Candida parapsilosis as a potent biocontrol agent against growth and aflatoxin production by aspergillus species

Aflatoxin contamination of food and feed stuff is a serious health problem and significant economic concerns. In the present study, the inhibitory effect of Candida parapsilosis IP1698 on mycelial growth and aflatoxin production in aflatoxigenic strains of Aspergillus species was investigated. Mycelial growth inhibitions of nine strains of aflatoxigenic and non-aflatoxigenic Aspergillus species in the presence of C. parapsilosis investigated by pour plate technique at different pH, temperature and time of incubation. Reduction of aflatoxin was evaluated in co-cultured fungi in yeast extract sucrose broth after seven days of incubation using HPLC method. The data were analyzed by SPSS 11.5. The presence of the C. parapsilosis at different pH did not affect significantly the growth rate of Aspergillm isolates. On the other hand, temperature and time of incubation showed to be significantly effective when compared to controls without C. parapsilosis [P<0.05]. In aflatoxigenic strains, minimum percentage of reductions in total aflatoxin and BI, 62, Gi, G2 fractions were 92.98, 92.54, 77.48, 54.54 and 72.22 and maximum percentage of reductions were 99.59, not detectable, 94.42, and not detectable in both GI and G2, respectively. C parapsilosis might employ as a good biocontrol agent against growth and aflatoxin production by aflatoxigenic Aspergillus species

Mycological microscopic and culture examination of 400 bronchoalveolar lavage [BAL] samples

The frequency of invasive opportunistic mycoses has increased significantly over the past decades especially in immunocompromised patients. Invasive aspergillosis [IA] has become a major cause of morbidity and mortality among these patients. As bronchoalveolar lavage [BAL] fluid samples are generally useful specimens in the diagnosis of invasive pulmonary aspergillosis [IPA], this study was designed to evaluate the incidence of fungal elements in at-risk patients by direct microscopy and culture of BAL samples. In a 16-month period, 400 BAL samples were obtained from several groups of different patients with pulmonary and respiratory disorders and examined by using both direct microscopy and culture. Of the 400 samples, 16 [4%] were positive direct examination with branching septate hyphae and 46 [11.5%] were positive culture: 25 [54%] Aspergillus flavus, 6 [13%] A. fumigatus, 5 [10.9%] A. niger, 1 [2.2%] A. terreus, 3 [6.5%] Penicillium spp. and 6 [13%] mixed A. flavus/A. niger. A. flavus was the most common cause of Aspergillus infection or colonization. Bone marrow transplant [BMT] recipients were the most susceptible group to fungal infection and/or colonization. Among Aspergillus species, A. flavus was the most common isolate in both infections and colonization in Iran. More studies are needed to clarify the epidemiological aspect of aspergillosis in Iran

Species identification and strain typing of candida isolates by PCR-RFLP and RAPD-PCR analysis for determining the probable sources of nosocomial infections

Since the incidence of nosocomial Candida infections have been increasing in parallel with the raising in the number of patients involving in predisposing factors, determining the sources and the ways of acquisition of infection is necessary as an efficient strategy for controlling the diseases. The aim of this study is identification and strain typing of Candida strains isolated from hospitals to facilitate tracing the sources of infections in hospitalized patients in addition to assess the discriminatory power of some random primers by using RAPD analysis. Samples were collected from patients who were hospitalized in oncology, intensive care unit [ICU], and organ transplants wards of the Shohadaye-Tajrish Hospital affiliated to Tehran University of Medical Sciences, Tehran, Iran, and their environments. The yeasts were isolated on CHRO Magar Candida. Species identification was performed by PCR-amplification of ITS1-5.8SrDNA-ITS2 region followed by restriction digestion with the enzyme MspI. To determine the probable origin of Candidia infections, in case of each patient whose the clinical and relevant environmental isolates were identified as the same species, a set of eight primers namely number 1 to 8, were applied in RAPD-PCR to find out the possible homogeny or variation within the isolated strains. One hundred and four Candida strains were identified. The most common species was C. albicans [57.5%] followed by C. tropicalis [13.5%], C. glabrata [12.5%], C. parapsilosis [8.65%], C. famata [3.8%], C. krusei [1.9%], C. guilliermondii [0.96%]. and C. lusitaniae [0.96%]. While the source of infection for three patients were not determined by RAPD analysis, interpretable results from RAPD-typing of Candida species isolated from 8/18 of cases implied that the infections might originate from the exogenous sources. Moreover, according to the function of each primer, primer No. 1 was determined as a best primer for typing of Candida albicans strains. The species of yeast isolates were determined by PCR-RFLP. It was found that RAPD assay can point out the genomic variability within the Candida species. Besides, the method could show a probable relationship between acquired infections and their sources

