Synthesis, characterization and antifungal activity of a novel formulated nanocomposite containing Indolicidin and Graphene oxide against disseminated candidiasis.

OBJECTIVE: Candidiasis is one of the most opportunistic fungal infections in immunocompromised patients. The emergence of multidrug-resistant Candida species necessitates the development of novel antifungal agents. Seeking to the discovery of natural antifungal agents, this study aimed to synthesize a novel formulated nanocomposite containing Indolicidin (IN), antimicrobial peptide, and Graphene oxide (GO), kind of nanomaterial, against Candida growth using in vitro and in vivo experiments for the first time. METHODS: The formulated nanocomposite (GO-IN) synthetized and was characterized using scanning electron microscopy, X-ray power diffraction, and fourier transform infrared method analysis. The in vitro antifungal activity of fluconazole (FLU), GO, IN, and GO-IN was determined against Candida albicans (C. albicans) compared to control groups, cell cytotoxicity assay on human intestinal epithelial cells (IEP) and hemolytic activities were performed. Moreover, in vivo experiments of nanocomposite were assessed in BALB/c mice. RESULTS: Our results showed that nanocomposite had the highest inhibitory effect against C. albicans (MIC 3.12µg/mL) compared with flu (MIC 4µg/mL), IN (MIC 12.5µg/mL), and GO (MIC 6.25µg/mL). Viability of human intestinal cell line at the MIC concentration (3.12µg/mL) of nanocomposite (GO-IN) was detected as 60% (P<0.05). The results of hemolytic activity showed that nanocomposite cause 2.73% of red blood cell membrane damage. For in vivo experiments, infected mice were successfully treated with GO-IN once a day within 7 days. GO-IN treated group eliminated the Candida infection in the spleen and liver of BALB/c mice (P=0.001) similar to fluconazole. There was no significant difference in histological manifestations between flu and GO-IN groups. CONCLUSION: This study suggests that synergistic combination of GO and IN provide a new option, representing a potential therapeutic efficiency against disseminated candidiasis in an animal model as well as might be used as adjunct therapy in the management of candidiasis. However, further investigation is needed to evaluate the efficacy of the nanocomposite.

Iranian HIV/AIDS patients with oropharyngeal candidiasis: identification, prevalence and antifungal susceptibility of Candida species.

Oropharyngeal candidiasis is the commonest mucocutaneous infection in HIV-positive individuals. Herein, samples were taken from oral cavities of 150 HIV-infected patients and cultured on Sabouraud-dextrose agar; 89 (59·3%) of 150 patients had positive culture for Candida and presented clinical sign of classical oral candidiasis. Totally, 102 morphologically distinct colonies were isolated from Candida positive cultures and subsequently identified by polymerase chain reaction and sequencing assay, presenting the following frequency: 54 C. albicans (52·9%), 16 C. dubliniensis (15·7%), 12 C. tropicalis (11·8%), 9 C. glabrata (8·8%), 7 C. kefyr (6·9%) and 4 C. africana (3·9%). Additionally, multiple Candida species were co-isolated from 13·5% (12/89) patients. Regarding the antifungal susceptibility test, which was performed by CLSI protocol (M27-A3/M27-S3), all Candida isolates were susceptible to amphotericin B and caspofungin, while some of them were resistant to fluconazole (17·6%; 16 C. albicans, 1 C. dubliniensis and 1 C. glabrata), itraconazole (16·7%; 15 C. albicans, 1 C. dubliniensis and 1 C. tropicalis) and voriconazole (5·9%; 5 C. albicans and 1 C. tropicalis). Collectively, our findings reinforce the urgent necessity to find new therapeutic agents to treat oral candidiasis in HIV-positive patients, especially due to the high incidence of azole-resistant Candida strains and the increased frequency of non-C. albicans species. SIGNIFICANCE AND IMPACT OF THE STUDY: The Candida species recovered from oral cavity of 150 Iranian HIV/AIDS patients and their antifungal susceptibility profiles were reported. Candida albicans was the commonest Candida species, followed by C. dubliniensis, C. tropicalis, C. glabrata, C. kefyr and C. africana. All Candida isolates were susceptible to amphotericin B and caspofungin, while resistance to azoles was detected. The growing drug-resistance profile reported in clinical isolates of C. albicans and non-C. albicans strains is a serious problem in hospitals worldwide. Consequently, the suitable antifungal choice to treat the HIV/AIDS population with oral candidiasis needs to be rethought and new therapeutic options must urgently arise.

