Stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes by human monocytes

Monocytes/macrophages play a critical role in the defense mechanisms against malaria parasites, and are the main cells responsible for the elimination of malaria parasites from the blood circulation. We carried out a microscope-aided evaluation of the stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes, by human monocytes. These cells were obtained from healthy adult individuals by means of centrifugation through a cushion of Percoll density medium and were incubated with erythrocytes infected with Plasmodium falciparum that had previously been incubated with a pool of anti-plasmodial immune serum. We described the stages of phagocytosis, starting from adherence of infected erythrocytes to the phagocyte membrane and ending with their destruction within the phagolisosomes of the monocytes. We observed that the different erythrocytic forms of the parasite were ingested by monocytes, and that the process of phagocytosis may be completed in around 30 minutes. Furthermore, we showed that phagocytosis may occur continuously, such that different phases of the process were observed in the same phagocyte.

Atividade antimicrobiana, antiaderente e antifúngica in vitro de plantas medicinais brasileiras sobre microrganismos do biofilme dental e cepas do gênero Candida

Avaliou-se in vitro a atividade antimicrobiana, antifúngica e antiaderente da aroeira-do-sertão, malva e goiabeira sobre microrganismos do biofilme dental e candidose oral. Os extratos mostraram-se eficazes, inibindo o crescimento das bactérias do biofilme dental e fungos da candidose oral, sugerindo a utilização dessas plantas como meio alternativo na terapêutica odontológica.

Produção de fatores de virulência in vitro por espécies patogênicas do gênero Candida

Avaliou-se, in vitro, a capacidade de crescimento em 39ºC e 42ºC, a produção de enzimas hidrolíticas e a atividade hemolítica de 21 cepas clínicas e de referência de sete espécies de Candida spp, Candida dubliniensis e Candida krusei demonstraram menor potencial de virulência e Candida albicans maior.

Comparison of in vitro activity of five antifungal agents against dermatophytes, using the agar dilution and broth microdilution methods

The purpose of this study was to compare the agar dilution and broth microdilution methods for determining the minimum inhibitory concentration (MIC) of fluconazole, itraconazole, ketoconazole, griseofulvin and terbinafine for 60 dermatophyte samples belonging to the species Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. The percentage agreement between the two methods, for all the isolates with < 2 dilutions that were tested was 91.6% for ketoconazole and griseofulvin, 88.3% for itraconazole, 81.6% for terbinafine and 73.3% for fluconazole. One hundred percent agreement was obtained for Trichophyton mentagrophytes isolates evaluated with ketoconazole and griseofulvin. Thus, until a reference method for testing the in vitro susceptibility of dermatophytes is standardized, the similarity of the results between the two methods means that the agar dilution method may be useful for susceptibility testing on these filamentous fungi.

Primeira descrição da caracterização fenotípica e susceptibilidade in vitro a drogas de leveduras do gênero Cryptococcus spp isoladas de pacientes HIV positivos e negativos, Estado de Mato Grosso

Foram avaliados 37 isolados de 10 pacientes HIV negativos e 26 positivos, em Mato Grosso. Exame direto, cultura e quimiotipagem de espécies foram realizados. Cetoconazol, itraconazol, voriconazol, fluconazol e anfotericina B foram avaliados. Foram identificadas 37 leveduras do gênero Cryptococcus spp sendo 26 de pacientes HIV- positivos (25 Cryptococcus neoformans e um Cryptococcus gattii) e 10 de HIV- negativos (cinco Cryptococcus neoformans e cinco Cryptococcus gattii). Considerando isolados clínicos (Cryptococcus neoformans) de HIV positivos observou-se resistência (8% e 8,7%) e susceptibilidade dose-dependência (20% e 17,4%) para fluconazol e itraconazol respectivamente. Para isolados de Cryptococcus neoformans oriundos de pacientes HIV negativos, observou-se susceptibilidade dose-dependência (40%) ao fluconazol. Os isolados de Cryptococcus gattii provenientes de pacientes HIV- negativos mostraram-se susceptíveis a todos os antifúngicos, exceto um isolado de Cryptococcus gattii que foi susceptível dose-dependente ao fluconazol (20%). O isolado proveniente do paciente HIV- positivo demonstrou resistência ao fluconazol (CIM > 256µg/mL) e itraconazol (CIM=3µg/mL).