Inhibitory effect of farnesol on biofilm formation by Candida tropicalis

Candidiasis associated with indwelling medical devices is especially problematic since they can act as substrates for biofilm growth which are highly resistant to antifungal drugs. Farnesol is a quorum-sensing molecule that inhibits filamentation and biofilm formation in Candida albicans. Since in recent years Candida tropicalis have been reported as an important and common non-albicans Candida species with high drug resistance pattern, the inhibitory effect of farnesol on biofilm formation by Candida tropicalis was evaluated. Five Candida tropicalis strains were treated with different concentration of farnesol [0, 30 and 300 micro M] after 0, 1 and 4 hrs of adherence and then they were maintained under biofilm formation condition in polystyrene, 96-well microtiter plates at 37°C for 48 hrs. Biofilm formation was measured by a semiquantitative colorimetric technique based on reduction assay of 2,3- bis -2H-tetrazolium- 5- carboxanilide [XTT]. The results indicated that the initial adherence time had no effect on biofilm formation and low concentration of farnesol [30 micro M] could not inhibit biofilm formation. However the presence of non-adherent cells increased biofilm formation significantly and the high concentration of farnesol [300 micro M] could inhibit biofilm formation. Results of this study showed that the high concentration of farnesol could inhibit biofilm formation and may be used as an adjuvant in prevention and in therapeutic strategies with antifungal drugs

Molecular characterization of cyclophilin protein gene in skin normal microflora: malassezia furfur

Malassezia are dimorphic, lipid-dependent yeasts, which are responsible for causing several cutaneous and systemic conditions. Although cyclophilins [CyPs] are highly conserved cytosolic proteins that catalyze the peptidyl-prolyl cis-trans isomerazation reaction before protein folding process, it has been suggestive of an allergen in a few numbers of fungi such as Aspergillus fumigatus and Malassezia species. Allergenic cyclophilins are IgE-binding components, which have been characterized in other species of Malassezia; and are considered as Mala s 6 in Malassezia sympodialis. In the present study we tried to identify the molecular characterization of cyclophilin gene in M. furfur. Pairs of oligonucleotide primers were designed from highly conserved regions of the gene counterparts in other fungi. The primers were then applied to amplify the primer-specific DNA fragment. Afterward, PCR product fragments were sequenced to be used in further analysis. About 573 nucleotides, encoding a polypeptide of 190 amino acids, have been sequenced. Sequence comparison was performed in Gene Bank, both for the nucleotides and their deduced amino acid sequence. It revealed a significant homology with cyclophilin genes and proteins of other eukaryotic cells. The amino acid sequence of the encoded protein was about 86% identical to the sequence of cyclophilin protein from other fungi. The molecular characterization of cyclophilin gene may open the way to disclosure of the functional characteristics of cyclophilin and is a fundamental step for understanding the molecular basis of its pathogenesis in AEDS disease