A wide diversity of zoonotic intestinal parasites in domestic and stray dogs in rural areas of Kermanshah province, Iran.

Dogs can act as reservoirs, carriers, and transmitters of several zoonotic intestinal parasites that can cause serious health problems for humans. The aim of this study was to determine the prevalence of intestinal parasites in dogs in Kermanshah Province, west of Iran. Faecal samples were collected from domestic and stray dogs from 30 rural areas of Kermanshah province from August 2014 to April 2015 and were analyzed by formalin-ether sedimentation, sucrose otation technique and the modied Ziehl-Neelsen method. Out of 301 dogs examined, 230 (76.4%) were infected with at least one parasite. The incidence on the different types of intestinal parasitic species recovered from domestic and stray dogs are as follows: Toxocara leonina (20.8% and 27.6%), T. canis (7.5% and 9.4%), Taenia spp. (9.2% and 9.4%), hookworm spp. (18.3% and 33.7%), Capillaria spp. (0.8 and 1.7%), Dicrocoelium dendriticum (0.8% and 3.3%), Fasciola spp. (0.8% and 2.2%), Acanthocephal spp. (3.3% and 5.5%), Trichuris vulpis (0.8% and 1.7%), Dipylidium caninum (4.2% and 3.3%), Physaloptera spp. (6.7% and 6.6%), Cryptosporidium spp.(21.7% and 25.4%), Eimeria spp. (35.0% and 34.3%), Giardia spp. (6.7% and 12.7%), Cystoisospora spp. (7.5% and 5.5%), Blastocystis spp. (18.3% and 20.4%) and Sarcocystis spp. (6.7% and 7.2%), respectively. Signicant difference in infection rates was observed between domestic and stray dogs (P>0.05). Hookworm and Eimeria spp. were the most common intestinal helminth and protozoa detected with 83 (27.6%) out of 301 and 104 (34.6%), out of 301, respectively. There was no significant difference in prevalence and distribution of intestinal parasites between male and female dogs (P>0.05). The wide range of zoonotic parasites indicated that people residing in rural areas in Kermanshah province are at risk of exposure to these pathogens. In this respect, appropriate implementation of control programs by public health authorities and veterinarians should be taken into account.

Overexpression of MDR-1 and CDR-2 genes in fluconazole resistance of Candida albicans isolated from patients with vulvovaginal candidiasis.

BACKGROUND AND PURPOSE: Candida albicans (C. albicans) is an opportunistic fungus that can colonize women's mucosal epithelial cell surfaces, causing vulvovaginitis in specific circumstances. The major genes contributing to drug resistance in C. albicans are the candida drug resistance (CDR) and multi drug resistance (MDR) genes. The purpose of this study was to evaluate the CDR-2 and MDR-1 gene expression patterns in C. albicans strains isolated from patients with recurrent vulvovaginal candidiasis. MATERIALS AND METHODS: In this study, 40 isolates of fluconazole-resistant C. albicans were cultured on Sabouraud dextrose agar. These isolates were collected from women with vulvovaginitis who were referred to a clinic in Tehran, Iran, and transferred to a mycology laboratory. Then, RNA was extracted from the isolates using phenol-chloroform and glass beads, and the complementary DNA (cDNA) was synthetized. To detect the semi-quantitative expression of CDR-2 and MDR-1 genes, the reverse transcriptase-PCR (RT-PCR) technique was performed using specific primers. RESULTS: Our findings indicated that of the 40 C. albicans isolates, 35 (87.5%) strains were positive for mRNA of the CDR-2 gene, 32 (80%) strains expressed mRNA of the MDR-1 gene, and 30 (75%) strains were confirmed to express mRNA of both the CDR-2 and MDR-1 genes simultaneously using the RT-PCR assay. CONCLUSION: According to the obtained results, the expression rates of CDR-2 and MDR-1 genes were high in fluconazole-resistant C. albicans isolates, which can cause treatments to fail and result in chronic infections. Inhibiting these important genes using novel or natural agents can help with the treatment of chronic and recurrent vaginitis.

