Diagnostic surveillance by Candida albicans germ tube antibody in intensive care unit patients.

BACKGROUND: The diagnosis of Invasive Candidiasis (IC) presents serious problems, mainly associated with the absence of pathognomonic symptoms of the disease and the difficulty of isolating the fungus in blood culture. Candida albicans germ tube antibody (CAGTA) provides a rapid and simple test for diagnosis of IC. The aim of this study was to evaluate the diagnostic role of the CAGTA in the monitoring of critically-ill patients at risk of developing IC. METHODS: During diagnostic surveillance in the intensive care units (ICU) CAGTA was performed twice a week if predetermined risk factors were present and a positive result was considered when a serum titer ≥1/160 was detected in at least one sample. RESULTS: Seventy critically ill patients were included in the study. Twenty-three patients with proven/probable IC were identified. The sensitivity, specificity, PPV, and NPV of CAGTA for the diagnosis of proven/probable IC in all 70 patients were 91.3%, 68.1%, 58.3%, and 94.1%, respectively. Statistically significant highest titers were found in patients with proven/probable IC as well as increasing titers more than 1/160. CONCLUSIONS: Our results suggest that detection of CAGTA could be a useful biomarker for the diagnosis of proven and probable IC in critical patients during prolonged ICU stay. During the monitoring it is opportune to evaluate the titers kinetics since the clinical diagnosis of proven/probable IC coincided with increase titer from negative (<1/160) to more than 1/160.

Enhancement of the antidermatophytic activity of silver nanoparticles by Q-switched Nd:YAG laser and monoclonal antibody conjugation.

The objective of this research was to investigate the effect of silver nanoparticles (AgNPs), free or conjugated with monoclonal antibody and mediated by Q-switched Nd:YAG laser on five dermatophytes. The laser was applied for 45 s at 532 nm and 0.8 J/cm2. The application of AgNPs combined with laser caused an increase in fungal susceptibility compared to application of AgNPs alone. The MIC50 and MIC100 recorded 3 and 9 µg/ml in the case of E. floccosum (the most susceptible species), 10 and 19 µg/ml for T. rubrum (the most tolerant species), respectively. A decrease in keratinase activity reaching 76.1, 67.1, and 62.4% was attained in the case of M. gypseum, T. rubrum, and T. mentagrophyte, respectively, on application of 10 µg/ml AgNPs combined with Nd:YAG laser. Under the same conditions of application, a steady increase in leaked materials coupled with reduction in ergosterol synthesis was reached. The structural alterations occurred to the fungus were more observed on the application of AgNPs in combination with laser where the conidia and hyphae lost their cellular integrity, become flaccid, permanently destructed, and completely killed. The monoclonal antibody conjugated AgNPs did not result in significant variation in in vitro experiments compared with that produced by nonconjugated nanoparticles. However, the conjugates achieved significantly more curing of M. canis-inoculated guinea pigs compared with nonconjugated nanoparticles.

Recombinant Phage Elicits Protective Immune Response against Systemic S. globosa Infection in Mouse Model.

Sporothrix globosa is a type of fungus that typically infects immunocompromised patients. Its prevention continues to pose a challenge. A 70-KDa glycoprotein (Gp70) of Sporothrix has been previously reported to protect host against infection from this fungus. Here, we displayed an epitope peptide (kpvqhalltplgldr) of Gp70 on the major coat protein (pIII), and investigated its efficiency as a vaccine for preventing S. globosa infection. The recombinant phage and the heat-killed S. globosa were used to immunize mice separately. In this study, we evaluated the humoral and cellular immune responses in the mice and demonstrated that recombinant phage could induce mice to produce a stronger immune response and generate antibodies to inhibit S. globosa infection. Furthermore, immunization with recombinant phage could increase the survival rate of S. globosa infection in mice. All these results together indicated that recombinant phages displaying kpvqhalltplgldr are a potential vaccine candidate against S. globosa infection.

Influence of IgG Subclass on Human Antimannan Antibody-Mediated Resistance to Hematogenously Disseminated Candidiasis in Mice.