Fungal nail infections in Tehran, Iran

Onychomycosis results from invasion of the nail plate by dermatophytes, yeasts or mould species of fungi. The objective was to determine the etiological agents of onychomycosis. A total of 549 patients clinically suspected of onychomycosis were examined for causative fungal agents. Both direct microscopy and the cultures of the nail material were performed to identify the causative agents between 2004-2005 in Tehran, Iran. Out of 549 cases examined, 263 [47.9%] were mycologically proven cases of onychomycosis [139 finger, 124 toe nails], among those 33 [6.09%] were only positive in direct microscopic examination. From an etiological point of view, 21.85% of nail infections were caused by yeasts, 10.55% were infected by dermatophytes and 15.5% by non-dermatopyte moulds.Candida albicans was the common yeast causative agent [16.73%] followed by A. flavus [11.78%], T. mentagrophytes [10.26%], [2.66%], Aspergillus spp [1.90%], each of Rhizopus spp and Cladosporium spp [1.52%], C.giulliermondii [1.14%], Scopolariopsis spp. [1.14%], each of C. famata, C.glabrata, C. krusei, S. lusitania, Acremonium spp. [0.76%] and C. homicola [0.38%], T. rubrum [4.94%]. Candida species were most common responsible agent for onychomycosis in female hands [74.1%] followed by 17.26% non-dermatophyte moulds. Dermatophytes caused tinea unguim of hand [8.63%] and peduum [37.1%] in males. The yeasts of the Genus Candida and non-dermatophyte moulds are dominant cause of female finger nail onychomycosis and dermathophytes are principal causes of both finger and toe nails in males in Tehran

Single strand conformation polymorphism analysis of PCR-amplified rDNA to differentiate medically important Aspergillus species

Aspergillus species are associated with allergic bronchopulmonary disease, mycotic keratitis, otomycosis, nasal sinusitis and invasive infection. In this study, we developed a PCR-Single Strand Conformational Polymorphism method to identify the most common Aspergillus species and we showed some advantages of this method comparing a PCR-Restriction Fragment Length Polymorphism with our designed restriction enzyme. We selected ITS2, as a short fragment within the rDNA region [length size: 330 bp] to be amplified as small size PCR product. We mixed 5 ml of the PCR product with an equal volume of loading buffer and followed by incubation for 5 min at 95°C and quenching in an ice bath. The mixture was applied to a 6%-12% Gradient Poly acryl amide gel to run in a vertical electrophoresis, then gel was stained with ethidium bromide and silver nitrate which followed by an ethidium bromide staining. Our results of restriction digestion showed a fine identification of 7 tested Aspergillus species during 5-6 hours after an overnight mycelial growth. As our results some of tested Aspergillus species: A. nidulans, A.fisheri, A. quadricincta, [A. fumigatus and A. niger] as a group and [A. flavus, A. tereus and A. ochraceus] as another group, can be discriminated. Moreover SSCP analysis enabled us to identify above Aspergillus species within 8-12 h after an over night growth without using an expensive restriction enzyme. It is concluded that Single Strand Conformational Polymorphism is a simple and rapid method for identification of some medically important Aspergillus

Detection of Aflr gene and toxigenicity of Aspergillus flavus group isolated from patients with fungal sinusitis

Aspergillus flavus is the second most important Aspergillus species causing human infections particularly fungal sinusitis. Since little is known about aflatoxin producing ability of clinical isolates, this study was undertaken to detect the aflatoxigenic isolates amongst these isolates. A total of 23 isolates of A. spp. which were recovered from patients proved to have fungal sinusitis by morphological and histological methods and also 5 additional aflatoxigenic and non-aflatoxigenic reference of A. flavus group strains were studied. The isolates were identified morphologically using Czapek Yeast Agar and A. flavus and parasiticus Agar [AFPA]. Aflatoxin producing ability of the isolates was confirmed by Thin Layer Chromatography. Existing of aflR gene the regulatory gene in aflatoxin biosynthesis, were studied in all isolates by PCR method. All twenty three Aspergillus isolates confirmed as A. flavus group by their macroscopic and microscopic features. One clinical isolate confirmed as A. oryzae by mycological methods. A. oryzae as well as A. flavus JCM2061 and NCPF2008 and 3 clinical isolates were not able to produce orange pigment on AFPA. From total of 23 isolates 4 [17.4%] confirmed to be aflatoxigenic by TLC method. A banding pattern which matched to aflR primers was amplified with approximate size of 800 bp in all 23 clinical A. flavus isolates. A larger banding pattern 1050 bp was revealed in clinical isolate; strain no.20 as well. Some clinical sinus isolates are able to produce aflatoxin and all of studied isolates including; A. oryzae, A. parasiticus and A. sojae were able to amplify aflR gene under our laboratory conditions

