaluated by the XTT reduction assay, whereas pathogen recognition, signal transduction and cytokine modulation of DAP on MNCs in response to MRSA biofilms were assessed by RT-PCR and ELISA methodology. The MIC50 of DAP to MRSA biofilms was 16-32 mg/L. Pre-treatment of MRSA with 1, 2 or 4 mg/L DAP caused a synergistic effect on PMN-mediated biofilm damage, being dependent on the effector-to-target ratio. MNCs responded to MRSA biofilms and DAP through Toll like receptor 2 (TLR2) upregulation and increased NLRP3 inflammasome production. DAP caused 2.5-fold greater TLR2 mRNA levels than those caused by MRSA biofilms. A predominantly inflammatory response was induced by either component, causing the release of significantly increased IFN-γ, TNF-α, IL-8 and IL-6 levels by MNCs exposed to the combination treatment. MRSA biofilms alone or combined with DAP caused low amounts of IL-10 production, but increased IL-1ß levels. DAP may condition MNCs towards an inflammatory response through TLR2 engagement and NLRP3 inflammasome activation, possibly controlling biofilm-associated pathogenicity.

A Longitudinal Study of Spontaneous Gut Decolonization of Carbapenem-resistant Gram-negative Bacteria in Neonatal and Pediatric Patients.

BACKGROUND: Antibiotic exposure may convert gut microbiome to reservoir of resistant organisms, including carbapenem-resistant Gram-negative bacteria (CRGNB). Little is known about natural history of spontaneous CRGNB decolonization of neonates/children and their risk to develop CRGNB infections. METHODS: Patients hospitalized in a tertiary care hospital (1 days to 16 years) found to be CRGNB colonized in weekly surveillance cultures during hospitalization (January 2018 to December 2019) were prospectively followed after discharge with monthly rectal cultures for 12 months after colonization until decolonization (3 consecutive negative rectal cultures, ≥1 week apart). Patient demographics, clinical characteristics and CRGNB infections were recorded. Polymerase chain reaction for carbapenemases was performed in patients colonized, after 3 negative cultures, at the day of the last negative and the day of the first new positive culture. RESULTS: One-hundred thirty patients (median age, 1.3 months; lower-upper quartile values, 0.8-6.9 months) were studied including 66 neonates (median age, 12.6 days; Q1-Q3, 5-18.5 days). Among patients >30 days old, 51.6% achieved decolonization within 6 months, and among neonates, 91% achieved decolonization within 6 months. By 12th month, 89% of >30 days and 100% of neonates were decolonized. Forty-four (33.9%) patients (59% >30 days and 9% neonates) developed CRGNB infection(s), mainly pneumonia (25%) and bloodstream infection (20.5%). Prolonged colonization (odds ratio [OR], 7.75; 95% confidence interval [CI], 2.10-28.58), duration of broad-spectrum antibiotic use (OR, 1.22; 95% CI, 1.11-1.34) and parenteral nutrition (OR, 4.53; 95% CI, 1.14-17.94) were associated with the development of CRGNB infection. Two patients (1.5%) were found by polymerase chain reaction colonized after 3 negative cultures. CONCLUSIONS: Spontaneous decolonization occurs in most CRGNB colonized >30 days and all neonates within 12 months. One-third of colonized patients develop CRGNB infection(s). These findings may help optimize duration of contact precautions and empirical antimicrobial therapy for CRGNB colonized pediatric patients.

Activities of triazole-echinocandin combinations against Candida species in biofilms and as planktonic cells.

Biofilm formation complicates the treatment of various infections caused by Candida species. We investigated the effects of simultaneous or sequential combinations of two triazoles, voriconazole (VRC) and posaconazole (PSC), with two echinocandins, anidulafungin (AND) and caspofungin (CAS), against Candida albicans and Candida parapsilosis biofilms in comparison to their planktonic counterparts. Antifungal activity was assessed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide (XTT) metabolic assay. Antifungal-agent interactions were analyzed by the Bliss independence model in the simultaneous-treatment studies and by analysis of variance (ANOVA) in the sequential-treatment studies. Against C. albicans planktonic cells, the simultaneous combination of PSC (32 to 128 mg/liter) and CAS (0.008 to 0.25 mg/liter) was synergistic; the combinations of PSC (128 to 1,024 mg/liter) with AND (0.03 to 0.5 mg/liter) and VRC (32 to 512 mg/liter) with AND (0.008 to 0.03 mg/liter) were antagonistic. Against C. parapsilosis planktonic cells, the interaction between VRC (32 to 1,024 mg/liter) and CAS (1 to 16 mg/liter) was antagonistic. All simultaneous antifungal combinations demonstrated indifferent interactions against biofilms of both Candida species. Damage to biofilms of both species increased (P<0.01) in the presence of subinhibitory concentrations of echinocandins (0.008 to 0.064 mg/liter), followed by the addition of PSC (512 mg/liter for C. albicans and 64 to 512 mg/liter for C. parapsilosis) or VRC (256 to 512 mg/liter for C. albicans and 512 mg/liter for C. parapsilosis). Triazole-echinocandin combinations do not appear to produce antagonistic effects against Candida sp. biofilms, while various significant interactions occur with their planktonic counterparts.

