galactomannan and Disease-Models--Animal

Invasive aspergillosis is a serious and lethal infection among immunocompromised patients, with reported mortality rates as high as 74-92%. The high mortality is related to the severe immunosuppression experienced by these patients as well as the difficulties for physicians in arriving at a timely diagnosis. Definitive diagnostic procedures (tissue biopsy for histopathology and culture) are often precluded by severe cytopenias and coagulation abnormalities. The development of minimally invasive, nonculture diagnostic methods is a major advance in the early diagnosis of invasive aspergillosis. Galactomannan is a heteropolysaccharide (mannan core and side residues of galactofuranosyl units) present in the cell wall of Aspergillus spp. The double sandwich enzyme immunoassay, which detects galactomannan in serum samples, has been available in Europe for almost a decade and in the USA since May 2003, for the diagnosis of invasive aspergillosis. However, availability of the double galactomannan enzyme immunoassay is center variable in the USA and, although its analytical performance in the diagnosis of invasive aspergillosis is well documented, its routine use in clinical practice is limited. As an adjunct in the diagnosis and management of invasive aspergillosis, incorporation of the galactomannan enzyme immunoassay into clinical trials will help to further define its role.

Topics: Animals; Antigens, Fungal; Aspergillosis; Disease Models, Animal; Galactose; Humans; Immunohistochemistry; Mannans; Molecular Weight

Isavuconazole, a novel triazole antifungal agent, has broad-spectrum activity against Aspergillus spp. and other pathogenic fungi. The isavuconazole exposure-response relationship in experimental invasive pulmonary aspergillosis using galactomannan index (GMI) suppression as a marker of disease clearance was explored.. The impact of exposure on GMI suppression in persistently neutropenic rabbits treated with isavuconazonium sulphate (isavuconazole-equivalent dosages of 20, 40 or 60 mg/kg every 24 h, after a 90 mg/kg loading dose) for 12 days was linked using mathematical modelling. Bridging to humans using population pharmacokinetic (PK) data from a clinical trial in invasive aspergillosis was performed using Monte Carlo simulations.. Mean plasma isavuconazole AUC/MIC (EC50) of 79.65 (95% CI 32.2, 127.1) produced a half-maximal effect in GMI suppression. The inhibitory sigmoid Emax curve dropped sharply after an AUC/MIC of ≥30 and was near maximum (EC80) at ∼130. Bridging the experimental PK/pharmacodynamic (PD) target to human population PK data was then used to return to the rabbit model to determine a clinically relevant PD endpoint. The clinical dosing regimen used in the trial would result in a mean GMI of 4.3 ± 1.8, which is a 50% reduction from the starting GMI in the experiment.. The clinical trial results showing the non-inferiority of isavuconazole to voriconazole for all-cause mortality further support the PK-PD endpoint, thereby demonstrating the usefulness of the rabbit model and endpoint for isavuconazole and implications on interpretive breakpoints. Importantly, the analysis supports this model as an important tool for development of antifungal agents.

Topics: Animals; Antifungal Agents; Aspergillus; Disease Models, Animal; Drug Monitoring; Female; Galactose; Humans; Invasive Pulmonary Aspergillosis; Mannans; Microbial Sensitivity Tests; Models, Theoretical; Monte Carlo Method; Nitriles; Pyridines; Rabbits; Triazoles

We observed false-positive results in the Platelia Aspergillus enzyme-linked immunoassay (EIA) for specimens from patients with histoplasmosis and mice with experimental infection. Platelia Aspergillus EIA-positive specimens were negative in the second-generation Histoplasma antigen EIA. Care must be taken to exclude histoplasmosis for patients with positive Platelia Aspergillus EIA results.

Topics: Animals; Antibodies, Fungal; Antigens, Fungal; Aspergillosis, Allergic Bronchopulmonary; Cross Reactions; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Galactose; Histoplasmosis; Humans; Mannans; Mice; Rabbits

Topics: Analgesics; Animals; Disease Models, Animal; Fabaceae; Foot Joints; Galactose; Iodoacetic Acid; Male; Mannans; Nociception; Osteoarthritis; Pain; Rats, Wistar; Seeds

Guar gum-derived galactomannan (GGGM) has been widely used in the food industry for a long time and its adverse impacts have been scarcely reported. Galactomannan is considered to have a structure similar to the surface components of certain pathogens, and the present study was thus conducted to investigate if oral administration of GGGM could cause physiological effects that were hypothesized to be related to intestinal inflammatory responses. The results showed that oral administration of GGGM resulted in compromises on growth performance, an increase of the relative weight of spleen and epididymal fat, and an elevation of the α1-acid glycoprotein content in both serum and livers of mice. With regard to energy metabolism-related indices, the activities of intestinal lactic dehydrogenase and succinic dehydrogenase were all increased by the GGGM treatment in both in vivo and in vitro experiments, the latter of which also showed an elevation in the consumption of reducing sugar by intestinal epithelial cells along with a reduced viability of these cells in response to the GGGM treatment. Notably, the GGGM treatment triggered intestinal inflammatory responses that were evidenced by the increased expression of intestinal inflammatory cytokines such as TNF-α and IL-6 both in vivo and in vitro, which were at least partially responsible for the increased energy expenditure in the intestine and the retardation of growth. The results of this study could expand our knowledge of GGGM administration and provide integrated insights into the consumption of GGGM-containing foods.

Topics: Animals; Disease Models, Animal; Energy Metabolism; Galactans; Galactose; Inflammation; Intestines; Male; Mannans; Mice; Plant Gums

Galactomannan (GAL), a polysaccharide present on the cell wall of several fungi, has shown an ability to modulate inflammatory responses through the dectin-1 receptor in human macrophages. However, studies evaluating the modulatory properties of this polysaccharide in in vivo inflammatory scenarios are scarce. We hypothesized that GAL pretreatment would modulate local and remote damage related to intestinal reperfusion after an ischemic insult.. Adult male Balb/c mice were subjected to intestinal ischemia-reperfusion injury by reversible occlusion of the superior mesenteric artery, consisting of 45 min of ischemia followed by 3 or 24 h of reperfusion. Intragastric GAL (70 mg/kg) was administered 12 h before ischemia, and saline solution was used in the control animals. Jejunum, lung, and blood samples were taken for the analysis of histology, gene expression, plasma cytokine levels, and nitrosative stress.. Intestinal and lung histologic alterations were attenuated by GAL pretreatment, showing significant differences compared with nontreated animals. Interleukin 1β, monocyte chemoattractant protein 1, and IL-6 messenger RNA expression were considerably downregulated in the small intestine of the GAL group. In addition, GAL treatment significantly prevented plasma interleukin 6 and monocyte chemoattractant protein 1 upregulation and diminished nitrate and nitrite levels after 3 h of intestinal reperfusion.. GAL pretreatment constitutes a novel and promising therapy to reduce local and remote damage triggered by intestinal ischemia-reperfusion injury. Further in vivo and in vitro studies to understand GAL's modulatory effects are warranted.

Topics: Animals; Disease Models, Animal; Galactose; Humans; Intestinal Mucosa; Ischemia; Jejunum; Male; Mannans; Mice; Reperfusion Injury

Invasive pulmonary aspergillosis is a life-threatening fungal disease principally caused by the ubiquitous mould Aspergillus fumigatus. This clinical entity is a major cause of morbidity and mortality (principally, but not restricted to, immunocompromised individuals). A few recent reports suggest in vitro fungicidal activity of sertraline against Aspergillus spp., but this activity has not yet been investigated in vivo.. To evaluate the antifungal activity of sertraline in two in vivo models of aspergillosis.. The antifungal activity of sertraline as monotherapy at three different doses (3, 10 and 15 mg/kg) was evaluated in Galleria mellonella and in a murine model of invasive pulmonary aspergillosis. Therapeutic efficacy parameters determined were larval survival and health index score for G. mellonella, whereas pulmonary fungal burden, galactomannan and lung histopathology were assessed in the murine model.. Sertraline treatments improved larval survival and health index score, especially at doses of 10 and 15 mg/kg. Moreover, 10 mg/kg sertraline was able to reduce pulmonary fungal burden with an efficacy comparable with that of 3 mg/kg amphotericin B and 10 mg/kg voriconazole.. To the best of our knowledge, this is the first in vivo study that evaluates the antifungal activity of sertraline against A. fumigatus, showing a possible promising option for the adjuvant treatment of pulmonary aspergillosis.

