Mycotoxins Detection and Fungal Contamination in Black and Green Tea by HPLC-Based Method.

The fungal contamination and total aflatoxins (AF) and ochratoxin A (OTA) of tea samples were examined. A total of 60 tea samples were extracted and treated with immunoaffinity columns. The amount of AF and OTA were determined by using high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). Tea samples were cultured and the fungi were identified. The results showed that 24 (40%) samples were contaminated with AFs and none of the tea samples were above the acceptable limit of AFs (≥10 µg/kg). All of the samples were contaminated with OTA where only 3 black tea samples (6.6%) and 1 green tea sample (6.7%) were detected to have more than the standard limits of toxin (10 µg·kg-1). The mean concentration of OTA in the black tea was higher than green tea. Aspergillus niger was the predominant fungi isolated from black and green tea samples. Considering the high contamination of mycotoxins in tea samples, regular monitoring in the tea process for improving quality is recommended.

Burden of musculoskeletal disorders in Iran during 1990-2017: estimates from the Global Burden of Disease Study 2017.

Musculoskeletal diseases (MSDs) are the leading cause of disability and facing them demands updated reports on their burden for efficient policymaking. We showed Iran had the highest female-to-male ratio and highest increase in the burden of musculoskeletal diseases, in the past three decades, worldwide. We further confirmed the role of population aging as the main cause. PURPOSE: MSDs comprise most of the top causes of years lived with disability (YLDs) worldwide and are rapidly increasing in lower- and middle-income countries. Here, we present disability and mortality due to MSDs in Iran at the national level from 1990 to 2017. METHODS: We used Global Burden of Disease (GBD) 2017 Study data and standard methodology and presented the burden of MSDs in rates of years of life lost (YLLs), YLDs, and disability-adjusted life years (DALYs) during 1990-2017, for population aged ≥ 5 years old. We further explored attributable risk factors and decomposed the changing trend in DALYs to assess underlying causes. RESULTS: In Iran, MSDs were responsible for 1.82 million (95%uncertainty interval [UI] 1.3-2.4) DALYs, in 2017. During the past 28 years, with 1.75% annualized percentage change (APC), Iran had the highest percentage increase in the all-ages MSD DALYs rate worldwide, while the age-standardized DALYs APC was negligible. Low back pain was the greatest contributor to DALYs and caused 4.5% of total DALYs. The female population is experiencing considerably higher burden of MSDs, with 115% and 48% higher all-ages YLLs and YLDs rates per 100,000, respectively (YLLs 28.7; YLDs 2629.1), than males (YLLs 13.2; YLDs 1766.1). However, due to wide UIs, difference was not significant. Only 17.6% of MSD YLDs are attributable to assessed risk factors. CONCLUSION: Despite that MSDs are rising as an important cause of disability in Iran, these conditions are not sufficiently addressed in health policies. There is urgent need for cross-sectoral engagement, especially addressing the MSDs in females.

Screening the antifungal activities of monoterpenes and their isomers against Candida species.

