Efficacy of Azithromycin and Miltefosine in Experimental Systemic Pythiosis in Immunosuppressed Mice.

We evaluated the efficacy of azithromycin (50 mg/kg, every 12 h [q12h] orally) and miltefosine (25 mg/kg, q24h orally) treatments in an experimental model of vascular/disseminated pythiosis in immunosuppressed mice. Azithromycin was the only treatment able to reduce mortality. The histopathological findings showed acute vascular inflammation, pathogen dissemination, necrotizing myositis, neuritis, and arteritis. The results suggest that azithromycin, but not miltefosine, may have clinical relevance in the treatment of vascular/disseminated pythiosis.

Screening of antibacterial drugs for antimicrobial activity against Pythium insidiosum.

We tested 25 isolates of Pythium insidiosum to investigate their susceptibility to antibacterial drugs that act through inhibition of protein synthesis or other mechanisms of action. We observed that tetracycline, erythromycin, linezolid, nitrofurantoin, Synercid (quinupristin and dalfopristin), chloramphenicol, clindamycin, cetrimide, and crystal violet had inhibitory activity against P. insidiosum. Those in vitro results suggest that antibacterials that inhibit protein synthesis should be the primary antimicrobials investigated for the treatment of pythiosis in animals and humans.

In vitro assessment of antifungal, antibacterial, and antiprotozoal drugs against clinical isolates of Conidiobolus lamprauges.

We have determined the in vitro activity of antifungal, antibacterial, and antiprotozoal drugs alone and in combination against seven Conidiobolus lamprauges clinical isolates. The assays were based on the M38-A2 protocol and the checkerboard microdilution method. The lowest inhibitory concentrations were observed for amphotericin B, miconazole (MCZ), terbinafine, and miltefosine (MTF) (MIC range 0.25-1; 2-8; 0.25-2; 2-16 µg/ml, respectively). The main synergism observed was through the combination of azithromycin (AZI)+MTF and dapsone (DAP)+MTF (100%), AZI+DAP (85.7%), AZI+MCZ (57.1%) as well as MCZ plus CTX and DAP (42.9%). The in vitro activities suggest that the combination of MTF and AZI or DAP are promising candidate therapies for conidiobolomycosis.

In vitro combination of antifungal agents against Malassezia pachydermatis.

The yeast Malassezia pachydermatis is a common commensal and occasional opportunistic pathogen of theskin microbiota of animals and humans. In this study, the susceptibility of M. pachydermatis isolates to fluconazole (FLC), itraconazole (ITZ), ketoconazole (KTZ), clotrimazole (CLZ), and miconazole (MCZ) alone and in combination with terbinafine (TRB), nystatin (NYS), and caspofungin (CSP) was evaluated in vitro based on the M27-A3 technique and the checkerboard microdilution method using Sabouraud dextrose broth with 1% tween 80 (SDB). Based on the mean FICI values, the main synergies observed were combinations of ITZ+CSP and CLZ+CSP (55.17%). The most significant combinations deserve in vivo evaluations because might provide effective alternative treatments against M. pachydermatis due to their synergistic interactions.

In vitro combination between antifungals and diphenyl diselenide against Cryptococcus species.

Cryptococcus species are an encapsulated fungal pathogen that cause cryptococcal meningitis. There are limited therapeutic options for this infection. The management includes the use of different antifungals such as amphotericin B, flucytosine, or fluconazole, either alone or in combination. However, numerous therapeutic failures, as well as the limited effectiveness of such therapeutics, have been described. Diphenyl diselenide is a chemically synthesised molecule with was found to have antimicrobial activity. In this study, we evaluated the antifungal activities of fluconazole, amphotericin B and flucytosine, in combination with diphenyl diselenide against 30 clinical isolates of Cryptococcus spp. using CLSI M27-A3 method and the checkerboard microdilution technique. Our results show that the combination of flucytosine and diphenyl diselenide displayed 100% of synergism. However, when we analysed (PhSe)2 plus AMB or FLZ we observed around 70% of indifference. Our results suggest that the combination of diphenyl diselenide with other antifungal agents deserves attention as a new option for the development of alternative therapies for cryptococcosis.

In Vitro Activities of Miltefosine and Antibacterial Agents from the Macrolide, Oxazolidinone, and Pleuromutilin Classes against Pythium insidiosum and Pythium aphanidermatum.