Topics: Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Colony Count, Microbial; Disease Models, Animal; Galactose; Histocytochemistry; Lepidoptera; Lung; Male; Mannans; Mice, Inbred BALB C; Sertraline; Survival Analysis; Treatment Outcome

The growing emergence of azole-resistant Aspergillus fumigatus strains worldwide is a major concern for current systemic antifungal treatment. Here we report antifungal activities of a novel inhaled triazole, PC1244, against a collection of multi-azole-resistant A. fumigatus strains.. MICs of PC1244 were determined for A. fumigatus carrying TR34/L98H (n = 81), TR46/Y121F/T289A (n = 24), M220 (n = 6), G54 (n = 11), TR53 (n = 1), TR463/Y121F/T289A (n = 2), G448S (n = 1), G432C (n = 1) and P216S (n = 1) resistance alleles originating from either India, the Netherlands or France. The effects of PC1244 were confirmed in an in vitro model of the human alveolus and in vivo in temporarily neutropenic, immunocompromised mice.. PC1244 exhibited potent inhibition [geometric mean MIC (range), 1.0 mg/L (0.125 to >8 mg/L)] of growth of A. fumigatus strains carrying cyp51A gene mutations, showing much greater potency than voriconazole [15 mg/L (0.5 to >16 mg/L)], and an effect similar to those on other azole-susceptible Aspergillus spp. (Aspergillus flavus, Aspergillus terreus, Aspergillus tubingensis, Aspergillus nidulans, Aspergillus niger, Aspergillus nomius, Aspergillus tamarii) (0.18-1 mg/L). In TR34/L98H and TR46/Y121F/T289A A. fumigatus-infected in vitro human alveolus models, PC1244 achieved superior inhibition (IC50, 0.25 and 0.34 mg/L, respectively) compared with that of voriconazole (IC90, >3 mg/L and >10 mg/L, respectively). In vivo, once-daily intranasal administration of PC1244 (0.56-70 μg/mouse) to the A. fumigatus (AF91 with M220V)-infected mice reduced pulmonary fungal load and serum galactomannan more than intranasal posaconazole.. PC1244 has the potential to become a novel topical treatment of azole-resistant pulmonary aspergillosis.

Topics: Animals; Antifungal Agents; Aspergillus; Colony Count, Microbial; Disease Models, Animal; France; Galactose; Humans; India; Lung; Mannans; Mice; Microbial Sensitivity Tests; Netherlands; Pulmonary Aspergillosis; Treatment Outcome; Triazoles

Topics: Animals; Antibodies, Fungal; Antibodies, Monoclonal; Antigens, Fungal; Aspergillosis; Aspergillus; Aspergillus flavus; Aspergillus fumigatus; Cell Wall; Cross Reactions; Disease Models, Animal; Epitopes; Female; Galactose; Immunologic Tests; Mannans; Mice; Mice, Inbred BALB C; Polysaccharides, Bacterial; Recombinant Proteins

Galactomannan and citrus pectin are considered 'super fibers' known for altering gut microbiota composition and improving glucose and lipid metabolism. The study aims to investigate the fiber's effect on a nonalcoholic steatohepatitis (NASH) model.. Two feeding experiments are carried out using groups of 7-8 week-old male C57BL/6J mice. The diets used are based on a high cholesterol/cholate diet (HCD), such as a nutritional NASH model. Mice are fed a diet with or without 15% fiber-citrus pectin (HCD-CP) or galactomannan (HCD-G) together with the HCD (first experiment), which commenced 3 weeks prior to the HCD (second experiment). Liver damage is evaluated by histological and biochemical parameters. Galactomannan leads to lesser weight gain and improved glucose tolerance, but increased liver damage. This is shown by elevated levels of liver enzymes compared to that with HCD alone. Fibers induce higher steatosis, as evaluated by liver histology. This intriguing result is linked to various changes in the gut microbiota, such as elevated Proteobacteria levels in the galactomannan group, which are correlated with disturbed metabolism and dysbiosis.. In a NASH mouse model, galactomannan increases liver damage but improves glucose metabolism. Changes in the microbiota composition may answer this enigmatic observation.

Topics: Animals; Body Weight; Cholesterol; Diet, High-Fat; Dietary Fiber; Disease Models, Animal; Fatty Acids, Volatile; Galactose; Gastrointestinal Contents; Gastrointestinal Microbiome; Glucose Tolerance Test; Lipid Metabolism; Liver; Male; Mannans; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Pectins

An oral dosage form containing floating bioadhesive gastroretentive microspheres forms a stomach-specific drug delivery system for the treatment of Helicobacter pylori.. To prepare and evaluate controlled release floating bioadhesive gastroretentive chitosan-coated amoxicillin trihydrate-loaded Caesalpinia pulcherrima galactomannan (CPG)-alginate beads (CCA-CPG-A), for H. pylori eradication.. CCA-CPG-A beads were prepared by ionotropic gelation, using 2(3) factorial design with quantity of drug, combination of CPG with sodium alginate and concentration of calcium chloride as variables. Beads facilitated mucoadhesion to gastric mucosa with floating nature caused by chitosan coating for wide distribution throughout GIT. Developed beads were evaluated for characteristics like beads size-morphology, entrapment efficiency, DSC, XRD, FTIR, swelling ratio, in vitro mucoadhesion, in vitro drug release, in vitro floating and in vitro H. pylori growth inhibition studies. CCA-CPG-A beads were studied in Wistar rats for in vivo gastric mucoadhesion, in vivo H. pylori growth inhibition studies using PCR amplification of isolated DNA, rapid urease test.. Developed beads possess drug release of 79-92%, entrapment efficiency of 65-89%, mucoadhesion of 61-89%. In vivo mucoadhesion study showed more than 85% mucoadhesion of beads even after 7th hour. In vitro-in vivo growth inhibition study showed complete eradication of H. pylori.. CPG-alginate and chitosan in beads interacts with gastric mucosubstrate surface for prolonged gastric residence with floating bioadhesion mechanism for H. pylori eradication in rats.. Floating bioadhesive CCA-CPG-A beads offer a promising drug delivery system for H. pylori eradication at lower dose, reduced adverse effect and enhance bioavailability.

Topics: Adhesiveness; Administration, Oral; Alginates; Amoxicillin; Animals; Anti-Bacterial Agents; Biological Availability; Caesalpinia; Calorimetry, Differential Scanning; Chitosan; Crystallography, X-Ray; Delayed-Action Preparations; Disease Models, Animal; Dosage Forms; Drug Carriers; Drug Compounding; Female; Galactose; Gastric Mucosa; Glucuronic Acid; Helicobacter Infections; Helicobacter pylori; Hexuronic Acids; Kinetics; Male; Mannans; Models, Chemical; Particle Size; Powder Diffraction; Rats, Wistar; Solubility; Spectroscopy, Fourier Transform Infrared; Technology, Pharmaceutical

The EuropeanAspergillusPCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated "in vivo" specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and β-d-glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and β-d-glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and β-d-glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and β-d-glucan proved optimal (area under the curve [AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests.