AIM: Increasing the occurrence of non-albicans Candida species with intrinsic or acquired resistance to antifungals as well as the emergence of multidrug Candida species coupled with the limited antifungal agents challenges the treatment of candidiasis. Consequently, a class of secondary metabolites of plants exhibiting decent antifungal activity. Therefore, this study aimed to evaluate the antifungal potential of various monoterpenes including Carvone, Limonene, Pinene, Menthone, Menthol, Camphor, Thujone, Citronellol, and Piperitone against standard and clinical isolates of Candida. METHODS AND RESULTS: Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of compounds were determined, using the broth Microdilution method based on M27-A3 protocol documented by clinical laboratory standard institute (CLSI). Amongst the tested monoterpenes, oxygenated terpenoids showed strong antifungal activity. Specifically, alcoholic terpenoids such as (±)-Citronellol possess more efficacy than the corresponding ketonic ones with MICs ranging from 0·03 to 2·00 µl ml-1 (0·16-10·80 mmol l-1 ). Among the examined yeasts, Candida tropicalis was the most susceptible species to (±)-Citronellol. Moreover, the examined monoterpenes successfully inhibited the growth of fluconazole-resistant Candida species. Moreover, statistical analysis showed no statistically significant difference between the (+) and (-) isomers, except for (±)-α-Pienene and (±) Menthone (ρ-value < 0·05). CONCLUSION: Among the tested monoterpenes, (±)-Citronellol was the most potent compounds followed by (+)-α-Pinene and Menthol. Considering the significant antifungal activity of the examined monoterpenes, they could be used in controlling or treating candidiasis. Those potent antifungal monoterpenes with GRAS status in addition to their pleasant taste and odour make them appropriate additive or preservative compounds in food and cosmetics products. Furthermore, these data might help researchers to predict EOs antifungal activities, after determining its constituents. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information about the antifungal activities of monoterpenes and their isomers presented widely in essential oils. Screening results against pathogenic yeasts confirm the correlation between the chemical structure of tested monoterpenes and their antifungal effects. The present findings might be helpful to anticipate the antifungal activity of essential oils.

Molecular identification of non-Cryptococcus yeasts associated with pigeon droppings in Shiraz, Southern Iran.

BACKGROUND: Birds are considered as a reservoir for pathogenic and non-pathogenic fungi. Pigeon droppings have the potential for spreading these fungi to the environment. Cryptococcus species are important fungi associated with pigeon droppings. In this regard, there are many types of yeast associated with guano that is important for human and animal health. AIMS: The main objective of this study is the identification of non-Cryptococcus yeasts isolated from pigeon dropping in Shiraz, Southern Iran. METHODS: A total of 100 unknown yeasts, which were previously screened and identified as non-Cryptococcus from pigeon guano through the conventional methods, were used in this study. Identification of the isolates was performed based on conventional methods and DNA sequence analysis of internal transcribed spacer (ITS) rDNA gene region. The sequence results were deposited in NCBI database using the Basic Local Alignment Search Tool (BLAST). RESULTS: A total of 16 species belonging to 7 genera were identified as Candida spp. 51% (8 species), Rhodotorula sp. 24%, Trichosporon spp. 21% (3 species), Rhodosporidium 2%, Saccharomyces 1%, Rhizoctonia 1%, and Meyerozyma 1%. The predominant isolates were Rhodotorula rubra (24%), Candida famata (20%), and Trichosporon asahii (13%). The other species were Rhodosporidium kratochvilovae 2 (2%), Saccharomyces cerevisiae 1 (1%), Rhizoctonia solani 1 (1%), and Meyerozyma caribbica 1 (1%). CONCLUSION: Pigeon excreta examined in this study were associated with several kinds of opportunistic yeasts which could cause diseases in prone human and animals.

Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism.

OBJECTIVE: Cryptococcosis is a major opportunistic fungal infection caused by members of the genus Cryptococcus, mainly those belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. Here, we report a comprehensive molecular epidemiological study of the environmental distribution of Cryptococcus isolates in Shiraz, Iran with review of litreature. METHOD: A total of 406 samples were obtained from Eucalyptus trees and 139 samples from pigeon droppings. Cryptococcus species identification and genotyping were performed by amplified fragment length polymorphism (AFLP) fingerprinting sequencing and sequencing of the ITS rDNA region. RESULTS: Majority of the isolates belonged to the Naganishia taxon (n=69) including N. albida (formerly C. albidus, n=62), N. globosa (formerly C. saitoi, n=4), N. adeliensis (formerly C. adeliensis, n=2), N. diffluens (formerly C. diffluens, n=1), and the identified C. neoformans isolates (n=25) belonged to genotype AFLP1/VNI (n=22) and AFLP1B/VNII (n=3). CONCLUSION: More research efforts should be employed to isolate C. gattii species complex from environmental niches in Iran and provide additional evidence related to novel molecular types.

Prevalence of oral Candida colonization in patients with diabetes mellitus.