Candida albicans is a yeast-like pathogen and can cause life-threatening systemic candidiasis. Its cell surface is enriched with mannan that is resistant to complement activation. Previously, we developed the recombinant human IgG1 antimannan antibody M1g1. M1g1 was found to promote complement activation and phagocytosis and protect mice from systemic candidiasis. Here, we evaluate the influence of IgG subclass on antimannan antibody-mediated protection. Three IgG subclass variants of M1g1 were constructed: M1g2, M1g3, and M1g4. The IgG subclass identity for each variant was confirmed with DNA sequence and subclass-specific antibodies. These variants contain identical M1 Fabs and exhibited similar binding affinities for C. albicans yeast and purified mannan. Yeast cells and hyphae recovered from the kidney of antibody-treated mice with systemic candidiasis showed uniform binding of each variant, indicating constitutive expression of the M1 epitope and antibody opsonization in the kidney. All variants promoted deposition of both murine and human C3 onto the yeast cell surface, with M1g4 showing delayed activation, as determined by flow cytometry and immunofluorescence microscopy. M1g4-mediated complement activation was found to be associated with its M1 Fab that activates the alternative pathway in an Fc-independent manner. Treatment with each subclass variant extended the survival of mice with systemic candidiasis (P < 0.001). However, treatment with M1g1, M1g3, or M1g4, but not with M1g2, also reduced the kidney fungal burden (P < 0.001). Thus, the role of human antimannan antibody in host resistance to systemic candidiasis is influenced by its IgG subclass.

Active immunizations with peptide-DC vaccines and passive transfer with antibodies protect neutropenic mice against disseminated candidiasis.

We previously report that peptide-pulsed dendritic cell (DC) vaccination, which targeting two peptides (Fba and Met6) expressed on the cell surface of Candida albicans, can induce high degree of protection against disseminated candidiasis in immunocompetent mice. Passive transfer of immune sera from the peptide immunized mice or peptide-related monoclonal antibodies demonstrated that protection was medicated by peptide-specific antibodies. In this study the efficacy of active and passive immunization against disseminated candidiasis was tested in mice with cyclophosphamide-induced neutropenia. Peptide-DC vaccines were given to mice prior to induction of neutropenia. We show active immunization with either Fba or Met6 peptide-DC vaccine significantly improved the survival and reduced the fungal burden of disseminated candidiasis in those immunocompromised mice. Importantly, we show that administration of two protective monoclonal antibodies also protect neutropenic mice against the disease, implying possibility of developing a successful passive immunotherapy strategy to treat the disease and protect against disseminated candidiasis. The results of this study are crucial as they address the fundamental questions as to whether the synthetic peptide vaccine induced immunity protects the host during a neutropenic episode. We anticipate that this peptide-vaccine study will serve as the foundation of future investigations into new peptide vaccines comprised of cell surface peptides from other medically important Candida species, as well as other fungi.

Therapeutic vaccine using a monoclonal antibody against a 70-kDa glycoprotein in mice infected with highly virulent Sporothrix schenckii and Sporothrix brasiliensis.

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungi that comprise the Sporothrix complex. The latter are widely distributed in nature, developing a saprophytic mycelial form on plant debris and soil. Formerly, the S. schenckii species was thought to be the only species capable of causing sporotrichosis. However, in recent years, the existence of a group of highly genotypically and phenotypically variable species has been reported as etiologic agents of this mycosis. Recently, it has become important to study aspects such as virulence and the immune response against key members of the Sporothrix complex and to observe the presence of glycoprotein (gp) 70 and efficacy of the P6E7 monoclonal antibody against more virulent strains. The data presented here demonstrate that the strain isolated from a case of feline sporotrichosis, that is, strain 5110 (American Type Culture Collection MYA-4823) is the most virulent and the only one able to secrete gp70. This glycoprotein is apparently an important factor in the virulence of Sporothrix spp. because treatment with MAb P6E7 resulted in the reduction of fungal burden in the analyzed organs. Additional studies of the role of gp70 in modulating the immune response of the host are needed to understand the pathology of sporotrichosis.