Topics: Animals; beta-Glucans; Blood; Diagnostic Tests, Routine; Disease Models, Animal; Galactose; Guinea Pigs; Invasive Pulmonary Aspergillosis; Male; Mannans; Molecular Diagnostic Techniques; Polymerase Chain Reaction; Proteoglycans; Sensitivity and Specificity; Time Factors

Invasive aspergillosis is an emerging threat to public health due to the increasing use of immune suppressive drugs and the emergence of resistance against antifungal drugs. To deal with this threat, research on experimental disease models provides insight into the pathogenesis of infections caused by susceptible and resistant Aspergillus strains and by assessing their response to antifungal drugs. However, standard techniques used to evaluate infection in a preclinical setting are severely limited by their invasive character, thereby precluding evaluation of disease extent and therapy effects in the same animal. To enable non-invasive, longitudinal monitoring of invasive pulmonary aspergillosis in mice, we optimized computed tomography (CT) and magnetic resonance imaging (MRI) techniques for daily follow-up of neutropenic BALB/c mice intranasally infected with A. fumigatus spores. Based on the images, lung parameters (signal intensity, lung tissue volume and total lung volume) were quantified to obtain objective information on disease onset, progression and extent for each animal individually. Fungal lung lesions present in infected animals were successfully visualized and quantified by both CT and MRI. By using an advanced MR pulse sequence with ultrashort echo times, pathological changes within the infected lung became visually and quantitatively detectable at earlier disease stages, thereby providing valuable information on disease onset and progression with high sensitivity. In conclusion, these non-invasive imaging techniques prove to be valuable tools for the longitudinal evaluation of dynamic disease-related changes and differences in disease severity in individual animals that might be readily applied for rapid and cost-efficient drug screening in preclinical models in vivo.

Topics: Animals; Aspergillus fumigatus; Colony Count, Microbial; Disease Models, Animal; Disease Progression; Galactose; Invasive Pulmonary Aspergillosis; Longitudinal Studies; Lung; Magnetic Resonance Imaging; Male; Mannans; Mice; Mice, Inbred BALB C; Spores, Fungal; Tomography, X-Ray Computed

Galactomannan and (1→3)-β-D-glucan are useful biomarkers of invasive pulmonary aspergillosis (IPA). However, the effects of immunosuppression on levels of galactomannan or (1→3)-β-D-glucan in IPA are not well understood or quantified. We therefore studied the simultaneous levels of galactomannan and (1→3)-β-D-glucan in two rabbit models of experimental IPA: (1) AraC-induced neutropenia in untreated (UC-AraC) and liposomal amphotericin B-treated (LAMB-AraC) rabbits; and (2) nonneutropenic cyclosporine-methylprednisolone immunosuppression in untreated (UC-CsA+M) and LAMB-treated (LAMB-CsA+M) rabbits. Simultaneous levels of galactomannan and (1→3)-β-D-glucan were determined in bronchoalveolar lavage (BAL) fluid and serial serum specimens and correlated with pulmonary host response. Serum galactomannan index (GMI) and (1→3)-β-D-glucan concentration-time-curves were higher in UC-AraC vs. UC-CsA+M (Mann-Whitney U-test, P < .05). Serum galactomannan and (1→3)-β-D-glucan in treatment groups demonstrated therapeutic responses with similarly lower levels in comparison to UC (P < .01) in both models. Host differences did not affect BAL fluid GMI or (1→3)-β-D-glucan but did affect galactomannan and (1→3)-β-D-glucan levels in serum. The higher serum GMI and (1→3)-β-D-glucan concentration-time-curves in UC-AraC correlated with extensive pulmonary infiltration by angioinvasive hyphae and minimal inflammation, while the lower concentration-time-curves in UC-CsA+M were associated with shorter and fewer hyphae in lung tissue and an intensive neutrophil response to Aspergillus hyphae. Thus, serum levels of galactomannan and (1→3)-β-D-glucan in IPA depended upon immunosuppression, which also affected severity of infection and hyphal morphology, while BAL fluid galactomannan and (1→3)-β-D-glucan were sensitive biomarkers not affected by host response.

Topics: Animals; Antifungal Agents; beta-Glucans; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Female; Galactose; Host-Pathogen Interactions; Invasive Pulmonary Aspergillosis; Lung; Mannans; Proteoglycans; Rabbits

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus, Aspergillus flavus, or Aspergillus niger is associated with high mortality. We evaluated the efficacy and compared the therapeutic effect differences of voriconazole (VRC) in combination with caspofungin (CAS) in transiently neutropenic rats infected by A. fumigatus, A. flavus, or A. niger.. Treatment groups consisted of VRC (10 mg/kg q12 h) monotherapy, CAS (1 mg/kg/day) monotherapy, combination of VRC (10 mg/kg q12 h) + CAS (1 mg/kg/day), and no drug for 10 consecutive days. The efficacy and the difference in the treatments were evaluated through prolongation of survival, reduction in serum galactomannan levels and residual fungal burden, and histological studies.. For all the strains, the combination of VRC and CAS led to significant prolongation in survival (P < 0.05) and reduction in residual fungal burden (P < 0.05) compared with CAS alone, and decrease in serum galactomannan levels (P < 0.05) compared with either agent alone. The survival in the combined therapy groups was significantly improved compared to VRC monotherapy for the strains of A. flavus and A. niger (P < 0.05), but no significant difference for the strains of A. fumigatus (P > 0.05).. Combination of VRC and CAS was synergistic in IPA by A. flavus and A. niger, but small efficacy benefits in IPA by A. fumigatus.

Topics: Animals; Antifungal Agents; Aspergillus flavus; Aspergillus fumigatus; Aspergillus niger; Caspofungin; Disease Models, Animal; Drug Therapy, Combination; Echinocandins; Galactose; Humans; Lipopeptides; Male; Mannans; Microbial Sensitivity Tests; Neutropenia; Pulmonary Aspergillosis; Pyrimidines; Rats; Rats, Sprague-Dawley; Treatment Outcome; Triazoles; Voriconazole

Since there is no consensus about the most reliable assays to detect invasive aspergillosis from samples obtained by minimally invasive or noninvasive methods, we compared the efficacy of an enzyme-linked immunosorbent assay (ELISA) for galactomannan (GM) detection and quantitative real-time PCR assay (qRT-PCR) for the diagnosis of invasive pulmonary aspergillosis. Neutropenic, male Sprague-Dawley rats (specific pathogen free; 8 weeks old; weight, 200 ± 20 g) were immunosuppressed with cyclophosphamide and infected with Aspergillus fumigatus intratracheally. Tissue and whole blood samples were harvested on days 1, 3, 5, and 7 post-infection and examined with GM ELISA and qRT-PCR. The A. fumigatus DNA detection sequence was detected in the following number of samples from 12 immunosuppressed, infected rats examined on the scheduled days: day 1 (0/12), day 3 (0/12), day 5 (6/12), and day 7 (8/12) post-infection. The sensitivity and specificity of the qRT-PCR assay was 29.2% and 100%, respectively. Receiver operating characteristic curve (ROC) analysis indicated a Ct (cycle threshold) cut-off value of 15.35, and the area under the curve (AUC) was 0.627. The GM assay detected antigen in sera obtained on day 1 (5/12), day 3 (9/ 12), day 5 (12/12), and day 7 (12/12) post-infection, and thus had a sensitivity of 79.2% and a specificity of 100%. The ROC of the GM assay indicated that the optimal Ct cut-off value was 1.40 (AUC, 0.919). The GM assay was more sensitive than the qRT-PCR assay in diagnosing invasive pulmonary aspergillosis in rats.

Topics: Animals; Aspergillus fumigatus; Diagnostic Tests, Routine; Disease Models, Animal; Galactose; Immunoenzyme Techniques; Invasive Pulmonary Aspergillosis; Mannans; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; ROC Curve; Sensitivity and Specificity

Aspergillus fumigatus, a thermotolerant fungus, is the main causative agent of invasive pulmonary aspergillosis (IPA) in immunocompromised patients that is associated with high mortality rates. Early diagnosis of IPA is crucial for mortality reduction and improved prognosis. An experimental inhalational model of IPA was developed in rats and the efficacy of three biomarkers, namely β-D-glucan (BDG), a panfungal marker, galactomannan (GM), a genus-specific marker, and A. fumigatus DNA, a species-specific marker was evaluated in serum and bronchoalveolar lavage (BAL) specimens at different time points postinfection for early diagnosis of IPA. BDG and GM were detected by using commercial Fungitell and Platelia Aspergillus EIA kits, respectively. A. fumigatus DNA was detected by developing a sensitive, single-step PCR assay. IPA was successfully developed in immunosuppressed rats and all animals until 5 days post-infection were positive for A. fumigatus by culture and KOH-calcofluor microscopy also showed A. fumigatus in 19 of 24 (79%) lung tissue samples. Fourteen of 30 (47%) and 27 of 30 (90%) serum and BAL specimens, respectively, were positive for all three biomarkers with 100% specificity (none of sera or BAL specimens of 12 control rats was positive for biomarkers). Our data show that BAL is a superior specimen than serum and combined detection of BDG, GM and A. fumigatus DNA provide a sensitive diagnosis of IPA in an experimental animal model. Moreover, combined detection of GM and DNA in BAL and detection of either GM or DNA in serum was also positive in 27 of 30 (90%) animals. For economic reasons and considering that the positive predictive value of BDG is low, the detection of GM and/or DNA in serum and BAL samples has the potential to serve as an integral component of the diagnostic-driven strategy in high-risk patients suspected for IPA.