OBJECTIVES OF THE STUDY: We aimed to assess the prevalence of oral Candida colonization in patients with diabetes and its relationship with factors such as Candida species, serum glucose level, and the susceptibility rate of isolated yeasts to antifungals. PATIENTS: Random samples were obtained from 113 patients with type 2 diabetes, 24 patients with type 1 diabetes, and 105 healthy controls. MATERIALS AND METHODS: The samples were taken by swabbing the oral mucosa of patients with diabetes mellitus and healthy individuals. Afterwards the samples were inoculated onto CHROMagar-Candida. The growing colonies were counted, and the isolated yeasts were identified by PCR-RFLP and RapID methods. Various isolated species of Candida were also subjected to susceptibility testing of antibiotic drugs. Blood samples were taken to evaluate glycosylated hemoglobin (HbA1c). RESULTS: Although the Candida carriage rate and density were statistically higher in diabetics than healthy individuals, no direct association was found between having high Candida-burden and glycosylated hemoglobin. The most commonly isolated species in both diabetics and controls was Candida albicans. Of the tested antifungal drugs, the highest rate of resistance was found against itraconazole, followed in frequency by ketoconazole and fluconazole. CONCLUSIONS: This study identified a significant association between the poor glycemic control and the higher prevalence rates of Candida carriage and density in diabetic patients. In addition, a high prevalence of C. dubliniensis in diabetic patients was found, which might be misdiagnosed with its morphologically related species, C. albicans.

Proteolytic activity and cooperative hemolytic effect of dermatophytes with different species of bacteria.

BACKGROUND AND PURPOSE: Globally, dermatophytes are the most common filamentous group of fungi causing cutaneous mycoses. Dermatophytes were shown to secrete a multitude of enzymes that play a role in their pathogenesis. There is limited data on co-hemolytic (CAMP-like) effect of different bacterial species on dermatophyte species. In this study, we sought to the evaluate exoenzyme activity and co-hemolytic effect of four bacteria on clinical dermatophytes isolated from patients in Shiraz, Iran. MATERIALS AND METHODS: A total of 84 clinical dermatophyte species were isolated from patients suffering dermatophytosis and identified by conventional methods. Hemolytic activity was evaluated with Columbia 5% sheep blood agar. Proteolytic activity was determined by plate clearance assay method, using gelatin 8% agar. CAMP-like factor was evaluated with four bacteria, namely, S. areus, S.saprophyticus, S.pyogenes, and S.agalactiae. Fisher's exact test was run for statistical analysis. RESULTS: T. mentagrophytes was the most predominant agent (27 [32.1%]) followed by T. verrucosum(20 [23.8%]), T. tonsurans (10 [11.9%]), Microsporum canis (7 [8.3%]), T. rubrum (6 [7.1%]), E. floccosum (6 [7.1%]), M. gypseum (5 [6%]), and T. violaceum (3[3.6%]). The most common clinical area of dermatophytosis was the skin. All the isolates expressed the zone of incomplete alpha hemolysis. All the isolates had CAMP- positive reaction with S. aureus and the other bacteria were CAMP-negative. All the isolates expressed proteolytic activity and no significant differences were noted among diverse genera of dermatophytes and severities of proteolytic activity. CONCLUSION: This study indicated that hemolysin and proteolytic enzymes potentially play a role in dermatophyte pathogenesis and S. aureus could be considered as a main bacterium for creation of co-hemolytic effect in association with dermatophyte species.

Molecular identification and in-vitro antifungal susceptibility testing of Candida species isolated from patients with onychomycosis.