Candida orthopsilosis fungemias in a Spanish tertiary care hospital: Incidence, epidemiology and antifungal susceptibility / Fungemias por Candida orthopsilosis en un hospital terciario español: incidencia, epidemiología y sensibilidad a antifúngicos

Background. Few studies exist on prevalence of fungemia by Candida orthopsilosis, with variable results. Aims. To study the incidence, epidemiology and antifungal susceptibility of C. orthopsilosis strains isolated from fungemias over two years at a tertiary hospital. Methods. Candidemia episodes between June 2007 and June 2009 in a university hospital (Puerta del Mar, Cádiz, Spain) were studied. The strains initially identified as Candida parapsilosis were genotypically screened for C. parapsilosis sensu stricto, C. orthopsilosis and Candida metapsilosis, and their antifungal susceptibility was evaluated. Results. In this period 52 cases of candidemia were documented. Of the 19 strains originally identified as C. parapsilosis, 13 were confirmed as C. parapsilosis sensu stricto and 6 as C. orthopsilosis. Of the 52 isolates, the most frequent species were Candida albicans (30.8%), C. parapsilosis sensu stricto (25%), C. orthopsilosis, Candida tropicalis and Candida glabrata in equal numbers (11.5%). C. orthopsilosis isolates were susceptible to amphotericin B, caspofungin, voriconazole and fluconazole, with no significant differences in MIC values with C. parapsilosis sensu stricto. The source of isolates of C. orthopsilosis were neonates (50%) and surgery (50%), and 100% were receiving parenteral nutrition; however C. parapsilosis sensu stricto was recovered primarily from patients over 50 years (69.2%) and 46.1% were receiving parenteral nutrition. Conclusions. These findings show that C. orthopsilosis should be considered as human pathogenic yeast and therefore its accurate identification is important. Despite our small sample size our study suggests that a displacement of some epidemiological characteristics previously attributed to C. parapsilosis to C. orthopsilosis may be possible (AU)

Antecedentes. Apenas se han publicado estudios sobre la prevalencia de los episodios de fungemia por Candida orthopsilosis, y sus resultados han sido variables. Objetivos. Examinar la incidencia, epidemiología y sensibilidad a antifúngicos de las cepas de C. orthopsilosis aisladas de fungemias en un periodo de 2 años en un hospital de asistencia terciaria. Métodos. Entre junio de 2007 y junio de 2009, en el Hospital Universitario Puerta del Mar (Cádiz, España) se estudiaron todos los episodios de fungemia. Las cepas identificadas inicialmente como Candida parapsilosis se genotipificaron para su clasificación como C. parapsilosis sensu stricto, C. orthopsilosis y Candida metapsilosis, y se testó su sensibilidad a los antifúngicos. Resultados. Durante este periodo, se documentaron 52 episodios de fungemia. De las 19 cepas identificadas originalmente como C. parapsilosis, 13 fueron C. parapsilosis sensu stricto, y 6 C. orthopsilosis. De los 52 aislamientos, las especies más frecuentes fueron Candida albicans (30,8%), C. parapsilosis sensu stricto (25%) y C. orthopsilosis (11,5%), y Candida tropicalis y Candida glabrata fueron aisladas en igual número. Todos los aislamientos de C. orthopsilosis fueron sensibles a anfotericina B, caspofungina, voriconazol y fluconazol, sin diferencias significativas en las concentraciones inhibitorias mínimas obtenidas con C. parapsilosis sensu stricto. Los aislamientos de C. orthopsilosis procedían de recién nacidos (50%) y de pacientes sometidos a cirugía (50%). El 100% de los pacientes recibía nutrición parenteral; sin embargo, el foco de C. parapsilosis sensu stricto procedía, ante todo, de pacientes de más de 50 años de edad (69,2%), y el 46,1% recibía nutrición parenteral. Conclusiones. Los resultados del presente estudio revelan que C. orthopsilosis debe considerarse una levadura patogénica para el ser humano y, por esta razón, es importante su identificación. A pesar del pequeño tamaño de la muestra, el presente estudio evidencia el desplazamiento a C. orthopsilosis de algunas características epidemiológicas atribuidas previamente a C. parapsilosis (AU)

Breaking the mould - novel diagnostic and therapeutic strategies for invasive pulmonary aspergillosis in the immune deficient patient.