Topics: Animals; Aspergillus fumigatus; beta-Glucans; Biological Assay; Biomarkers; Bronchoalveolar Lavage; Disease Models, Animal; Female; Galactose; Immunocompromised Host; Inhalation; Invasive Pulmonary Aspergillosis; Lung; Mannans; Polymerase Chain Reaction; Rats; Rats, Wistar

Invasive aspergillosis (IA) in immunocompromised host is a major infectious disease leading to reduce the survival rate of world population. Aspergillus niger is a causative agent causing IA. Cassia surattensis plant is commonly used in rural areas to treat various types of disease. C. surattensis flower extract was evaluated against the systemic aspergillosis model in this study. Qualitative measurement of fungal burden suggested a reduction pattern in the colony forming unit (CFU) of lung, liver, spleen and kidney for the extract treated group. Galactomannan assay assessment showed a decrease of fungal load in the treatment and positive control group with galactomannan index (GMI) value of 1.27 and 0.25 on day 28 but the negative control group showed high level of galactomannan in the serum with GMI value of 3.58. Histopathology examinations of the tissues featured major architecture modifications in the tissues of negative control group. Tissue reparation and recovery from infection were detected in extract treated and positive control group. Time killing fungicidal study of A. niger revealed dependence of the concentration of C. surattensis flower extract.

Topics: Animal Structures; Animals; Antifungal Agents; Aspergillus niger; Cassia; Colony Count, Microbial; Disease Models, Animal; Flowers; Galactose; Invasive Pulmonary Aspergillosis; Mannans; Mice, Inbred BALB C; Microbial Sensitivity Tests; Plant Extracts; Treatment Outcome

Interest in lateral-flow devices (LFDs) as potential point-of-care assays for the diagnosis of infectious diseases has increased. Our objective was to evaluate the interlaboratory and interstudy reproducibility and the effects of antifungal therapy on an LFD developed for invasive pulmonary aspergillosis (IPA) detection. An established neutropenic guinea pig model of IPA caused by Aspergillus fumigatus was used. At predetermined time points (1 h and 3, 5, and 7 days postinoculation), blood and bronchoalveolar lavage (BAL) fluid were collected from infected and uninfected animals. In a separate experiment, guinea pigs were treated with posaconazole (10 mg/kg of body weight orally [p.o.] twice a day [BID]), voriconazole (10 mg/kg p.o. BID), liposomal amphotericin B (10 mg/kg intraperitoneally [i.p.] once a day [QD]), or caspofungin (2 mg/kg i.p. QD), and samples were collected on days 7 and 11. Each laboratory independently evaluated the IgG monoclonal antibody-based LFD. Galactomannan and (1 → 3)-β-D-glucan were also measured using commercially available kits. Good interlaboratory agreement was observed with the LFD, as the results for 97% (32/33) of the serum and 78.8% (26/33) of the BAL fluid samples from infected animals were in agreement. Good interstudy agreement was also observed. The serum sensitivity of each surrogate-marker assay was reduced in animals treated with antifungals. In contrast, these markers remained elevated within the BAL fluids of treated animals, which was consistent with the fungal burden and histopathology results. These results demonstrate that the LFD assay is reproducible between different laboratories and studies. However, the sensitivity of this assay and other markers of IPA may be reduced with serum in the presence of antifungal therapy.

Topics: Animals; Antifungal Agents; Aspergillus; Bronchoalveolar Lavage Fluid; Colony Count, Microbial; Disease Models, Animal; Galactose; Glucans; Guinea Pigs; Humans; Invasive Pulmonary Aspergillosis; Male; Mannans; Point-of-Care Systems; Reproducibility of Results; Time Factors

The purpose of this study was to investigate the performance of non-invasive diagnostic tests such as galactomannan enzyme immunoassay and quantitative PCR in the early diagnosis of invasive aspergillosis (IA), and how these tests are impacted upon by the use of different classes of antifungal agents in an in-vivo model of IA.. A standardised rat inhalation model of IA was used to examine the effects of an azole, posaconazole, a polyene, amphotericin B and an echinocandin caspofungin. Daily blood samples were collected for subsequent analysis using a commercially available galactomannan assay and an inhouse qPCR assay.. No significant differences were observed in the CE/g of Aspergillus fumigatus in the lungs of each group. qPCR was statistically more sensitive than galactomannan for both the early detection of infected controls (p=0.045) and for overall detection (p=0.018). However, antifungal treatment significantly reduced the overall sensitivity of qPCR (p=0.020); these effects were due to posaconazole and caspofungin. In the latter stages of infection (days 4 and 5) there were no significant differences in the numbers of infections detected by galactomannan and qPCR; however, the antifungal class used caused significant qualitative differences (p=0.041). Galactomannan showed improved detection in posaconazole-treated animals.. Previous exposure to antifungal therapy must be considered when interpreting either qPCR or galactomannan-based IA diagnostics as this study has shown that individual classes of antifungal agents impact upon the dynamics of antigen and DNA release into the circulation.

Topics: Amphotericin B; Animals; Antifungal Agents; Aspergillus fumigatus; Biomarkers; Caspofungin; Disease Models, Animal; DNA, Fungal; Early Diagnosis; Echinocandins; Galactose; Immunoenzyme Techniques; Invasive Pulmonary Aspergillosis; Lipopeptides; Mannans; Microbiological Techniques; Mycological Typing Techniques; Predictive Value of Tests; Rats; Real-Time Polymerase Chain Reaction; Time Factors; Triazoles

We developed and assessed the diagnostic value of a novel quantitative nested real-time (QNRT) PCR assay targeting the internal transcribed spacer region of ribosomal DNA (rDNA) in a guinea pig model of invasive pulmonary aspergillosis. Groups of 5 immunosuppressed animals that were infected using an aerosol chamber with Aspergillus fumigatus conidia were humanely terminated 1 h postinoculation and at days 3, 5, 7, and 11 postchallenge, and lung tissue, bronchoalveolar lavage (BAL) fluid, whole blood, and serum samples were collected. The QNRT PCR results obtained with the serum and BAL fluid were compared to those achieved with galactomannan and (1→3)-β-d-glucan assays. High fungal burden levels were detected by QNRT PCR in both lung tissue and BAL fluid in all infected animals at each time point, and the sensitivity of each assay in BAL fluid was 100% by day 3 and remained so through the remainder of the study. The sensitivity of detection of fungi in whole blood and serum samples was significantly lower, and some samples remained negative by all three assays despite the advanced stage of the infection. From these data, we can conclude that this novel QNRT PCR method was highly sensitive for the detection of A. fumigatus from different types of samples in this model. In addition, BAL fluid samples appeared to be the most suitable for the early diagnosis of invasive pulmonary aspergillosis. When testing serum, the use of a combination of available assays may increase the possibility of early detection of this opportunistic mycosis.