Fractionation and identification of the allergic proteins in Aspergillus species.

BACKGROUND AND PURPOSE: Allergy is an undesired immune response to non-pathogenic agents. However, some opportunistic microorganisms such as fungi can also cause allergy. Among those fungi, hyphae form of Aspergillus strains including A. fumigatus, A. flavus, and A. niger could be mentioned. In this study, we aimed to separate allergic proteins from Aspergillus strains and determine their identity. MATERIALS AND METHODS: Standard species of Aspergillus strains were cultivated in optimized conditions and the mycelium was separated by centrifugation. The fungal cells were lysed through physical methods such as freeze-thawing and grinding to prepare a suitable protein extract. The protein concentration was measured by Bradford method and the electrophoretic pattern of the extract was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were fractionated by ammonium sulfate precipitation and anion exchange chromatography using fast protein liquid chromatography (FPLC) system. The IgE immunoreactivity of the sensitized patients and controls was studied using the fractionated proteins by enzyme-linked immunosorbent assay (ELISA). Following SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes and the strips were blotted with allergic patients' and controls' sera. The immunoreactive bands were excised from colloidal coomassie-stained SDS-PAGE gels and studied by mass spectroscopy methods. RESULTS: Among the studied species, A. fumigatus showed stronger IgE reactivity and more IgE reactive protein bands than others did. The proteins with higher molecular weights showed stronger immunoreactivity in Western blotting. Receiver operating characteristic curve analysis demonstrated a correlation between the results of the applied ELISA methods. One of the most prominent IgE-reactive proteins was confirmed to be 45 kDa mycelia catalase. CONCLUSION: Our findings confirmed that high molecular weight proteins might play a major role in allergy and IgE reactivity to Aspergillus species. Moreover, the results showed that precipitation and chromatographic methods are applicable for fractionation of fungal proteins such as mycelial catalase.

The effect of nanochitosans particles on Candida biofilm formation.

BACKGROUND AND PURPOSE: In people wearing dentures, the growth of various Candida species under the prosthesis leads to the formation of biofilm, which can play the role of a reservoir for Candida and other kinds of microbes. Since nano-chitosan particles can cause lasting antimicrobial activity, a more recent approach that utilizes acrylic resins with nano-chitosan particles is proposed. Therefore, we aimed to study the inhibitory effect of nano-chitosan particles on the biofilm formation of Candida species in acrylic resins. MATERIALS AND METHODS: In this analytical in-vitro study, acrylic resins with nano-chitosan particles with concentrations of 0, 1%, 5%, and %10 were put adjacent to the suspension of Candida cells isolated from the individuals' mouth and biofilm formation on resins was measured and compared. Finally, the data were analyzed using Kruskal-Wallis and Chi-square tests. RESULTS: The observed differences between unmodified acrylic resin (control) and acrylic resin with nano-chitosan particles in terms of biofilm formation were significant (P<0.05) but no significant difference was found in the formation of biofilm species on resins. CONCLUSION: Biofilm formation of Candida species depends on acrylic resin type, in a way that by adding nano-chitosan particles to acrylic resins, biofilm formation of Candida species was significantly reduced. To decrease the organization of biofilm and denture stomatitis, the use of acrylics with nano-chitosan particles in producing dentures is recommended.

Characterization and identification of candiduria due to Candida species in diabetic patients.