BACKGROUND AND PURPOSE: Candida species are the most opportunistic fungi affecting the nails and resulting in onychomycosis. In this study, we identified and evaluated in-vitro susceptibility of the recovered isolates against fluconazole (FLC), voriconazole (VRC), and clotrimazole (CLT) using the Clinical and Laboratory Standards Institute (CLSI) M27-A3 document. MATERIALS AND METHODS: From patients with either clinically or mycologically proven onychomycosis, 97 isolates comprising of seven Candida species were isolated, which were identified by both conventional and molecular techniques such as polymerase chain reaction-restriction fragment length polymorphism. In addition, Candida dubliniensis was confirmed by restriction endonuclease analysis. Antifungal susceptibility of each isolate against the three azoles applied in this study was determined using the CLSI microdilution reference method M27-A3. RESULTS: Candida parapsilosis(C. parapsilosis) was the most frequently isolated species (n=44), followed by C. albicans (n=23), C.tropicalis (n=13), C.glabrata (n=7), C.krusei (n=6), C.guilliermondii (n=3), and C. dubliniensis (n=1). All the isolates were susceptible to CLT. VRC had lower minimum inhibitory concentration (MIC) values for the isolates compared to FLC. Geometric mean MIC values of VRC, FLC, and CLT for C. parapsilosis isolates were 0.07 µg/ml, 0.8 µg/ml, and 0.35 µg/ml, respectively. Collectively, all species exhibited greater susceptibility to VRC in comparison to C. albicans (P≤0.001). CONCLUSION: This study showed that non-albicansCandida species were the most common etiologic agents of non-dermatophyte onychomycosis. The major antifungal agents used in clinics to empirically treat yeast onychomycosis are FLC and CLT. Our data suggested that CLT is a better choice for the treatment of Candida onychomycosis, especially in drug resistant cases.

Phospholipase, esterase and hemolytic activities of Candida spp. isolated from onychomycosis and oral lichen planus lesions.

OBJECTIVE OF THE STUDY: Candidiasis is an opportunistic fungal infection caused by many species of Candida that affects different sites of the body. Secretion of exoenzymes plays an important role in virulence and pathogenesis. The aim of this study was to evaluate the potential role of phospholipase, esterase and hemolytic activity of Candida species isolated from onychomycosis and oral lichen planus patients in candidiasis. METHODS: A total of 84 Candida isolates including 24 C. albicans and 46 C. parapsilosis from onychomycosis, and 14 C. albicans from oral lichen planus patients were included in our study. Egg yolk agar, Tween 80 opacity medium and blood agar plate assays were used for determining phospholipase, esterase and hemolytic activities, respectively. Statistical analyses were performed using the Fischer exact test. RESULTS: C. parapsilosis isolates had the least exoenzyme activity among the isolates (P≤0.001). C. albicans isolates from lichen planus showed less exoenzyme activity in comparison to the onychomycosis isolates. Only 16.08% of C. parapsilosis isolates had phospholipase activity. This difference between C. albicans and C. parapsilosis was statistically significant (P≤0.001). All of the C. albicans isolates from onychomycosis patients had beta hemolysin activity. There was more hemolytic activity in the C. albicans isolates when compared with C. parapsilosis and between C. albicans isolates, no significant difference was seen. All of the C. albicans isolates produced esterase enzyme on day three and no significant differences were seen between the two groups of C. albicans for esterase activity. Two strains of C. albicans had no phospholipase or esterase activity. CONCLUSION: This study showed that most of the isolates tested had different enzymatic patterns and C. parapsilosis strains had less phospholipase activity.

Synthesis and antifungal activity of benzimidazole, benzotriazole and aminothiazole derivatives.

In recent years, the use of antifungal drugs in human medicine has increased, especially with the advent of AIDS epidemic. Efforts have focused on the development of new, less toxic and more efficacious antifungal drugs with novel mechanism of action. The purpose of this study was to synthesize of some new benzimidazole, benzotriazole and aminothiazole derivatives and to evaluate their activity against some species of Candida, Aspergillus and dermatophytes. The desired compounds were synthesized by the reaction of benzimidazole and benzotriazole with bromoalkanes and also by the reaction of an amide derivative of aminothiazole with 2-piperazino-1-ethanol in an efficient solvent in the presence of tetraethyl ammounim bromide or triethylamine) as catalyst. Chemical structures of all the new compounds were confirmed by spectrophotometric methods. Antifungal activities of the new compounds were evaluated by broth micro dilution method as recommended by CLSI. Among the tested compounds, 1-nonyl-1H-benzo[d]imidazole and 1-decyl-1H-benzo[d]imidazole exhibited the best antifungal activities. Of the examined synthetic compounds in different categories, benzimidazole derivatives established better antifungal activities than benzotriazole derivatives, and the piperazine analogue had no significant antifungal effect.