Topics: Animals; Aspergillus fumigatus; beta-Glucans; Bronchoalveolar Lavage Fluid; Colony Count, Microbial; Disease Models, Animal; DNA, Fungal; DNA, Ribosomal Spacer; Galactose; Guinea Pigs; Invasive Pulmonary Aspergillosis; Lung; Male; Mannans; Mycology; Polymerase Chain Reaction; Proteoglycans; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity

Mortality associated with invasive aspergillosis (IA) remains high, partly because of delayed diagnosis. Detection of microbial exoantigens, released in serum and other body fluids during infection, may help timely diagnosis. In course of IA, Aspergillus galactomannan (GM), a well established polysaccharide biomarker, is released in body fluids including urine. Urine is an abundant, safely collected specimen, well-suited for point-of-care (POC) testing, which could play an increasing role in screening for early disease. Our main objective was to demonstrate GM antigenuria as a clinically relevant biological phenomenon in IA and establish proof-of-concept that it could be translated to POC diagnosis. Utilizing a novel IgM monoclonal antibody (MAb476) that recognizes GM-like antigens from Aspergillus and other molds, we demonstrated antigenuria in an experimental animal IA model (guinea pig), as well as in human patients. In addition, we investigated the chemical nature of the urinary excreted antigen in human samples, characterized antigen detection in urine by immunoassays, described a putative assay inhibitor in urine, and indicated means of alleviation of the inhibition. We also designed and used a lateral flow immunochromatographic assay to detect urinary excreted antigen in a limited number of IA patient urine samples. In this study, we establish that POC diagnosis of IA based on urinary GM detection is feasible. Prospective studies will be necessary to establish the performance characteristics of an optimized device and define its optimal clinical use.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Fungal; Aspergillosis; Aspergillus fumigatus; Cross Reactions; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Galactose; Humans; Mannans; Mice

The diagnosis of invasive aspergillosis remains very difficult, and there are limited treatment options for the disease. Pre-clinical models have been used to evaluate the diagnosis and treatment of Aspergillus infection and to assess the pathogenicity and virulence of the organism. Extensive efforts in Aspergillus research have significantly expanded the genomic information about this microorganism. The standardization of animal models of invasive aspergillosis can be used to enhance the evaluation of genomic information about the organism to improve the diagnosis and treatment of invasive aspergillosis. One approach to this process has been the award of a contract by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health to establish and standardize animal models of invasive aspergillosis for the development of new diagnostic technologies for both pulmonary and disseminated Aspergillus infection. This work utilizes molecular approaches for the genetic manipulation of Aspergillus strains that can be tested in animal-model systems to establish new diagnostic targets and tools. Studies have evaluated the performance characteristics of assays for cell-wall antigens of Aspergillus including galactomannan and beta-D-glucan, as well as for DNA targets in the organism, through PCR. New targets, such as proteomic and genomic approaches, and novel detection methods, such as point-of-care lateral-flow devices, have also been evaluated. The goal of this paper is to provide a framework for evaluating genomic targets in animal models to improve the diagnosis and treatment of invasive aspergillosis toward ultimately improving the outcomes for patients with this frequently fatal infection.

Topics: Animals; Antifungal Agents; Aspergillosis; Aspergillus; Biomarkers; Disease Models, Animal; DNA, Fungal; Galactose; Humans; Invasive Pulmonary Aspergillosis; Mannans; Predictive Value of Tests; Prognosis; Severity of Illness Index; Virulence Factors

Cutaneous models have proven useful in studies of the pathogenesis and treatment of Gram-positive bacterial infections. Because cutaneous invasive aspergillosis (IA) occurs in the clinical setting, we sought to develop a nonlethal murine cutaneous model of IA. We induced cutaneous IA in cyclophosphamide-treated nude BALB/c mice by subcutaneous injection of Aspergillus fumigatus conidia. Skin lesion areas correlated well with tissue fungal burdens, allowing dynamic visual monitoring of cutaneous infections. The cutaneous model accurately reflected alterations in A. fumigatus pathogenicity resulting from deletions of recognized virulence genes (pabaA, sidA, and pksP). Moreover, analysis of the roles of conidial and mycelial catalases revealed that the former is required for the initiation of cutaneous aspergillosis, whereas the latter contributes to its propagation. Finally, posaconazole treatment reduced skin lesion areas relative to those of untreated and fluconazole-treated controls. This novel cutaneous model system should be applicable to comparative studies of the pathogenesis, treatment, and tissue specificity of IA.

Topics: Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Catalase; Cyclophosphamide; Dermatomycoses; Disease Models, Animal; Female; Galactose; Hyphae; Immunocompromised Host; Immunosuppressive Agents; Mannans; Mice; Mice, Inbred BALB C; Mice, Nude; Mutagenesis; Skin; Spores; Triazoles; Virulence; Virulence Factors

The incidence of aspergillosis which has high mortality rates, has increased gradually. Since invasive aspergillosis (IA) is one of the leading causes of death in immunocompromized and neutropenic patients, early and accurate diagnosis of IA is of crucial importance. The aims of this study were to compare the results of culture, real-time polymerase chain reaction (RtPCR), galactomannan (GM) antigen and glucan (GC) antigen detection tests and to evaluate their performances in view of rapid and accurate diagnosis of IA in neutropenic rat model. Female Wistar albino rats were included in the study with the consent of Animal Searching Ethical Committee and classified into three groups as healthy controls (n= 6), neutropenic controls (n= 10) and pulmonary aspergillosis (n= 35) groups. Rats were immunosuppressed with 5-flourourasil and then Aspergillus fumigatus conidia were inoculated intranasally. On the seventh day of the infection, blood, bronchoalveolar lavage (BAL) and lung tissue samples were collected from the animals, and control and aspergillosis groups were compared in terms of infection markers. All of the tests (culture, RtPCR, GM and BG tests) were found to be negative in controls. At the end of the study 22 rats in aspergillosis group survived. Lung tissue samples from those 22 animals were all positive for the presence of hypha on pathological preparations, while 20 (91%) yielded Aspergillus colonies on the cultures. Aspergillus DNA was detected in 7 of the 12 BAL samples (58.3%), 7 of 19 blood samples (36.8%) and 4 of 22 lung tissue samples (18%) using RtPCR method. GM antigen was detected in 7 of 20 serum samples (35%) with a commercial kit (Platelia® Aspergillus ELISA, BioRad, France). Quantitative detection of betalucan levels were investigated by using a commercial kit (Fungitell™, Cape Cod, USA) in serum and BAL samples and positive results were obtained in 11 of 22 serum (50%) and 9 of 17 BAL (52.9%) samples. In this study it was demonstrated that PCR performed in BAL samples is the most sensitive method compared to the others, for the diagnosis of IA in the rat model. The sensitivity rates were as follows when culture method accepted as the gold standard: 58.3% for BAL-PCR, 41.2% for blood-PCR, 20% for tissue-PCR, 38.9% for serum GM, 55% for serum GC and 52.9% for BAL-GC. It was also concluded that detection of GC activity in serum was more sensitive than GM detection in serum (sensitivity of GM was %38.9, sensitivity of GC was %55, whi

Topics: Animals; Antigens, Fungal; Aspergillus fumigatus; beta-Glucans; Bronchoalveolar Lavage Fluid; Disease Models, Animal; DNA, Fungal; Enzyme-Linked Immunosorbent Assay; Female; Fluorouracil; Galactose; Immunosuppression Therapy; Immunosuppressive Agents; Invasive Pulmonary Aspergillosis; Lung; Mannans; Neutropenia; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity

The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.