Invasive pulmonary aspergillosis (IPA) caused by the ubiquitous environmental fungus Aspergillus is a frequently fatal lung disease of immunocompromised humans accounting for more than 200,000 infections each year, with an associated mortality rate of 30-90%. This review addresses the current status of IPA diagnosis and treatment and the urgent need to develop accurate, non-invasive strategies for identifying pulmonary infections in the ever-expanding population of immune deficient patients at risk of acquiring opportunistic fungal infections including hematological malignancy and hematopoetic stem cell transplant patients. Recent advances in the use of an Aspergillus-specific monoclonal antibody, JF5, for point-of-care diagnosis of IPA using lateral-flow technology is examined, as is its use in PET/MRI bioimaging and radio-immunotherapy using radionuclide-labeled single chain antibody fragments, Fab fragments, and a fully humanized JF5 derivative.

Radiolabeled Antibodies for Therapy of Infectious Diseases.

Novel approaches to the treatment of infectious diseases are urgently needed. This need has resulted in renewing the interest in antibodies for therapy of infectious diseases. Radioimmunotherapy (RIT) is a cancer treatment modality that utilizes radiolabeled monoclonal antibodies. During the last decade we have translated RIT into the field of experimental fungal, bacterial, and HIV infections. In addition, successful proof of principle experiments with radiolabeled pan-antibodies that bind to antigens shared by major pathogenic fungi have been performed in vitro. The armamentarium of pan-antibodies would result in reducing our dependence on microorganism-specific antibodies and thus would speed up the development of RIT for infections. We believe that the time is ripe for deploying RIT in the clinic to combat infectious diseases.

The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance

Antecedentes: Durante a˜nos, el fluconazol se ha utilizado para tratar las infecciones por Candida. Sin embargo, el uso indiscriminado de este antimicótico ha favorecido la aparición de cepas resistentes. Se han descrito mutaciones en el gen ERG11 como uno de los principales mecanismos de resistencia en especies de Candida. Objetivos En el presente estudio se investigaron las mutaciones de sentido erróneo en genes ERG11 de aislamientos de Candida albicans, glabrata y tropicalis previamente examinados mediante pruebas de sensibilidad a fluconazol. Métodos: La detección de las mutaciones de este gen se realizó en 19 aislamientos clínicos de Candida (8 C. albicans, 5 C. glabrata y 6 C. tropicalis) sensibles y resistentes a fluconazol. El gen se amplificó mediante reacción en cadena de la polimerasa (PCR) con cebadores específicos para cada especie de Candida y se analizaron mediante secuenciación automatizada. Resultados: Se identificaron 14 mutaciones de sentido erróneo diferentes, 5 de las cuales no habían sido descritas previamente. Entre ellas, se identificó una nueva mutación L321F en un aislamiento de C. albicans resistente a fluconazol y que fue analizada mediante una estructura tridimensional teórica de ERG11p. Conclusión: La mutación L321F del gen ERG11 de C. albicans puede asociarse a resistencia a fluconazol (AU)

Background. For many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species. Aims. In this study we investigated missense mutations in ERG11 genes of Candida albicans, Candida glabrata and Candida tropicalis isolates previously evaluated by susceptibility testing to fluconazole. Methods. Screening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing. Results. We identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p. Conclusion. The L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance (AU)

Actividad antifúngica sinérgica de combinaciones de estatinas y azólicos revelada mediante bioanálisis con Saccharomyces cerevisiae y Candida utilis y cuantificación de ergosterol / Synergistic antifungal activity of statin–azole associations as witnessed by Saccharomyces cerevisiae- and Candida utilis-bioassays and ergosterol quantification