BACKGROUND AND PURPOSE: The presence of Candida yeasts in urine, known as candiduria, is an indicator of infection or colonization of the urinary tract by Candida species. This condition in diabetic patients can be hazardous due to diminished immune system response. The objective of this study was to investigate the incidence of candiduria in diabetic patients and to identify its causative agents. Furthermore, the demographic and laboratory (HbA1c, urine glucose and pH, urine culture colony count, and fasting blood sugar) data and their possible associations with candiduria were investigated. MATERIALS AND METHODS: This cross-sectional, descriptive study was performed on 305 diabetic patients referred to the diabetes research center, Hamedan, Iran, during April 2015 to September 2015. Urine and blood specimens were collected and urine analysis, urine culture, FBS, and HbA1c tests were performed. Positive cases were subjected to colony count and the causative agents were subsequently identified through the routine identification tests, as well as colony color in CHROMagar Candida medium, and the assimilation patterns in API 20 C auxanographic method. RESULTS: Among the 305 cases, 38 (%12.5) were positive for candiduria. Causative agents were identified as Candidaglabrata (n=19, 50%), C. albicans (n=12, 31.6%), C. krusei (n=4, 10.5%), C. tropicalis (n=2, 5.3%), andC. kefyr (n=1, 2.6%). According to the results of the statistical analyses, there were significant association between candiduria and female gender, high FBS and urine glucose, uncontrolled diabetes (HbA1c ≥8), and acidic urine pH (P<0.05). CONCLUSION: Considering the high incidence rate of candiduria in diabetic patients, control of diabetes, predisposing factors, and causal relationships between diabetes and candiduria should be highlighted.

Evaluation of the antifungal activities of various extracts from Pistacia atlantica Desf.

BACKGROUND AND PURPOSE: Despite the availability of various treatments for fungal diseases, there are some limitations in the management of these conditions due to multiple treatment-related side-effects. The present study was designed to investigate the antifungal properties of different extracts from Pistacia atlantica Desf. MATERIALS AND METHODS: Different parts of P. atlantica (i.e., dried fruit, fresh fruit and dried leaf) were separately extracted via percolation method with 80% methanol and water. Gas chromatography/mass spectrometry (GC/MS) analysis was performed to determine the main constituents of leaf and fruit extracts from P. atlantica. In vitro anti-Candida activities of the extracts against Candida albicans, Candida glabrata and Saccharomyces cerevisiae were studied. For this purpose, the minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) were determined, using broth microdilution method, according to the modified M27-A3 protocol on yeasts, proposed by the Clinical and Laboratory Standards Institute (CLSI). RESULTS: Based on GC/MS analysis, the main constituents of P. atlantica fruit extracts were ß-myrcene (41.4%), α-pinene (32.48%) and limonene (4.66%), respectively, whereas the major constituents of P. atlantica leaf extracts were trans-caryophyllene (15.18%), α-amorphene (8.1%) and neo-allo-ocimene (6.21%), respectively. As the findings indicated, all the constituents exhibited both fungistatic and fungicidal activities, with MICs ranging from 6.66 to 26.66 mg/mL and MFCs ranging from 13.3 to 37.3 mg/mL, respectively. Among the evaluated extracts, the methanolic fresh fruit extract of P. atlantica was significantly more effective than other extracts (P<0.05). CONCLUSION: Based on the findings of the present study, novel antifungal agents need to be developed, and use of P. atlantica should be promoted in the traditional treatment of Candida infections.

A survey of the etiological agents of scalp and nail dermatophytosis in Yazd, Iran in 2014-2015.