Chemical composition and antimicrobial activities of the essential oils from three ecotypes of Zataria multiflora.

BACKGROUND: Zataria multiflora Boiss. is a traditional and popular spice in Iran. The effects of 3 ecotypes (ECTPs) of Z. multiflora essential oils (EOs) against most common causes of food-borne and nosocomial infections were evaluated. MATERIALS AND METHODS: The antimicrobial activities of the EOs were examined by broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI). The chemical compositions of the EOs from 3 ECTPs of Z. multiflora have been analyzed by gas chromatography-mass spectrometry. RESULTS: Analysis of the EOs indicated that 3 chemotypes were present in Z. multiflora, including carvacrol, thymol-carvacrol, and linalool, whereas previous studies have only found carvacrol and thymol. Inhibition studies showed that the tested EOs entirely inhibited the growth of yeasts at concentrations of less than 1 µL/mL. Moreover, the oils exhibited significant bacteriostatic and bactericidal activities against Gram-positive and Gram-negative bacteria at concentrations ranging from 0.12 to 8 µL/mL. CONCLUSION: These results suggest that the EOs from Z. multiflora should be investigated further for possible use in antimicrobial products and food preservatives.

Atypical presentation of Old-World cutaneous leishmaniasis, diagnosis and species identification by PCR.

BACKGROUND: The diagnosis of cutaneous leishmaniasis (CL) is traditionally based on microscopic demonstration of amastigote forms in tissue biopsies or smears. However, this method usually presents low sensitivity, and in atypical forms, CL may be overlooked because of similarity to other dermal diseases. Thus, it is necessary to apply specific diagnostic methods as polymerase chain reaction (PCR). OBJECTIVE: To evaluate the possible advantage of PCR in the diagnosis and species identification of CL in patients with atypical clinical presentation. METHODS: Fifty-one patients clinically suspected of CL with positive and negative controls were tested. After microscopic examination, extraction of DNA was performed on their smears and analysed by two specific PCR assays for diagnosis and species identification. For these methods, conserved and variable regions of kinetoplastic DNA (KDNA) of Leishmania species have been amplified, respectively. Atypical forms of CL were evaluated among PCR-positive patients. RESULTS: PCR results were positive in 37 out of 51 cases (72.5%), among whom microscopic examination revealed Leishmania amastigotes in only 3 (5.9%). Among these patients, 10 (27%) had atypical presentation of CL; using species-specific primers, 6 patients had Leishmania major, 3 had Leishmania tropica and 1 patient had no species diagnosis. None of the samples of other dermal diseases revealed positive results (specificity, 100%). All patients were successfully treated by CL-specific drug regimens. DISCUSSION: The results showed that KDNA PCR methods have a higher sensitivity compared with microscopic method. Moreover, PCR could identify the parasite species for specific therapy. Microscopic method had low sensitivity and less value in chronic and atypical CL cases.

Synthesis of novel azo Schiff bases and their antibacterial and antifungal activities.

Ten new azo Schiff bases 5a-h and 7a-b were prepared in excellent yields via the condensation of different aromatic amines and a new azoaldehyde, 2-hydroxy-3- methoxy-5-(4-methoxyphenylazo)benzaldehyde (4) by two different methods. All new compounds were tested against five microorganisms: Staphylococcus aureus (Gram positive and methicillin resistant), Bacillus subtilis (Gram positive), Kelebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli (all Gram negative). Compounds 4, 5a, 5c, 5d and 5g were moderately active against Staphylococcus aureus and Bacillus subtilis. Compound 7b was highly active against Bacillus subtilis and moderately active against Staphylococcus aureus. Other compounds were inactive against these strains of bacteria. The antifungal activities of these compounds were also tested against eight different fungal species. None of them were active against the fungi species tested.