Aspectos micológicos e suscetibilidade in vitro de leveduras do gênero Candida em pacientes HIV-positivos provenientes do Estado de Mato Grosso

INTRODUÇÃO: A candidíase é uma das infecções fúngicas mais frequentes entre os pacientes infectados pelo vírus da imunodeficiência humana. O presente estudo objetivou a caracterização das leveduras do gênero Candida de distintas amostras clínicas, provenientes de pacientes HIV - positivos, assim como a determinação do perfil de suscetibilidade in vitro a cinco drogas antifúngicas. MÉTODOS: A caracterização dos isolados de Candida sp foi realizada através da metodologia clássica, testes bioquímicos (zimograma e auxanograma) e morfológicos (prova do tubo germinativo e microcultivo em lâmina). Também, foram realizadas a técnica genotípica (PCR) e identificação pelo método comercial API 20C AUX (BioMeriéux). Para a determinação do perfil de suscetibilidade in vitro, foram utilizadas cinco drogas antifúngicas (cetoconazol, fluconazol, itraconazol, voriconazol e anfotericina B), através do método comercialmente disponível - Etest. RESULTADOS: Foram identificados 105 isolados de leveduras do gênero Candida provenientes de 102 pacientes infectados pelo vírus HIV. Destes, foram caracterizadas 82 (78,1%) Candida albicans, 8 (7,6%) Candida parapsilosis, 8 (7,6%) Candida tropicalis, 4 (3,8%) Candida krusei, 2 (1,9%) Candida glabrata e 1 (1%) Candida guilliermondii. CONCLUSÕES: Considerando o perfil geral de sensibilidade, 60% dos isolados foram suscetíveis a todos os antifúngicos testados, porém as espécies C. tropicalis e C. krusei demonstraram uma tendência a valores mais elevados de CIMs para os azóis do que os encontrados paraC. albicans, sugerindo resistência.

Ultrastructural study on the morphological changes to male worms of Schistosoma mansoni after in vitro exposure to allicin

INTRODUCTION: Garlic has a wide range of actions, including antibacterial, antiviral, antifungal, antiprotozoal and anthelmintic actions. This antiparasitic activity has been attributed to allicin, which is the main constituent of garlic. The present study aimed to investigate the in vitro activity of allicin on the tegument of adult Schistosoma mansoni worms using scanning electron microscopy. METHODS: Swiss Webster mice were infected with S. mansoni cercariae (100 per mouse) and sacrificed 50 days later to acquire the adult worms. These worms were collected by perfusion and placed in RPMI medium 1,640 at 37°C before transferring to RPMI media containing 0 (control), 5, 10, 15 and 20mg/mL of allicin, where they were incubated for 2h. The worms were fixed in 2.5% glutaraldehyde solution, washed twice, post-fixed in osmium tetroxide, washed twice and then dehydrated with ascending grades of ethanol. The samples were air-dried, mounted on stubs, gold coated in an ion sputtering unit and viewed using a scanning electron microscope. RESULTS: A concentration of 5mg/mL caused wrinkling in the tegument; a concentration of 10mg/mL resulted in changes to tubercles and loss or modification of spines. With 15 and 20mg/mL increasing damage to the tegument could be seen, such as vesicle formation and the presence of ulcers. CONCLUSIONS: These findings demonstrate the effect of allicin on adult S. mansoni worms and indicate that most of the changes occur at concentrations greater than that normally indicated for treatment.