Fundamento. La frecuencia de micosis oportunistas y la resistencia a los antimicóticos convencionales han fomentado la búsqueda de nuevas alternativas terapéuticas, como las combinaciones de antimicóticos. Objetivos. El presente estudio trató de detectar el sinergismo antifúngico entre las estatinas y los azólicos mediante un bioanálisis de difusión en pocillos de agar, utilizando Saccharomyces cerevisiae (S. cerevisiae) ATCC 32051 y Candida utilis (C. utilis) PR1-2 como cepas de control. Métodos. Los efectos antifúngicos sinérgicos se examinaron mediante la adición simultánea de una concentración sub-inhibitoria (CSI) de estatina (atorvastatina, lovastatina, pravastatina, rosuvastatina o simvastatina) más una concentración mínima inhibitoria (CMI) de un azólico (clotrimazol, fluconazol, itraconazol, ketoconazol o miconazol) a placas de agar YNB con las levaduras sembradas por inclusión. Un resultado positivo correspondió a un diámetro del halo de inhibición del crecimiento de la levadura mayor que el producido por la CMI del azólico exclusivo. Para confirmar el sinergismo estatina-azólico, se cuantificó el ergosterol de la membrana celular de las levaduras con cromatografía líquida de alto rendimiento (HPLC-RP). Para valorar la inhibición de la síntesis de ergosterol inducida por estatinas, se emplearon bioanálisis de rescate de ergosterol. Resultados. La inhibición del crecimiento aumentó significativamente cuando se combinaron clotrimazol, fluconazol, itraconazol, ketoconazol y miconazol con atorvastatina, lovastatina, rosuvastatina y simvastatina. Los mayores incrementos de la inhibición del crecimiento se observaron en S. cerevisiae (77,5%) y C. utilis (43,2%) con una CSI de simvastatina y una CMI de miconazol de 4+2,4mg/ml o 20+4,8mg/ml, respectivamente. Para pravastatina apenas se identificaron efectos significativos (incremento de la inhibición del 0-7,6%). Los mayores cocientes de interacción correspondieron a la combinación de simvastatina y miconazol y fueron indicativos de sinergismo. Este también se confirmó por la mayor disminución de los niveles celulares de ergosterol (S. cerevisiae, 40% y C. utilis, 22%). La inhibición de la síntesis de ergosterol inducida por estatinas se corroboró mediante bioanálisis de rescate de ergosterol, donde la inhibición por pravastatina se abolió con facilidad, mientras que la de rosuvastatina fue la más refractaria. Conclusiones. Las combinaciones seleccionadas de estatinas y azólicos podrían ser alternativas viables para el manejo terapéutico de las micosis, en dosis más bajas o con una mayor eficiencia(AU)

Background. Frequent opportunist fungal infections and the resistance to available antifungal drugs promoted the development of new alternatives for treatment, like antifungal drug combinations. Aims. This work aimed to detect the antifungal synergism between statins and azoles by means of an agar-well diffusion bioassay with Saccharomyces cerevisiae ATCC 32051 and Candida utilis Pr1–2 as test strains. Methods. Synergistic antifungal effects were tested by simultaneously adding a sub inhibitory concentration (SIC) of statin (atorvastatin, lovastatin, pravastatin, rosuvastatin or simvastatin) plus a minimal inhibitory concentration (MIC) of azole (clotrimazole, fluconazole, itraconazole, ketoconazole or miconazole) to yeast-embedded YNB agar plates, and a positive result corresponded to a yeast growth inhibition halo higher than that produced by the MIC of the azole alone. Yeast cell ergosterol quantification by RP-HPLC was used to confirm statin–azole synergism, and ergosterol rescue bioassays were performed for evaluating statin-induced ergosterol synthesis blockage. Results. Growth inhibition was significantly increased when clotrimazole, fluconazole, itraconazole, ketoconazole and miconazole were combined with atorvastatin, lovastatin, rosuvastatin and simvastatin. Highest growth inhibition increments were observed on S. cerevisiae (77.5%) and C. utilis (43.2%) with a SIC of simvastatin plus a MIC of miconazole, i.e. 4+2.4mg/ml or 20+4.8mg/ml, respectively. Pravastatin showed almost no significant effects (0–7.6% inhibition increase). Highest interaction ratios between antifungal agents corresponded to simvastatin–miconazole combinations and were indicative of synergism. Synergism was also confirmed by the increased reduction in cellular ergosterol levels (S. cerevisiae, 40% and C. utilis, 22%). Statin-induced ergosterol synthesis blockage was corroborated by means of ergosterol rescue bioassays, pravastatin being the most easily abolished inhibition whilst rosuvastatin being the most ergosterol-refractory. Conclusions. Selected statin–azole combinations might be viable alternatives for the therapeutic management of mycosis at lower administration doses or with a higher efficiency(AU)