Topics: Animals; Aspergillosis; Aspergillus fumigatus; Body Temperature; Cell Proliferation; Cell Wall; Disease Models, Animal; Drug Resistance, Fungal; Female; Furans; Galactose; Gene Expression Regulation, Fungal; Immunocompromised Host; Intramolecular Transferases; Mannans; Mice; Mice, Inbred BALB C; Opportunistic Infections; Virulence

Early diagnosis of invasive pulmonary aspergillosis is problematic in some patient groups due to the lack of rapid, sensitive, specific, and reliable diagnostic tests. Fungal burden and therapeutic efficacy were assessed by survival, quantitative culture (CFU counts), galactomannan enzyme immunoassay (GM-EIA), and quantitative PCR (qPCR) in a new guinea pig model of invasive pulmonary aspergillosis using an aerosol challenge. At 1 day postinfection, qPCR determined that the pulmonary fungal burden was 2 log(10) higher than that determined by CFU counting and increased significantly (P < 0.03) over time. In contrast, the tissue burden assessed by CFU counting did not rise over the course of the study. Therapy with the antifungal drug voriconazole produced statistically significant decreases in pulmonary fungal burden, as detected by CFU counting (P < 0.02), qPCR, and GM-EIA (both P < 0.0002). Daily assessment of the progression of fungal infection in serum was performed by qPCR and GM-EIA. GM-EIA demonstrated a statistically significant reduction in the fungal load on days 6 and 7 in voriconazole-treated animals compared to time-matched controls (P < 0.02). Confirmation of fungal tissue burden by two or more methods should provide a more precise account of the burden, allowing improved assessment of diagnostic and therapeutic strategies in invasive pulmonary aspergillosis.

Topics: Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Base Sequence; Colony Count, Microbial; Disease Models, Animal; DNA Primers; DNA, Fungal; Galactose; Guinea Pigs; Humans; Immunoenzyme Techniques; Lung; Lung Diseases, Fungal; Male; Mannans; Mycology; Polymerase Chain Reaction; Pyrimidines; Triazoles; Voriconazole

The purpose of this study was to assess the antifungal activity, pharmacokinetics, and tissue distribution of amphotericin B (AmpB) following the administration of Abelcet and AmBisome alone and in combination with Caspofungin to rats infected with Aspergillus fumigatus. Aspergillus fumigatus inoculum (2.1-2.5 x 10(7) colony forming units [CFU]) was injected via the jugular vein; 48 h later male albino Sprague-Dawley rats (350-400 g) were administered either a single intravenous (i.v.) dose of Abelcet (5 mg AmpB/kg; n = 6), AmBisome (5 mg AmpB/kg; n = 6), Caspofungin (3 mg/kg; n = 5), Abelcet (5 mg AmpB/kg) plus Caspofungin (3 mg/kg) (n = 6), AmBisome (5 mg AmpB/kg) plus Caspofungin (3 mg/kg) (n = 7), or physiologic saline (non-treated controls; n = 6) once daily for 4 days. Antifungal activity was assessed by organ CFU concentrations and plasma galactomannan levels. Plasma and tissue samples were taken from each animal for AmpB pharmacokinetic analysis and tissue distribution determinations. Abelcet treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 73% compared to non-treated controls. Ambisome treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 69% compared to non-treated controls. Caspofungin treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 80% compared to non-treated controls. Abelcet plus Caspofungin treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 81% compared to non-treated controls. Ambisome plus Caspofungin treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 98% compared to non-treated controls. Abelcet treatment significantly decreased plasma galactomannan levels by 50 and 75% 96 h following the initiation of treatment in the absence and presence of Caspofungin co-therapy, respectively. AmBisome treatment significantly decreased plasma galactomannan levels by 73 and 78% 96 h following the initiation of treatment in the absence and presence of Caspofungin co-therapy, respectively. Co-administration of Caspofungin with Abelcet and AmBisome did not significantly alter the plasma concentration-time profile, pharmacokinetic parameters, and tissue distribution of AmpB. Taken together, our findings suggest that an alternative mechanism, possibly at t

Topics: Amphotericin B; Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Caspofungin; Colony Count, Microbial; Disease Models, Animal; Drug Administration Schedule; Drug Combinations; Drug Therapy, Combination; Echinocandins; Galactose; Injections, Intravenous; Lipopeptides; Male; Mannans; Peptides, Cyclic; Phosphatidylcholines; Phosphatidylglycerols; Rats; Rats, Sprague-Dawley; Tissue Distribution

Viscosupplementation efficacy has been related to the high molecular weight of hyaluronic acid-like compounds, as well as to gel formulation. We evaluated the effect of a galactomannan polysaccharide derived from Guar gum (GG) in joint pain in an osteoarthritis (OA) model. Wistar rats (six animals/group) were subjected to anterior cruciate ligament transection (ACLT-OA group). The OA group was compared to a false-operated group (sham). Joint pain was recorded daily, using the articular incapacitation test, until 7 days after ACLT. Solutions or gel preparations of GG (100 microg) or Hylan G-F 20 (100 microg), used as a comparator, were given intraarticularly (i.a.) at day 4 after ACLT. Controls received saline i.a. The OA group had significantly increased joint pain as compared to sham (P<0.001). GG, either as a gel or solution, significantly inhibited joint pain similar to the inhibition achieved with Hylan G-F20. This is the first demonstration that a galactomannan derived from GG reduces joint pain in experimental OA. This analgesia is independent of the colloidal state. We propose that the analgesic benefit of viscosupplementation may be due to an intrinsic carbohydrate-mediated mechanism rather than to the rheologic properties of the material.

Topics: Analgesics; Animals; Arthralgia; Disease Models, Animal; Galactose; Injections, Intra-Articular; Male; Mannans; Osteoarthritis, Knee; Plant Extracts; Rats; Rats, Wistar

To assess the value of Aspergillus galactomannan (GM) double-direct sandwich enzyme-linked immunosorbent assay (ELISA) in the diagnosis of invasive pulmonary aspergillosis (IPA).. Ninety adult SD rats were randomly divided into 5 groups, including 4 groups of pulmonary infection by Aspergillus fumigatus (A. fumigatus), Candida albicans, mucor, and Streptococcus pneumoniae, respectively, and a colonization group by A. fumigatus in the pharynx oralis (n = 18 each). For the infection models, suspensions of pathogenic bacteria and fungi were instilled into the lungs of the rats by tracheal intubation. For the colonization model, the suspension of A. fumigatus was applied to the nasal cavity and pharynx oralis of the rats. The animals were sacrificed on days 3, 7, and 12 after inoculation, and blood samples were obtained by cardiac puncture and used for GM detection. The lung tissues were prepared for routine pathology examination, and hexamethylene tetramine silver staining was used to detect the fungi. A double-direct sandwich ELISA was employed to detect GM optical density index in the serum samples.. The lung tissues of rats infected with A. fumigatus, Candida albicans, mucor and Streptococcus pneumoniae all showed remarkable inflammatory reactions, and hyphae were observed in rats with fungal infection (including A. fumigatus and mucor), and spores in rats infected with Candida albicans. The lung tissues of the A. fumigatus colonization rats showed no inflammatory reactions. The serum GM optical density index of the groups infected with A. fumigatus, Candida albicans, mucor and Streptococcus pneumoniae, and the A. fumigatus colonization group were 1.69 +/- 0.29, 0.89 +/- 0.46, 0.87 +/- 0.39, 0.77 +/- 0.34 and 0.90 +/- 0.49, respectively. The serum GM optical density index of the IPA group was higher than those of the other 4 groups (P < 0.05). If the cutoff ODI was 1.5, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for diagnosis of IPA in the rats were 78.6%, 87.5%, 57.9% and 94.9%, respectively. The sensitivity for A. fumigatus infection on days 3, 7, and 12 was 60.0%, 80% and 100%, and the specificity was 95.5%, 81.0% and 85.7%, respectively.. GM detection could distinguish Aspergillus infection from Candida albicans, mucor, Streptococcus pneumoniae infection and Aspergillus colonization. The sensitivity of the test for the diagnosis of IPA tended to be higher with longer duration of infection. A cutoff ODI of 1.5 showed the best sensitivity and specificity for IPA in this rat model.