In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida

INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3%) of isolates tested were phospholipase positive and 16 (44.4%) were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%). Statistically significant differences were observed in relation to production of phospholipases among species (p<0,0001) and among the strains from different sites of origin (p=0.014). Regarding the production of acid protease, the isolates of C. parapsilosis tested presented a larger number of producers (69.2%). Among the species analyzed, the percentage of protease producing isolates did not differ statistically (χ2=1.9 p=0.5901 (χ2=1.9 p=0.5901). CONCLUSIONS: The majority of C. non-albicans and all C. albicans isolates were great producers of hydrolytic enzymes and, consequently, might be able to cause infection under favorable conditions.

Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period

INTRODUCTION: In this study, we aimed at identifying Candida isolates obtained from blood, urine, tracheal secretion, and nail/skin lesions from cases attended at the Hospital Universitário de Londrina over a 3-year period and at evaluating fluconazole susceptibilities of the isolates. METHODS: Candida isolates were identified by polymerase chain reaction (PCR) using species-specific forward primers. The in vitro fluconazole susceptibility test was performed according to EUCAST-AFST reference procedure. RESULTS: Isolates were obtained from urine (53.4%), blood cultures (19.2%), tracheal secretion (17.8%), and nail/skin lesions (9.6%). When urine samples were considered, prevalence was similar in women (45.5%) and in men (54.5%) and was high in the age group >61 years than that in younger ones. For blood samples, prevalence was high in neonates (35%) and advanced ages (22.5%). For nail and skin samples, prevalence was higher in women (71.4%) than in men (28.6%). Candida albicans was the most frequently isolated in the hospital, but Candida species other than C. albicans accounted for 64% of isolates, including predominantly Candida tropicalis (33.2%) and Candida parapsilosis (19.2%). The trend for non-albicans Candida as the predominant species was noted from all clinical specimens, except from urine samples. All Candida isolates were considered susceptible in vitro to fluconazole with the exception of isolates belonging to the intrinsically less-susceptible species C. glabrata. CONCLUSIONS: Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.

In vitro antifungal activity of fatty acid methyl esters of the seeds of Annona cornifolia A.St.-Hil. (Annonaceae) against pathogenic fungus Paracoccidioides brasiliensis

INTRODUCTION: Fatty acids are abundant in vegetable oils. They are known to have antibacterial and antifungal properties. METHODS: Antifungal susceptibility was evaluated by broth microdilution assay following CLSI (formerly the NCCLS) guidelines against 16 fungal strains of clinical interest. RESULTS: In this work, fatty acid methyl esters (FAME) was able to inhibit 12 clinical strains of the pathogenic fungus Paracoccidioides brasiliensis and were also active in the bioautographic assay against Cladosporium sphaerospermum. CONCLUSIONS: FAME was a more potent antifungal than trimethoprim-sulfamethoxazole against P. brasiliensis under the experimental conditions tested.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

The influence of carbohydrates in the interaction of Paracoccidioides brasiliensis with CCL-6 cells in vitro

INTRODUCTION: Little is known about the early events in the interaction between Paracoccidioides brasiliensis and its host. To understand the effect of carbohydrates in the interaction between the fungus and epithelial cell in culture, we analyzed the influence of different carbohydrate solutions on the adhesion of P. brasiliensis yeast cells to CCL-6 cells in culture. METHODS: Fungal cells were cultivated with the epithelial cell line, and different concentrations of D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine, D-galactosamine, sorbitol and fructose were added at the beginning of the experiment. Six hours after the treatment, the cells were fixed and observed by light microscopy. The number of P. brasiliensis cells that were adhered to the CCL-6 monolayer was estimated. RESULTS: The number of adhesion events was diminished following treatments with D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine and D-galactosamine as compared to the untreated controls. Sorbitol and fructose-treated cells had the same adhesion behavior as the observed in the control. P. brasiliensis propagules were treated with fluorescent lectins. The FITC-labeled lectins WGA and Con-A bound to P. brasiliensis yeast cells, while SBA and PNA did not. CONCLUSIONS: The perceptual of adhesion between P. brasiliensis and CCL-6 cells decreased with the use of D-mannose, N-acetyl-glucosamine and D-glucosamine. The assay using FITC-labeled lectins suggests the presence of N-acetyl-glucosamine, α-mannose and α-glucose on the P. brasiliensis cell surface. An enhanced knowledge of the mediators of adhesion on P. brasiliensis could be useful in the future for the development of more efficient and less harmful methods for disease treatment and control.