Identification of aspergillus species using morphological characteristics

Although molecular methods continue to improve and become more rapidly available, microscopy and culture remain commonly used and essential tools for identification of Aspergillus spp. In this study we emphasize on morphological methods including; macroscopic and microscopic characteristics for identification of Aspergillus species isolated from environmental and clinical specimens. We used four differential media: czapek dox agar [CZ], czapek yeast agar [CYA], malt extract agar [MEA], and czapek yeast 20% sucrose agar. Morphological features of colonies on above culture media as well as microscopically characteristics for the major strains were studied and then compared with those of standard Aspergillus strains. Our major subjects were Iranian Aspergillus strains isolated from clinical and environmental specimens. Standard Aspergillus strains for study development included; A. fumigatus, [JCM 10253], A. flavus [JCM 2061], A. niger [JCM 10254], A. nidulans [JCM 02728], A. tereus [JCM 10227]. Morphological features of Aspergillus cultures were studied, the major and remarkable macroscopic features in species identification were the colony diameter, color [conidia and reverse], exudates and colony texture. Microscopic characteristics for the identification were conidial heads, stipes, color and length vesicles shape and seriation, metula covering, conidia size, shape and roughness also colony features including diameter after 7 days, color of conidia, mycelia, exudates and reverse, colony texture and shape. Finally we compared the morphological characteristics of tested Aspergillus isolates with those of the standard species Aspergillus isolates were identified in the level of species using the differential culture media. A total of 205 Aspergillus isolates studied included: 153[75%] environmental Aspergilli and 52 [25%] clinical isolates. Within 11 Aspergillus species identified, A.flavus [55%], A.niger [31.7%] and A. fumigatus [8.7%] were the most common Aspergillus isolates from all of the specimens. In our view morphological method using the differential media is the most reliable and sensitive assay to identify more medically important Aspergillus species

Performance of five phenotypical methods for identification of Candida isolates from clinical materials

Although Candida albicans is the most common etiologic agent of candidiasis, C. dubliniensis, has been emerged, as another pathogen resembles C. albicans in many phenotypic aspects and noted for its in vitro potential for fluconazole resistance. Since there was no evidence of any report about detection of this organism in Iran, this study was designed to use of five different tests for identification of Candida species with special reference to C. dubliniensis among 313 suspected Candida isolates in Tehran, capital of Iran. Overall, 199 [63.6%] C. albicans and 114 [36.6%] Candida spp. were identified. All 199 C. albicans isolates were found germ tube and chlamydospore positive. Different shades of green color colonies were yielded on CHROMagar Candida of which 23 [11.6%] showed dark green color indicative of C. dubliniensis. All but four C. albicans isolates grew well at 45 °C. These 4 isolates beyond to 23 dark green colony producers were suspected of being C.dubliniensis, later examined by API 20C AUX system. The results indicated that all 27 isolates were able to assimilate both xylose and alpha-methyl-D-glucoside, therefore these isolates were identified as C. albicans. Overall, C. dubliniensis had not been found in present study. It must be concluded that no single phenotypic test has proven to be highly effective, and the use of several tests may be necessary of these two closely related Candida species for definitive identification