Topics: Animals; Aspergillosis; Disease Models, Animal; Female; Galactose; Lung Diseases, Fungal; Male; Mannans; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity

Bronchoalveolar lavage (BAL) is widely used for evaluation of patients with suspected invasive pulmonary aspergillosis (IPA). However, the diagnostic yield of BAL for detection of IPA by culture and direct examination is limited. Earlier diagnosis may be facilitated by assays that can detect Aspergillus galactomannan antigen or DNA in BAL fluid. We therefore characterized and compared the diagnostic yields of a galactomannan enzyme immunoassay (GM EIA), quantitative real-time PCR (qPCR), and quantitative cultures in experiments using BAL fluid from neutropenic rabbits with experimentally induced IPA defined as microbiologically and histologically evident invasion. The qPCR assay targeted the rRNA gene complex of Aspergillus fumigatus. The GM EIA and qPCR assay were characterized by receiver operator curve analysis. With an optimal cutoff of 0.75, the GM EIA had a sensitivity and specificity of 100% in untreated controls. A decline in sensitivity (92%) was observed when antifungal therapy (AFT) was administered. The optimal cutoff for qPCR was a crossover of 36 cycles, with sensitivity and specificity of 80% and 100%, respectively. The sensitivity of qPCR also decreased with AFT to 50%. Quantitative culture of BAL had a sensitivity of 46% and a specificity of 100%. The sensitivity of quantitative culture decreased with AFT to 16%. The GM EIA and qPCR assay had greater sensitivity than culture in detection of A. fumigatus in BAL fluid in experimentally induced IPA (P+/-0.04). Use of the GM EIA and qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis and more-timely initiation of specific therapy.

Topics: Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Female; Galactose; Immunoenzyme Techniques; Lung Diseases, Fungal; Mannans; Polymerase Chain Reaction; Rabbits; Sensitivity and Specificity

In view of the poor therapy outcomes of invasive aspergillosis, the objective of this study was to evaluate the efficacy of combination treatment consisting of the polyene amphotericin-B-intralipid, the echinocandin caspofungin and granulocyte-colony stimulating factor (G-CSF) in experimental murine systemic aspergillosis. With inhibition of synthesis of 1,3-beta-d-glucan in the fungal cell wall by caspofungin and an effect on the cell membrane by amphotericin-B-intralipid, this treatment may result in a synergic effect against Aspergillus fumigatus. Addition of G-CSF may further contribute to therapy of aspergillosis.. ICR mice were immunosuppressed by intraperitoneal administration of cyclophosphamide. Three days later, the mice were inoculated intravenously (iv) with A. fumigatus conidia. Infection and treatment were evaluated during an observation period of 30 days in terms of mortality (survival rate and mean survival time) and morbidity (quantitative determination of fungal burden, histopathology, and detection of serum galactomannan).. Combination of caspofungin + G-CSF or addition of G-CSF to the combination of caspofungin + amphotericin-B-intralipid increased the survival rate of infected mice up to 78.9% and prolonged their mean survival time to 25 days. These combinations also resulted in a reduction in fungal burden in organs, and a decrease in serum galactomannan.. The successful results obtained in the experimental model may possibly open the way to more effective management of aspergillosis in humans.

Topics: Amphotericin B; Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Caspofungin; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Echinocandins; Female; Galactose; Granulocyte Colony-Stimulating Factor; Immunocompromised Host; Kidney; Lipopeptides; Liposomes; Lung; Mannans; Mice; Mice, Inbred ICR; Peptides, Cyclic; Polyenes; Survival Rate

The performance of different in vitro diagnostic tests for the diagnosis of invasive aspergillosis (IA) was investigated in a transiently neutropenic rat model. Rats were immunosuppressed with cyclophosphamide and then inoculated intravenously with 1.5 x 10(4) CFU Aspergillus fumigatus spores. Animals were then either treated with caspofungin acetate, 1 mg/kg/day for 7 days, or not treated. PCR-enzyme-linked immunosorbent assay (ELISA), real-time PCR, and galactomannan (GM) detection were performed on postmortem blood samples, along with culture of liver, lung, and kidney homogenate. Caspofungin-treated animals showed a decrease in residual tissue burden of A. fumigatus from organ homogenate compared to untreated animals (P < 0.002). PCR-ELISA returned positive results for 11/17 animals treated with antifungal agents and for 10/17 untreated animals. Galactomannan was positive in 8/17 caspofungin-treated animals and 4/17 untreated animals. Real-time PCR was positive in 2/17 treated and 3/17 untreated animals. This study demonstrates that PCR-ELISA is a more sensitive test than either GM detection (P = 0.052) or real-time PCR (P < 0.01) for diagnosis of IA but that any of the three tests may return false-negative results in cases of histologically proven disease. Galactomannan indices from animals treated with antifungal agents showed a trend (P = 0.1) towards higher levels than those of untreated animals, but no effect was observed with PCR-ELISA indices (P = 0.29). GM detection, as previously described, may be enhanced by the administration of caspofungin, but PCR-ELISA appears not to be affected in the same way. We conclude that PCR-ELISA is a more sensitive and reliable method for laboratory diagnosis of IA.

Topics: Animals; Antifungal Agents; Aspergillosis; Caspofungin; Disease Models, Animal; Echinocandins; Enzyme-Linked Immunosorbent Assay; Galactose; Lipopeptides; Liver Diseases; Male; Mannans; Neutropenia; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction

Invasive aspergillosis (IA) is a frequent complication of blood or marrow transplantation. Previous studies have reported that the Aspergillus galactomannan enzyme immunoassay (GM EIA) may be a useful diagnostic tool for IA, but its sensitivity is variable. We examined the performance of the GM EIA in 986 serum samples from 67 patients. Results demonstrated that decreasing the index cutoff for positivity to 0.5 increased its sensitivity, with minimal loss of specificity. The low cutoff increased the duration of test positivity before diagnosis by clinical means. Sensitivity was highest in patients who did not receive preventative mold-active antifungals (87.5%). A rabbit model demonstrated that the level of circulating antigen correlated with the tissue fungus burden. A quantifiable response to antifungal therapy in clinical samples and the rabbit model supports the development of this assay for early diagnosis and therapeutic monitoring. The 0.5 cutoff may allow for better performance as an early diagnostic test.

Topics: Adolescent; Adult; Aged; Animals; Antigens, Fungal; Aspergillosis; Aspergillus; Bone Marrow Transplantation; Child; Child, Preschool; Disease Models, Animal; Female; Fungemia; Galactose; Humans; Immunoenzyme Techniques; Lung Diseases, Fungal; Male; Mannans; Middle Aged; Rabbits; Sensitivity and Specificity

Mouse models of systemic and gastrointestinal infection with the yeast Candida albicans were used to investigate the ability of a commercial mannan antigen enzyme immunoassay and a commercial (1-->3) beta-D-glucan limulus assay to detect systemic infection and to differentiate between colonization and infection. Both assays were positive in all i.v. infected mice and negative in all uninfected control mice. In gastrointestinal infection both tests were positive whenever organ cultures were positive. In colonized mice with no detectable dissemination, there were mostly negative results with the glucan assay whereas the mannan assay was positive or intermediate in all colonized mice. Therefore, in the mouse model used, glucan detection appeared to be superior for differentiation between colonization and dissemination.

Topics: Animals; Candida albicans; Candidiasis; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Fungemia; Galactose; Gastrointestinal Diseases; Glucans; Linear Models; Mannans; Mice; Mice, Inbred BALB C; Probability; Sensitivity and Specificity

The antifungal efficacy, pharmacokinetics, and safety of caspofungin (CAS) were investigated in the treatment and prophylaxis of invasive pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Antifungal therapy consisted of 1, 3, or 6 mg of CAS/kg of body weight/day (CAS1, CAS3, and CAS6, respectively) or 1 mg of deoxycholate amphotericin B (AMB)/kg/day intravenously for 12 days starting 24 h after endotracheal inoculation. Prophylaxis (CAS1) was initiated 4 days before endotracheal inoculation. Rabbits treated with CAS had significant improvement in survival and reduction in organism-mediated pulmonary injury (OMPI) measured by pulmonary infarct score and total lung weight (P < 0.01). However, animals treated with CAS demonstrated a paradoxical trend toward increased residual fungal burden (log CFU per gram) and increased serum galactomannan antigen index (GMI) despite improved survival. Rabbits receiving prophylactic CAS1 also showed significant improvement in survival and reduction in OMPI (P < 0.01), but there was no effect on residual fungal burden. In vitro tetrazolium salt hyphal damage assays and histologic studies demonstrated that CAS had concentration- and dose-dependent effects on hyphal structural integrity. In parallel with a decline in GMI, AMB significantly reduced the pulmonary tissue burden of A. fumigatus (P < or = 0.01). The CAS1, CAS3, and CAS6 dose regimens demonstrated dose-proportional exposure and maintained drug levels in plasma above the MIC for the entire 24-h dosing interval at doses that were > or =3 mg/kg/day. As serial galactomannan antigen levels may be used for therapeutic monitoring, one should be aware that profoundly neutropenic patients receiving echinocandins for aspergillosis might have persistent galactomannan antigenemia despite clinical improvement. CAS improved survival, reduced pulmonary injury, and caused dose-dependent hyphal damage but with no reduction in residual fungal burden or galactomannan antigenemia in persistently neutropenic rabbits with invasive pulmonary aspergillosis.