BACKGROUND AND PURPOSE: Tinea capitis and tinea unguium are regarded as global public health concerns. The purpose of the present study was to identify the etiological agents of tinea capitis and tinea unguium in patients, referring to the Central Laboratory of Yazd University of Medical Sciences, Yazd, Iran. MATERIALS AND METHODS: This study was conducted during 2014-2015. Skin scraping, scalp hair, and nail clipping specimens were collected from 134 patients (80 males and 54 females) with clinical features suggesting fungal involvement. Direct microscopic examinations were carried out, using potassium hydroxide 10%, while culture studies were performed on Sabouraud dextrose agar, containing chloramphenicol and cycloheximide at 28°C for four weeks. Fungal colonies were identified based on their macroscopic and microscopic characteristics, as well as supplementary diagnostic tests. RESULTS: Among 134 patients, 12 cases showed positive results on direct examination and culture studies. The frequency of infections was equal among male and female subjects. Among 12 affected cases, the frequency of tinea capitis and tinea unguium was 91.6% and 8.4%, respectively. Microsporum canis (50%) was the most prevalent species, followed by Trichophyton verrucosum (25%) and Trichophyton mentagrophytes (25%). Also, tinea unguium, caused by T. mentagrophytes, was found in a female patient. CONCLUSION: The etiological agents of scalp and nail dermatophytosis have changed in Yazd over the past 13 years. In the present study, replacement of anthropophilic dermatophytes by zoophilic species was noteworthy, highlighting the necessity of efficient surveillance for the management and prevention of infections.

A study on etiologic agents and clinical manifestations of dermatophytosis in Yazd, Iran.

BACKGROUND AND PURPOSE: Dermatophytosis is one of the most common infections of skin, hair, and nails, caused by a group of keratinophilic fungi known as dermatophytes. Species identification of these fungi is of great significance from epidemiological and therapeutic points of view. The objective of the present study was to investigate dermatophytosis and its causative agents in patients, referring to the Central Mycology Laboratory of Yazd University of Medical Sciences, Yazd, Iran. MATERIALS AND METHODS: In total, 139 clinically suspected cases of dermatophytosis were examined during 12 months from February 2014 to February 2015. Skin scrapings were assessed through direct microscopic examinations and culture studies. Dermatophyte isolates were identified based on colony morphology on potato dextrose agar and dermatophyte test medium, nutritional requirements, urease and hair perforation tests, and microscopic characteristics on slide cultures. RESULTS: Dermatophytosis was mycologically confirmed in 26 (18.70%) out of 139 cases. Although there was a statistically insignificant difference between male and female subjects, men were dominantly affected. Infection was significantly common in the age group of ≤ 29 years (P<0.043). The most common clinical manifestation of dermatophytosis was tinea corporis (69.2%), followed by tinea cruris (15.4%), tinea manuum (11.5%), and tinea pedis (3.8%). Trichophyton mentagrophytes complex was the main etiologic agent (38.5%), followed by T. rubrum (23%), T. violaceum (15.5%), T. verrucosum (11.5%), Microsporum canis (7.7%), and Epidermophyton floccosum (3.8%). CONCLUSION: In comparison with previous research, epidemiology of dermatophytosis has changed in Yazd over the past decades. Therefore, periodical investigations on the epidemiological aspects of this infection are required for efficient control and prevention of this cutaneous dermatophytic disease.

Identification of Prototheca zopfii from Bovine Mastitis.

BACKGROUND: The aim of this study was identification of the epidemiology of Prototheca zopfii species from the milk samples of dairy cattle in Isfahan, central Iran. METHODS: Milk samples were obtained from 230 dairy cattle, 130 with and 100 without mastitis, in Isfahan. The samples were cultured in Prototheca Isolation Medium (PIM) and Sabouraud's dextrose agar. All P. zopfii isolates were identified by morphological and biochemical methods. Then, as a confirmatory test they were examined by genotype-specific PCR. RESULTS: Four P. zopfii strains (3.07%) were isolated from the 130 samples of dairy cattle with clinical mastitis and there was no isolation from totally 100 samples of healthy bovines without mastitis. Specific PCR product (about 946 bp) was detected in four isolates. CONCLUSION: It seems that P. zopfii genotype II plays a key role in affecting bovine mastitis that confirmed other previous studies. Our study was the first, which identified the Prototheca species by traditional and molecular methods in Iran and Middle East as well.

Characteristics of dermatophytoses among children in an area south of Tehran, Iran.

The aim of this study was to determine the prevalence and etiological agents of dermatophytoses, and also their distribution according to age, gender, and body site among children in an area south of Tehran. A total of 382 children aged