Fungi as Causative agent of nasal polyps

Nasal polyposis is an inflammatory condition of unknown etiology that involves nasal and sinus mucous membrane. These polyps can impair a person's quality of life by nasal obstruction, recurrent sinusitis, persistent postnasal drainage, hyposmia, anosmia, changes in sense of taste and even bony destruction. It has been shown that chronic inflammation causes a reactive hyperplasia of the intranasal mucosal membrane which results in the formation of polyps. Recently, fungal elements were suspected to be the causative agent of chronic rhinosinusitis and a fungal etiology has been proposed to underlie severe nasal polyposis. The present study was undertaken to determine the role of fungi in development of nasal polyps. In this study resected polyps from 100 patients were examined by mycological and pathological methods for the presence of fungi. Fungal elements were shown in 9 samples by mycological methods and isolated fungi were Aspergillus flavus, Aspergillus fumigatus and Rhizopus sp. Tissue invasion by fungi also was seen by histopathological examination in 6 patients. Therefore, fungi could be considered as the causative factor in the development of nasal polyposis in those patients and since medical treatment of nasal polyps have become increasingly recognized in recent years, the present study also implying the benefits of topical antifungal therapy in such cases

Study on the sources of nosocomial fungal infections at intensive care unit and transplant wards at a teaching hospital in Tehran

The incidence of nosocomial fungal infections has increased dramatically during the past two decades as the consequence of continuous increase in the number of severely immunocompromised patients. This study was done to determine the presumptive sources of nosocomial fungal infections at the intensive care unit and transplant wards [in a university- based teaching hospital in Tehran] during a 10-month period. Totally 583 samples were obtained from the air, surfaces, health care workers and also from the patients at those wards. Mycological culture of the samples yielded growth of 25 different genus and species of fungi and the most common isolated fungi were Candida albicans, Penicillium spp., Aspergillus niger, and Cladosporium spp., respectively. It was noted that health care workers were carrying fungi on their hands [50%], nasal mu-cosa [57.6%], in oral cavity [38.6%] and also by their shoes [92.3%] and uniforms [92.7%]. Environmental fungal contamination was shown and it was more prominent at the intensive care unit. Hospitalization also had more significant effect on colonization of fungi in the patients at the latter ward. Therefore, the highly susceptible patients in present study were at the greatest risk of developing fungal infections and preventive measures were critical for prevention and control of these life-threatening fatal infections

Anti-Cryptococcal-Globulin-Latex production for rapid detection of cryptococcus neoformans polysaccharide antigen in cryptococcosis

Cryptococcosis has become the fourth leading life-threatening opportunistic infection in patients with AIDS, but also occurs in non-AIDS patients. In view of the increasing numbers of infection during last decade in Iran, use of rapid, sensitive and specific test for diagnosis of cryptococcal disease has become important than ever. We aimed to produce the reagents for latex cryptococcal antigen test. The antigen was prepared from NCPF 3168 strain of Cryptococcus neoformans. Anticapsular antiserum of C. neoformans raised in rabbits and latex carboxylate- modified beads were coated with antiserum. The maximally- reactive globulin dilution was obtained at dilution of 1:400. For evaluation of efficacy of reagents, challenged 38 BALB/C mice and other 38 mice were used as controls. The mice were bled and autopsied. Brain, heart and lung were checked by direct, histopathological and cultural examination for cryptococcosis. The sera from case and control mice were tested with Immunomycologic [Immy] kit and also our produced reagents [OPR] for detection of cryptococcal antigen. Moreover, 15 cerebrospinal fluid and 15 serum samples from patients with cryptococcal meningitis, 30 with aspergillosis, 30 with suspected other fungal infections, and 30 from healthy individuals were tested as well. The results showed that the sensitivity [97.3%] and specificity [100%] of OPR was quite comparable with those of Immy kit. Therefore, it could be regarded as a substitute for commercial kits

[Prevalence and identification of etiological agents of funguria in foley catheterized patients]