Topics: Animals; Anti-Bacterial Agents; Antibiotic Prophylaxis; Antifungal Agents; Aspergillosis; Aspergillus; Caspofungin; Disease Models, Animal; Echinocandins; Female; Galactose; Lipopeptides; Lung Diseases, Fungal; Mannans; Microbial Sensitivity Tests; Neutropenia; Peptides; Peptides, Cyclic; Rabbits; Treatment Outcome

The antifungal efficacy, safety, and pharmacokinetics of posaconazole (SCH 56592) (POC) were investigated in treatment and prophylaxis of primary pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Antifungal therapy consisted of POC at 2, 6, and 20 mg/kg of body weight per os; itraconazole (ITC) at 2, 6, and 20 mg/kg per os; or amphotericin B (AMB) at 1 mg/kg intravenously. Rabbits treated with POC showed a significant improvement in survival and significant reductions in pulmonary infarct scores, total lung weights, numbers of pulmonary CFU per gram, numbers of computerized-tomography-monitored pulmonary lesions, and levels of galactomannan antigenemia. AMB and POC had comparable therapeutic efficacies by all parameters. By comparison, animals treated with ITC had no significant changes in outcome variables in comparison to those of untreated controls (UC). Rabbits receiving prophylactic POC at all dosages showed a significant reduction in infarct scores, total lung weights, and organism clearance from lung tissue in comparison to results for UC (P < 0.01). There was dosage-dependent microbiological clearance of A. fumigatus from lung tissue in response to POC. Serum creatinine levels were greater (P < 0.01) in AMB-treated animals than in UC and POC- or ITC-treated rabbits. There was no elevation of serum hepatic transaminase levels in POC- or ITC-treated rabbits. The pharmacokinetics of POC and ITC in plasma demonstrated dose dependency after multiple dosing. The 2-, 6-, and 20-mg/kg dosages of POC maintained plasma drug levels above the MICs for the entire 24-h dosing interval. In summary, POC at > or =6 mg/kg/day per os generated sustained concentrations in plasma of > or =1 microg/ml that were as effective in the treatment and prevention of invasive pulmonary aspergillosis as AMB at 1 mg/kg/day and more effective than cyclodextrin ITC at > or =6 mg/kg/day per os in persistently neutropenic rabbits.

Topics: Animals; Antibiotic Prophylaxis; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Disease Models, Animal; Female; Galactose; Itraconazole; Lung Diseases, Fungal; Mannans; Neutropenia; Rabbits; Treatment Outcome; Triazoles

Intravenous (i.v.) infection of immunocompetent mice with Aspergillus fumigatus was used to investigate the ability of a commercial galactomannan enzyme-linked immunosorbent assay (ELISA) to monitor the course of organ infection after dissemination. The test detected 100% of the fungemias which occurred for up to 5 days after infection. When blood-cultures became negative but there was a high load of fungi in the parenchymal organs and a positive culture from the brain, the ELISA was again positive in all animals. However, when blood cultures as well as brain cultures were negative and lower amounts of fungi demarcated by immune cells were present in the liver and kidneys which was the case between day 5 and 30 of infection, the test was negative in most of the animals. Therefore, the test was excellent for detection of early i.v. infection with Aspergillus fumigatus but not suited for detection of limited organ infection in immunocompetent mice.

Topics: Animals; Antibodies, Fungal; Antigens, Fungal; Aspergillosis; Aspergillus fumigatus; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fungemia; Galactose; Injections, Intravenous; Mannans; Mice; Mice, Inbred BALB C

Two diagnostic tests, an Aspergillus-specific PCR and an enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of galactomannan, were compared for diagnosing and monitoring invasive pulmonary aspergillosis. Persistently neutropenic rats with left-sided invasive pulmonary aspergillosis were sacrificed at regular intervals after inoculation. Blood samples and bronchoalveolar lavage (BAL) fluid were cultured and tested by PCR as well as by ELISA. Disseminated fungal infection in extrapulmonary organs was determined. The sensitivity of the ELISA was higher than that of the PCR on all days of measurements, in both blood and BAL fluid. Positive PCR or ELISA results in blood were not significantly associated with disseminated fungal infection. Serial testing in a separate group of rats showed consistently increasing concentrations of circulating galactomannan during the course of disease, while a positive PCR could be followed by negative results. The concentration of galactomannan was highly predictive for the time of survival (P < 0.0001). It was concluded that, in this model, quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis.

Topics: Animals; Aspergillosis; Aspergillus; Bronchoalveolar Lavage Fluid; Disease Models, Animal; DNA, Fungal; Enzyme-Linked Immunosorbent Assay; Female; Galactose; Lung Diseases, Fungal; Mannans; Polymerase Chain Reaction; Rats; Reproducibility of Results

The G test containing factor G, fractioned from the Limulus lysate, was used to detect (1-3)-beta-D-glucan in a rat model of aspergillosis. Aspergillus fumigatus strain MF-13, 1 x 10(4) conidia, were inoculated transtracheally into rats treated with cortisone acetate (100 mg/kg) and fed a low-protein (8%) diet. Increased serum (1-3)-beta-D-glucan was found on the sixth day after inoculation in concentrations of 370 +/- 178 pg/ml (mean +/- SD) in untreated controls, and 154 +/- 43 pg/ml in rats treated with 0.5 mg/kg of amphotericin B. On day 11 (1-3)-beta-D-glucan concentrations were 2,590 +/- 2,940 pg/ml and 448 +/- 442 pg/ml, respectively. The elevation in levels of (1-3)-beta-D-glucan increased in correlation with the elevation of galactomannan antigen titers; (1-3)-beta-D-glucan is thus measurable during experimental aspergillosis in rats.

Topics: Amphotericin B; Animals; Aspergillosis; Aspergillus fumigatus; beta-Glucans; Disease Models, Animal; Galactose; Glucans; Horseshoe Crabs; Lung; Male; Mannans; Rats; Rats, Sprague-Dawley

The performance of Pastorex Aspergillus, a new latex agglutination test for the detection of circulating galactomannan in the serum of patients with invasive aspergillosis, was evaluated in a blind trial in standardized guinea-pig models of invasive aspergillosis and other invasive mycoses. In these animal models, the invasive nature of the fungal infection was confirmed by re-isolation of the etiologic agent from the organs of every animal. Ninety-two plasma samples from 42 animals with invasive aspergillosis were submitted to the test. In 41 of these animals, at least one plasma sample was positive with the latex test (sensitivity 97.6%), titers ranging from 1/1 to 1/512. In general, antigen titers increased as a function of time, reaching the highest values shortly before death. Guinea-pigs infected with Penicillium marneffei also yielded positive agglutination reactions but antigen titers were lower (maximal titer 1/8). Plasma samples from animals with invasive candidosis (23), disseminated trichophytosis (11) and cryptococcosis (23) were all negative with the latex test. In 80 guinea-pigs without fungal infection, 3 false positive results (titers 1/1) were observed, which means a specificity of 96.2% in this control group.

Topics: Animals; Antigens, Fungal; Aspergillosis; Aspergillus fumigatus; Disease Models, Animal; Galactose; Guinea Pigs; Latex Fixation Tests; Male; Mannans; Reagent Kits, Diagnostic