Impact of liposomal amphotericin B on renal function in critically ill patients with renal function impairment

Objetivo: Evaluar la tolerabilidad de anfotericina B liposomal (L-AmB) en pacientes críticos con concentraciones elevadas de creatinina sérica (Cr) (> 1,5 mg/dL) al inicio del tratamiento con L-AmB. Métodos: Estudio retrospectivo, multicéntrico y comparativo de dos cohortes de pacientes críticos tratados con L-AmB durante tres o más días, que se diferenciaban según el nivel de creatinina al inicio del tratamiento. Se estableció como punto de corte un valor de Cr de 1,5 mg/dL. Se excluyeron los pacientes con técnicas de depuración extrarrenal antes o 48 horas después del inicio de L-AmB. La variable principal fue la diferencia entre el valor de creatinina al final comparado con el inicio de tratamiento con L-AmB. Otros parámetros secundarios fueron: abandonos relacionados con el tratamiento, necesidad de técnicas de depuración extrarrenal (TDE) y acontecimientos adversos graves (AAG) relacionados con el tratamiento. Se recogieron datos demográficos, enfermedad subyacente, motivo de prescripción, factores concomitantes de riesgo de nefrotoxicidad y estado vital al alta de UCI y del hospital. Resultados: Se reclutaron 122 pacientes en 26 UCI (Cr > 1,5 g/dL, n= 16; Cr normal, n= 106). Los motivos principales por los que se indicó L-AmB en ambos grupos fueron el amplio espectro y la presencia de inestabilidad hemodinámica. Se administró como tratamiento de 1ª línea en el 68,8% de los pacientes con Cr elevada y en el 52,8% con Cr normal. La puntuación APACHE II al ingreso en UCI fue 25 en pacientes con Cr elevada y 17 en aquellos con Cr normal (p< 0,001). La duración del tratamiento con L-AmB fue 16 y 12 días en pacientes con Cr elevada y normal y una dosis media de 3,5 vs 3,9 mg/kg/día. El uso concomitante de otros fármacos nefrotóxicos, la tasa de mortalidad, y la estancia en UCI y hospitalaria fueron similares en ambas cohortes. En los pacientes con función renal alterada al inicio de L-AmB se observó una reducción absoluta de Cf-Ci de 1,08 mg/dl (P<0,001). La Cr bajó a valores normales en el 50% de los pacientes, descendió pero sin llegar a valores normales en el 37,5% y sólo se elevó en 1 (6,25%) paciente. En ningún paciente se suspendió L-AmB por nefrotoxicidad ni se precisaron técnicas de depuración extrarrenal. No se reportaron AAG relacionados con el tratamiento. Conclusiones: El tratamiento con L-AmB en pacientes críticos con función renal deteriorada tuvo un impacto mínimo en la función renal. L-AmB puede utilizarse para el tratamiento de infecciones fúngicas en pacientes críticos independientemente de la función renal al inicio del tratamiento(AU)