Background: Nosocomial infections are a group of infectious diseases acquired during hospitalization. Catheter-associated urinary tract infection is the most common nosocomial disease in catheterized patients. The first sign of this infection is symptomatic or asymptomatic bacteriuria or funguria. The aim of this study was to determine the point prevalence of funguria and urinary tract infection as well as their etiological agents in foley catheter users

Patients and Methods: Over a seven- month period, a total of 509 urine samples from 101 catheterized patients at Shariati Hospital, Tehran, were collected. Uncentrifuged urine samples were used for colony count. Culture, direct examination and leucocyte count were done on urine sediments. Identification of etiological agents was performed by conventional methods and API 20C-Aux commercial system

Results: Bacteriuria, funguria and mixed infections were observed in 33.7 percent [34 cases], 11.9 percent [12 cases] and 16.8 percent [17 cases] of the cases, respectively. The prevalence rate of funguria was 21.27 percent for males and 35.1 percent for females. The etiological agents were: Candida albicans [24 cases], Candida glabrata [4 cases] and Cryptococcus laurentii [1 case]. The highest number of colony forming units in 1 milliliter of the urine was 185000. None of the patients showed signs of urinary tract infection. Nine patients developed funguria after replacement of the catheter

Conclusion: The presence of yeasts in the urine should not be considered as a sign of urinary tract infection, particulary in the presence of foley catheters, however, it may increase the risk of urinary tract infection

Fungal Involvement in Patients with Paranasal Sinusitis

Fungal involvement of the paranasal sinuses is frequently observed in the immunocompromised host and it can become lifethreatening if it is not diagnosed. Definitive diagnosis is made by tissue biopsy and culture. In this study biopsy materials of maxillary, ethmoidal and frontal sinuses of 60 patients with clinical manifestation of sinusitis and no response to medical therapy were assessed by mycological and pathological methods for the presence of fungi. Invasive fungal sinusitis was diagnosed in 3 patients and etiologic agents were C and ida albicans, Rhizopus sp. and Aspergillus fumigatus. Predisposing factors in these patients were leukemia, diabetes mellitus and previous sinus and polyp surgery, respectively. Allergic fungal sinusitis also was seen in one patient and Alternaria sp. isolated from the biopsy material. Only the patient with allergic form of disease survived but all the patients with invasive form of fungal infection were expired. This clearly underscores the need of early recognition of fungal sinusitis in at risk population in order to start urgent treatment. In this study Nocardia asteroids also was isolated from the biopsy sample in a patient with sinunasal adenocarcinoma

epidemiological approach to the zoophilic dermatophytoses in Iran

Dermatophytosis in domestic animals constitutes a constant source of infection for persons in contact with them. To have an epidemiological picture of zoophilic dermatophyte infections in Iran, a study has been carried out during a period of three years [1986-1989] in an attempt to find the causative dermatophytes which infect cats and cattle and also infected human subjects in contact with them. For this purpose, 9850 samples of hair and skin were collected from suspected cattle, 953 from suspected cats, and 2326 from infected human subjects. Clinical diagnosis was confirmed by direct microscopic examination and culture. The species isolated from all cattle were Trichophyton verrucosum; from cats, Microsporum canis and man, M. canis, 1583[68.1%] and T. verrucosum 743[31.9%]. From the infected human cases, mostly Tinea capitis and Tinea corporis were detected among the age groups of 1-9 and 20-29 years old, respectively. The incidence rate observed in winter and fall was higher than spring and summer

case report of cryptococcal meningitis

Case of cryptococcal meningitis is reported in a 26 year old man by the Medical Mycology Department of the School of Public Health, Tehran University. The patient with the symptom of meningitis was admitted to the hospital with the symptom of meningitis was admitted to the hospital. Lumbar puncture was performed in the hospital. After direct examination and culture of cerebrospinal fluid, Cryptococcus neoformans were isolated