Additive antifungal activity of anidulafungin and human neutrophils against Candida parapsilosis biofilms.

OBJECTIVES: To investigate the activities of two newer triazoles and two echinocandins combined with human phagocytes against Candida parapsilosis biofilms. METHODS: An in vitro model of C. parapsilosis biofilms was used. Biofilms were grown on silicone elastomer discs in 96-well plates at 37°C for 72 h. Biofilms or planktonic cells were incubated with voriconazole, posaconazole, caspofungin or anidulafungin, at clinically relevant concentrations, and human phagocytes (neutrophils or monocytes) alone or in combination with each of the antifungal agents for a further 22 h. Fungal damage induced by antifungal agents and/or phagocytes was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide] metabolic assay. RESULTS: Each of the antifungal agents alone and in combination with human phagocytes induced less damage against C. parapsilosis biofilms compared with planktonic cells. No antagonistic interactions between antifungal agents and phagocytes were found. Furthermore, anidulafungin, but not caspofungin, and neutrophils exerted additive activity against C. parapsilosis biofilms. CONCLUSIONS: Besides a lack of antagonistic interactions between newer antifungal agents and phagocytes, anidulafungin exerts additive immunopharmacological activity against C. parapsilosis biofilms.

Effects of interferon-γ and granulocyte colony-stimulating factor on antifungal activity of human polymorphonuclear neutrophils against Candida albicans grown as biofilms or planktonic cells.

Candida albicans is a leading cause of biofilm-related infections. As Candida biofilms are recalcitrant to host defenses, we sought to determine the effects of interferon-γ and granulocyte colony-stimulating factor, two pro-inflammatory cytokines, on the antifungal activities of human polymorphonuclear neutrophils (PMNs) against C. albicans biofilms, using an in vitro biofilm model. Priming of PMNs by these cytokines augmented fungal damage of planktonic cells; however, priming of PMNs did not have the same effect against Candida biofilms. Biofilm phenotype appears to play an important role in protecting C. albicans from the innate immune system.

Differential transcriptional profiles induced by amphotericin B formulations on human monocytes during response to hyphae of Aspergillus fumigatus.

Amphotericin B formulations possess diverse immunomodulatory properties that may contribute to the activity of phagocytes against invasive aspergillosis. In this work we provide a novel set of data on different gene transcriptional profiles of monocytes exposed to the combination of Aspergillus fumigatus and amphotericin B formulations. We used pathway-specific microarray analysis, RT-PCR analysis and enzyme-linked immunosorbent assays to compare the effects of amphotericin B deoxycholate (DAMB) at 1 µg/ml and amphotericin B lipid complex (ABLC) at 5 µg/ml to assess gene expression of immune molecules of THP-1 cells exposed to A. fumigatus hyphae (AF) for 4 h. A. fumigatus hyphae at effector/target ratio 10/1 induced mostly chemotactic factors for monocyte recruitment. DAMB at 1 µg/ml in the presence or absence of AF induced the most pronounced changes in pro-inflammatory and chemokine gene expression, while ABLC under the same conditions caused less dramatic effect. There was a reciprocal response of increased expression of the genes encoding IL-1ß and IL-20 and decreased expression of IL-10, IL-2 and IL-3 in response of monocytes to both the hyphae and antifungal agents. These results demonstrate that amphotericin B formulations exert differential effects on genes encoding pro-inflammatory molecules, immunoregulatory molecules and chemokines by human monocytes during response to A. fumigatus and that these molecules may affect antifungal activity.

Immunomodulatory properties of antifungal agents on phagocytic cells.

Phagocytic cells, particularly neutrophils and monocytes/macrophages, are the first line and the most effective form of innate host defence against pathogenic fungi. During antifungal therapy these phagocytic cells are also exposed to antifungal agents. In the phagocyte-fungus-antifungal agent interplay, drugs may directly interact with phagocytes through specific pattern recognition receptors, leading to altered antifungal activities. Antifungal agents, through modulation of fungal virulence, may initiate different immune response programs in the phagocytes, leading to antifungal synergism/antagonism or up-regulation of gene expression for a pro-inflammatory response. Additionally, indirect modulation of phagocyte behavior by pretreatment of neutrophils, monocytes, and macrophages with cytokines and exposure to antifungal agents have shown promising findings for combined drug-cytokine therapy that may improve treatment of life-threatening fungal diseases. In this review, we discuss the main in vitro and in vivo immunomodulatory effects of antifungal agents on phagocytes in response to pathogenic fungi, as well as we address underlying immunopharmacologic mechanisms and their potential impact on clinical outcome.