We tested 29 isolates of Pythium insidiosum and one isolate of Pythium aphanidermatum to investigate their susceptibility to miltefosine and antibacterial drugs from the macrolide, oxazolidinone, and pleuromutilin classes. We found that miltefosine, azithromycin, clarithromycin, josamycin, linezolid, sutezolid, retapamulin, tiamulin, and valnemulin had inhibitory and cidal activity against the pathogens at concentrations ranging from 0.25 to 64 µg/ml. Our results suggest that these antimicrobials are promising candidates for future studies on pythiosis in animals and humans.

An experimental murine model of otitis and dermatitis caused by Malassezia pachydermatis.

We report a malasseziosis model in immunocompromised Swiss mice. For this model, the mice were immunosuppressed with a combination of cyclophosphamide at 150 mg/kg and hydrocortisone acetate at 250 mg/kg. Two groups were formed according to the site of inoculation. Dermatitis group received an intradermal injection of 5 × 106 cell/mouse at a shaved dorsal region, while the otitis group received the same inoculum in the middle ear. Five animals/group were euthanised at different times, and the skin and ear were histopathologically analysed. During the first euthanasia, which occurred after inoculation, microscopic examination showed that all mice presented budding yeast-like in a tissue sample. The presence of yeasts decreased over time being undetected on the 17th day (dermatitis group) and the 21st day (otitis group) after inoculation. This is the first murine model for malasseziosis that can be useful for evaluating new treatment approaches.

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection.

Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of 4 zoospore concentrations into the egg allantoic cavity at 3 embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 hours after inoculation, and 95% embryos died within 72 hours. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only 1 liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis.

Oxidative stress in rats experimentally infected by Sporothrix schenckii.

The aim of this study was to evaluate whether oxidative stress occurs in rats experimentally infected by Sporothrix schenckii, and its possible effect on disease pathogenesis. Thirty rats were divided into two groups: the group A (uninfected, n = 18) and the group B (infected by S. schenckii, n=21). Blood samples were collected on days 15, 30 and 40 post-infection (PI). At each sampling time, six rats of the group A, and seven of the group B were bled. TBARS (thiobarbituric acid reactive substances) levels in serum samples were measured to evaluate lipid peroxidation. In addition, catalase (CAT) and superoxide dismutase (SOD) activities, known as biomarkers of antioxidants levels, were verified in whole blood. Seric pro-inflammatory cytokine levels were measured (IFN-γ, TNF-α, and IL-6), which showed that these inflammatory mediators were at higher levels in the infected rats (P < 0.001). In comparison to uninfected animals, rats with sporotrichosis showed significantly higher (p < 0.01) levels of TBARS on day 40 PI; CAT activity was significantly increased (p < 0.01) on days 30 and 40 PI; and SOD activity was increased (p < 0.01) on day 40 PI. Infected rats showed larger testicles and granulomas in the testicular capsule, as well as hepatic granulomas and splenic follicular hyperplasia. All tissues (testicle, spleen, and liver) showed inflammation associated with numerous fungal structures. These results demonstrated that the intense inflammatory response (seric and tissue) in sporotrichosis is a likely mechanism for redox imbalance, and consequently cause the oxidative stress in experimentally infected rats.

Nanoparticle formulation increases Syzygium cumini antioxidant activity in Candida albicans-infected diabetic rats.

CONTEXT: Syzygium cumini (L.) Skeels (Myrtaceae) is a medicinal plant widely used in folk medicine for the treatment of diabetes mellitus (DM). However, studies on the use of this plant and of nanoparticle formulations against DM-related fungal infections are scarce. OBJECTIVE: To evaluate the effect of the treatments with aqueous seed extract of S. cumini (ASc) and ASc-loaded polymeric nanoparticles (NPASc) on biochemical parameters in Candida albicans-infected diabetic rats. MATERIALS AND METHODS: Male Wistar rats were divided into eight groups: Control, DM, C. albicans, C. albicans + ASc, C. albicans + NPASc, DM + C. albicans, DM + C. albicans + ASc and DM + C. albicans + NPASc. Rats were daily treated with ASc or NPASc (100 mg/kg) for 21 days. Biochemical parameters in serum and urine, advanced oxidation protein product (AOPP) and TBARS levels in the serum, kidney, liver and pancreas and N-acetyl-ß-d-glucosaminidase (NAG) activities in kidney and urine were evaluated. RESULTS: Biochemical and oxidative stress parameters increased in rats with DM and/or candidiasis. NPASc was more effective than ASc in decreasing glucose (56%), cholesterol (33%) and creatinine (51%) levels; serum (16%) and pancreatic (46%) AOPP and renal (48%) TBARS levels when compared with DM + C. albicans group. In C. albicans group, both treatments decreased NAG activity but did not decrease creatinine levels. CONCLUSIONS: These data suggest that the use of nanotechnology is able to improve plant extract properties such as antioxidant activity that may be useful in diabetes-related complications.