Objetive: To assess the tolerability of liposomal amphotericin B (L-AmB) in critically ill patients with elevated serum creatinine concentrations (Cr) (> 1.5 mg/dL) at starting L-AmB therapy. Methods: Retrospective, multicenter, comparative study of two cohorts of critically ill patients treated with L-AmB during 3 or more days, the difference between them was the level of Cr at the beginning of treatment. A cutoff value of Cr of 1.5 mg/dL was established. Patients undergoing extrarenal depuration procedures before or 48 hours after starting L-AmB were excluded. The primary endpoint was the difference between Cr values at the end of treatment as compared with Cr at starting L-AmB. Secondary endpoints were treatment-related withdrawals, need of extrarenal depuration techniques, and treatment-related severe adverse events. Demographic data, underlying illness, indication of L-AmB therapy, concomitant risk factors of nephrotoxicity, and vital status at ICU and hospital discharge were recorded. Results: A total of 122 patients admitted to 26 ICUs (16 with Cr > 1.5 g/dL; 106 with normal Cr levels) were recruited. Main reasons for the use of L-AmB in both groups were the broad spectrum of the drug and the presence of hemodynamic instability. L-AmB was administered as first-line treatment in 68.8% of patients with elevated Cr and in 52.8% with normal Cr. The APACHE II score on ICU admission was 25 in patients with elevated Cr and 17 in those with normal Cr values (p < 0.001). Duration of treatment with L-AmB was 16 and 12 days in patients with elevate and normal Cr values, respectively, with a mean dose of 3.5 vs 3.9 mg/kg/day. The use of concomitant nephrotoxic drugs, mortality rate, and ICU and hospital length of stay were similar in both cohorts. In patients with renal function impairment at the initiation of L-AmB treatment, an absolute decrease of Cf-Ci of 1.08 mg/dL was observed (P < 0.001). A decrease of Cr levels to normal limits was observed in 50% of the patients; in 37.5% of patients there was a decrease but normal levels were not achieved, whereas a Cr increased occurred in only one (6.25%) patient. None of the patients required withdrawal of L-AmB or use of extrarenal depuration procedures. Treatment-related severe adverse events were not reported. Conclusions: In critically ill patients with impaired renal function, the impact of L-AmB on renal function was minimal. L-AmB can be used for the treatment of fungal infections in critically ill patients independently of renal function at the initiation of treatmen(AU)

Toward developing a universal treatment for fungal disease using radioimmunotherapy targeting common fungal antigens.

BACKGROUND: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall. METHODS: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls. RESULTS: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing. CONCLUSION: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Plant production of anti-ß-glucan antibodies for immunotherapy of fungal infections in humans.

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting ß-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the ß-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially ß1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

Radioimmunotherapy is more effective than antifungal treatment in experimental cryptococcal infection.

Radioimmunotherapy (RIT) prolongs the survival of mice infected with Cryptococcus neoformans. To compare the efficacy of RIT with that of amphotericin B, we infected AJ/Cr mice intravenously with either nonmelanized or melanized C. neoformans cells. Infected mice were either left untreated or treated 24 h after infection with (213)Bi-18B7 antibody, amphotericin B, or both. Melanization before infection did not increase resistance of C. neoformans to RIT in vivo. (213)Bi-18B7 treatment almost completely eliminated colony-forming units from the lung and brain, whereas amphotericin B did not decrease the number of colony-forming units. We conclude that RIT is more effective than amphotericin B against systemic infection with C. neoformans.

Potential of anti-Candida antibodies in immunoprophylaxis.

The need for new options for the treatment of invasive candidiasis has fuelled the use of antibodies in combination with conventional antifungal therapy. After a long period of time in which antibodies were considered irrelevant in the resistance against invasive candidiasis, it was demonstrated that a number of antibodies or their engineered derivatives directed against Candida albicans cell-wall polysaccharides and glycopeptides, as well as against some protein epitopes, confer protection against invasive candidiasis. This has confirmed this approach as a new strategy for the prophylaxis of invasive candidiasis. Of particular interest is Mycograb, a human recombinant monoclonal antibody that inhibits heat shock protein 90, and has been administrated in combination with lipid-associated amphotericin B to patients with invasive candidiasis, and the fungicidal anti-beta-glucan antibodies induced by the glycoconjugate vaccine composed of a beta-glucan polysaccharide conjugated with the diphtheria toxoid CRM 197. However, despite the promising data obtained in vitro and in animal models, at present there is very little clinical experience on the use of antibodies in Candida immunoprophylaxis.

Antifungal immunotherapy and immunomodulation: a double-hitter approach to deal with invasive fungal infections.