In vitro predatory activity of conidia of fungal isolates of the Duddingtonia flagrans on Angiostrongylus vasorum first-stage larvae

INTRODUCTION: Angiostrongylus vasorum is a nematode that parasitizes molluscs, dogs, and even man. METHODS: The objective was to evaluate the predatory activity of the conidia of two fungal isolates of Duddingtonia flagrans (AC001 and CG722) on first-stage larvae (L1) of A. vasorum in laboratory conditions. RESULTS: At the end of the experiment, there were significant reductions (p<0.01) of 74.5% and 63.2%, on average, in the A. vasorum L1 recovered in the AC001 and CG722 treatment conditions, respectively. CONCLUSIONS: The two isolates of fungi were efficient in the capture and destruction of A. vasorum L1.

In vitro detection of hepatitis C virus in platelets from uninfected individuals exposed to the virus

IntroductionDespite hepatocytes being the target cells of hepatitis C virus (HCV), viral ribonucleic acid RNA has been detected in other cells, including platelets, which have been described as carriers of the virus in the circulation of infected patients. Platelets do not express cluster differentiation 81 CD81, the main receptor for the virus in hepatocytes, although this receptor protein has been found in megakaryocytes. Still, it is not clear if HCV interacts with platelets directly or if this interaction is a consequence of its association with megakaryocytes. The aim of this study was to evaluate the interaction of HCV with platelets from non-infected individuals, after in vitro exposure to the virus.MethodsPlatelets obtained from 50 blood donors not infected by HCV were incubated in vitro at 37°C for 48h with serum containing 100,000IU∕mL of genotype 1 HCV. After incubation, RNA extracted from the platelets was assayed for the presence of HCV by reverse transcription – polymerase chain reaction RT-PCR.ResultsAfter incubation in the presence of virus, all samples of platelets showed HCV RNA.ConclusionsThe results demonstrate that, in vitro, the virus interacts with platelets despite the absence of the receptor CD81, suggesting that other molecules could be involved in this association.

In vitro activity of antimicrobial combinations against multidrug-resistant Pseudomonas aeruginosa

Introduction Pseudomonas aeruginosa isolates related to nosocomial infections are often resistant to multiple antibacterial agents. In this study, antimicrobial combinations were evaluated to detect in vitro synergy against clinical isolates of P. aeruginosa. Methods Four clinical P. aeruginosa isolates were selected at random among other isolates from inpatients treated at the public University hospital in Ribeirão Preto, SP, Brazil. Two isolates were susceptible to imipenem (IPM-S) and several other antimicrobials, while the other two isolates were imipenem and multidrug resistant (IPM-R). The checkerboard method was used to assess the interactions between antimicrobials. Results Combinations of imipenem or other anti-Pseudomonas drugs with complementary antibiotics, such as aminoglycosides, fosfomycin and rifampin, reached synergy rates of 20.8%, 50%, 62.5% and 50% for the two IPM-S and two IPM-R Pseudomonas isolates, respectively. Imipenem, piperacillin-tazobactam and ceftazidime yielded a greater synergy rate than cefepime or ciprofloxacin. Synergist combinations were more commonly observed when the complementary drug was tobramycin (65%) or fosfomycin (57%). Conclusions Some antibacterial combinations led to significant reductions of the minimum inhibitory concentrations of both drugs, suggesting that they could be clinically applied to control infections caused by multidrug-resistant P. aeruginosa.