In Vitro Synergism between Azithromycin or Terbinafine and Topical Antimicrobial Agents against Pythium insidiosum.

We describe here in vitro activity for the combination of azithromycin or terbinafine and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate. With the exception of potassium permanganate, the remaining antimicrobial drugs were active and had an MIC90 between 2 and 32 µg∕ml. The greatest synergism was observed for the combination of terbinafine and cetrimide (71.4%). In vivo experimental evaluations will clarify the potential of these drugs for the topical treatment of lesions caused by Pythium insidiosum.

In Vitro and In Vivo Antimicrobial Activities of Minocycline in Combination with Azithromycin, Clarithromycin, or Tigecycline against Pythium insidiosum.

The present study investigated the in vitro and the in vivo interactions among azithromycin, clarithromycin, minocycline, and tigecycline against Pythium insidiosum. In vitro antimicrobial activities were determined by the broth microdilution method in accordance with CLSI document M38-A2, and the antibiotic interactions were assayed using the checkerboard MIC format. In vivo efficacy was determined using a rabbit infection model. The geometric mean MICs of azithromycin, clarithromycin, minocycline, and tigecycline against P. insidiosum were, respectively, 1.91, 1.38, 0.91, and 0.79 µg/ml. By checkerboard testing, all combinations resulted in in vitro synergistic interactions (>60%). Antagonism was not observed. The in vivo studies showed that azithromycin (20 mg/kg/day twice daily) alone or in combination with minocycline (10 mg/kg/day twice daily) significantly decreased the fungal burden. This study demonstrates that azithromycin possesses potent curative efficacy against subcutaneous pythiosis in the rabbit model.

Cholinergic enzymes and inflammatory markers in rats infected by Sporothrix schenckii.

The aim of this study was to evaluate the cholinesterase activity in serum, whole blood, and lymphocytes, as well as to verify its relation to immune response in rats experimentally infected by Sporothrix schenckii. For this study, 63 Wistar rats (Rattus norvegicus), male, adult were divided into three groups: the negative control group (GC: n = 21), the group infected subcutaneously (GSC: n = 21), and the group infected intraperitoneally (GIP: n = 21). The groups were divided into subgroups and the following variables were evaluated at 15, 30, and 40 days post-infection (PI): acetylcholinesterase (AChE) activity in lymphocytes and whole blood, butyrylcholinesterase (BChE) activity in serum, cytokines levels (IL-1, IL-6, TNFα, and INF-γ), immunoglobulins levels (IgA, IgG, IgM, and IgE), and protein profile by electrophoresis. Both infected groups showed increased levels of inflammatory parameters (P < 0.05) in tissue and inflammatory infiltrates. The activities of AChE in lymphocytes and BChE in serum increased (P < 0.05) significantly in animals from the GSC group on day 40 PI compared to the GC group. Regarding the GIP, there was a marked increase in the AChE activity in lymphocytes on days 30 and 40 PI, and in whole blood on days 15, 30, and 40 PI compared to GC. Furthermore, IL-10, an anti-inflammatory cytokine, was also present in high levels during chronic systemic S. schenckii infections in animals. Therefore, it is concluded that cholinesterase has an important modulatory role in the immune response during granulomatous infection by S. schenckii.

In vitro interaction of antifungal and antibacterial drugs against Cryptococcus neoformans var. grubii before and after capsular induction.

This study evaluated the synergistic interactions between amphotericin B (AMB) and azithromycin (AZM), daptomycin (DAP), linezolid (LNZ), minocycline (MINO), fluconazole (FLZ), flucytosine (5FC), linezolid (LZD), or tigecycline (TIG) against clinical isolates of Cryptococcus neoformans var. grubii before and after capsule induction. High synergism (>75%) was observed for the combinations, AMB+5FC, AMB+TIG, AMB+AZM, AMB+LZD and AMB+MINO but only in the strains after capsule induction. The results show that the presence of the capsule may lower the minimum inhibitory concentrations (MICs) of antifungal agents, but antimicrobial activity can be improved by combining antifungal and antibacterial agents.