In recent years, the incidence of life-threatening fungal infections has dramatically increased. Despite significant developments in antifungal chemotherapy, its efficacy remains limited by the inability to sterilize infected organs and the tendency to induce resistance and cause side effects. In response to these challenges, it is now recognized that several aspects of antifungal immunity can be modulated to better deal with fungal infections. Extensive work was carried out on the development and testing of preventive and therapeutic antifungal vaccines. The potential use of cytokines, adoptive T-cell transfer, monoclonal antibodies (MoAb) and antimicrobial peptides (AMP) as solo or adjunctive therapies is also receiving much attention. Although each of these immune-based treatment strategies has many advantages and some shortcomings, none on its own, has proven satisfactorily effective to deal with invasive fungal infections. Appropriate combinations that optimize the advantages and minimize the disadvantages of immune-based antifungals are still lacking mainly due to the immense difficulty in sorting out candidate combinations given the long list of choices. In this review, immune-based antifungals are divided into two general categories on the basis of the intended target being the host (immunomodulation through vaccines, cytokines, adoptive T-cell transfer or MoAb) or the pathogen (immunotherapy through MoAb or AMP). Potential advantages and disadvantages of immunotherapy and immunomodulation are tentatively discussed so as to facilitate the design of future studies that aim at devising more potent immune-based antifungal treatment combinations.

Fungal vaccines: real progress from real challenges.

Among viral, bacterial, and fungal diseases, the latter are the only branch of infectious diseases without a vaccine for any of their causative agents. This is at odds with a disease burden that remains unabated by conventional chemotherapy and infection control measures. Since most fungal infections occur in immunocompromised patients, the generation of tools relying on host immunity for effectiveness is a notable challenge. Nevertheless, with improved knowledge of the host-fungus relation, and the spectacular advances in genome sequencing, genetic engineering, and proteomics, strong progress in fungal vaccine research is being made. Some vaccines induce the generation of directly fungicidal antibodies; others are protective in animals carrying major risk factors for fungal infections, such as CD4+ T-cell-deficiency or neutropenia. Together with the demonstrated efficacy of various antibodies in passive vaccination approaches, there is growing confidence in the future availability of safe and efficacious immunological tools to combat deadly microbes in a weak host.

An anti-beta-glucan monoclonal antibody inhibits growth and capsule formation of Cryptococcus neoformans in vitro and exerts therapeutic, anticryptococcal activity in vivo.

In this study we tested the in vitro and in vivo anti-Cryptococcus neoformans activity of an antilaminarin (anti-beta-glucan) monoclonal antibody (MAb 2G8) (immunoglobulin G2b) which was previously shown to inhibit the growth of beta-glucan-exposing Candida albicans cells. Here we show that MAb 2G8 binds to the cell wall of C. neoformans and inhibits its growth to an extent comparable to that observed for C. albicans. Binding and growth inhibition were detected almost equally for encapsulated and acapsular C. neoformans strains. In addition, at subinhibitory concentrations, MAb 2G8 reduced the capsule thickness without affecting protease or phospholipase production. Acapsular fungal cells, but not encapsulated fungal cells, were opsonized by the antibody and more efficiently phagocytosed and killed by human monocytes and by murine peritoneal macrophages. A single administration of MAb 2G8 resulted in a reduction in the fungal burden in the brains and livers of mice systemically infected with a highly virulent, encapsulated C. neoformans strain. This protective effect was also detected in neutropenic mice. Overall, these findings demonstrate that cell wall beta-glucan of encapsulated C. neoformans is accessible to antibodies which can exert remarkable anticryptococcal activities in vitro and in vivo.

Immunomodulators as an antimicrobial tool.

The spectrum of infectious diseases has shifted in the past 50 years to include those caused by microbes that cause disease predominantly in immunocompromised individuals. This phenomenon has underscored the dependence of microbial virulence on the immune status of the host. The limited efficacy of the available antimicrobial armamentarium in immunocompromised individuals, combined with increasing resistance to these agents, has led to an urgent need for new therapies for infectious diseases. Immunomodulation represents a novel approach to antimicrobial therapy that depends on bolstering host immunity, rather than direct antimicrobial activity. Immunomodulators can be divided into those that are specific to pathogens (pathogen-specific) and those that are not specific to pathogens (non-specific). However, to date only a few immunomodulators have been evaluated for their efficacy as antimicrobial tools.