New insights into the in vitro susceptibility of Pythium insidiosum.

We have determined the in vitro activity of several antibacterial and antifungal drugs against Pythium insidiosum using broth microdilution (BMD), disk diffusion, and Etest methods. The largest zones of inhibition (disk diffusion) and the lowest BMD and Etest MICs were observed for azithromycin, clarithromycin, linezolid, mupirocin, doxycycline, minocycline, and tigecycline. The in vitro activities observed suggest that antibacterials, which act by inhibiting protein synthesis, are promising candidate therapies for the treatment of pythiosis.

In vitro synergism observed with azithromycin, clarithromycin, minocycline, or tigecycline in association with antifungal agents against Pythium insidiosum.

We describe here the in vitro activities of azithromycin, clarithromycin, minocycline, or tigecycline alone and in combination with amphotericin B, itraconazole, terbinafine, voriconazole, anidulafungin, caspofungin, or micafungin against 30 isolates of the oomycete Pythium insidiosum. The assays were based on the CLSI M38-A2 technique and the checkerboard microdilution method. The main synergisms observed were through the combination of minocycline with amphotericin B (73.33%), itraconazole (70%), and micafungin (70%) and of clarithromycin with micafungin (73.33%).

Scutia buxifolia Reissek essential oil: in vitro antioxidant and antimicrobial activities.

The volatile oil from the stem bark of Scutia buxifolia (Rhamnaceae) has been obtained by hydrodistillation and analyzed by GC-MS. Twenty-one components were identified representing 99.93 % of the total oil composition, spathulenol (35.87%), ß-cubebene (17.26%), germacrene D (6.43%), linalool (5.19%), carvacrol (4.05%) were the main components of S. buxifolia essential oil. Antioxidant and antimicrobial properties of the essential oil were evaluated by free radical scavenging (DPPH) assay and micro broth dilution method, respectively. S. buxifolia essential oil presented interesting radical scavenging activity (IC50 = 15.03 ± 0.11 µg/mL). The antibacterial assay showed that S. buxifolia stem bark essential oil was moderately active against the Staphylococcus aureus and Micrococcus sp. (MIC = 500 µg/mL) and Escherichia coli (250 µg/mL). To the best of our knowledge, this is the first study on the composition, antioxidant and antimicrobial activities of essential oil from the S. buxifolia collected from Brazil.

Toll-deficient Drosophila is susceptible to Pythium insidiosum infection.

There is a paucity of animal models of pythiosis, a life-threatening disease of humans and animals, the immunopathogenesis of which is poorly understood. A pythiosis model was developed by injecting Toll (Tl)-deficient Drosophila melanogaster flies with Pythium insidiosum zoospores. The infected Tl mutant flies had significantly lower survival rates (73.7%) than did control flies. This study reveals the important role of Tl pathway activation in fly immune response to pythiosis.

Canine gastrointestinal pythiosis treatment by combined antifungal and immunotherapy and review of published studies.

Pythium insidiosum is an oomycete, a fungal like microorganism, which infects mammals, causing pythiosis in animals and humans, especially in tropical and subtropical regions around the world. The treatment for this infection is very difficult, and therapeutic options commonly comprise surgery, immunotherapy and antimicrobial drugs. The present report describes the clinical healing of a dog with gastrointestinal pythiosis by treatment with a combination of antifungals and immunotherapy, as well as reviews the cases reported in the literature that used some type of therapy for canine pythiosis. A 2.5-year-old male beagle initially showed sporadic vomiting episodes, and this symptom became more frequent 5 months after the onset of clinical signs. Celiotomy procedure found thickness of the stomach wall extending to the pylorus and duodenum. A biopsy was performed, and the diagnosis of pythiosis was made by mycological, histopathological analyses and molecular identification. Therapy was based on an association of terbinafine plus itraconazole during 12 months and immunotherapy for 2.5 months. The healing of the dog reported here allows us to propose the use of immunotherapy associated with antifungal therapy to treat canine gastrointestinal pythiosis. However, additional studies should be performed on a larger number of patients to establish a standard treatment protocol for canine pythiosis.