[Show abstract][Hide abstract] ABSTRACT: Identification of uncommon species of the clinical isolates of the yeasts by ITS-sequencing
By using advanced detection/identification methods, the list of emerging uncommon opportunistic yeast infections is rapidly expanding worldwide. In the present study, our aim was identifying the rare clinically yeast isolates. Forty nine out of 855 (5.7%) yeast isolates which formerly remained unidentified by PCR-RFLP method were subjected to sequence analysis of internal transcribed spacers (ITS) regions. These species include: Hanseniaspora uvarum, Saccharomyces cerevisiae, Sporidiobolus salmonicolor, Pichia fabianii, Pichia fermentans, Candida famata, Candida inconspicua, Candida maqnoliae, Candida guilliermondii, Candida kefyr, Candida rugosa, Candida lusitaniae, Candida orthopsilsis, and Candida viswanathii. Opportunistic infections caused by rare yeasts have increased in recent decade and some of these yeasts may resistant to some antifungals. Conventional methods could not definitely identify all yeast species, thus correct identification of yeasts using modern and reliable methods is essential.
58th Annual Meeting of JSMM, Yokohma, Japan; 11/2014
[Show abstract][Hide abstract] ABSTRACT: Dermatophytosis is a fungal infection caused by dermatophytes. These fungi are taxonomically classified in the genera Trichophyton, Microsporum, and Epidermophyton. Pleomorphism, cultural variability, slow growth and sporulation, and the need for additional physiological tests make dermatophytes notoriously difficult to identify. The present study aimed to compare the results of morphological and molecular identification of certain groups of clinical isolates of dermatophytes with a view to evaluating the accuracy of molecular methods.
Journal de Mycologie Médicale / Journal of Medical Mycology. 11/2014;
[Show abstract][Hide abstract] ABSTRACT: Introduction and objectives: Pulmonary aspergillosis (PA) is one of the most serious complications in different patients, in particular among immunocompromised individuals. In contrast, PA occurs very infrequently in immunocompetent patients. However, in this study, immunocompetent patients were evaluated for PA by conventional and molecular methods.
Materials and methods: Two hundred and fifteen BAL specimens obtained from the immunocompetent patients. The specimens were evaluated by routine mycological methods, nested-PCR on internal transcribed spacer (ITS) region, and TaqMan real-time PCR targeted beta-tubulin (β–TUB) gene.
Results: Of the 215 specimens, 6 (2.8%) were positive (septate hyphae) in direct examination, 21 (9.8%) had positive culture including 10 A. flavus, 3 A. fumigatus, one A. niger, one A. terreus, 5 mixed A. flavus/A. niger and one Penicillium spp. sixty (27.9%) specimens had positive nested-PCR with A. flavus and 33 (15.3%) with A. fumigatus specific primers. Only 6 (2.8%) had positive real-time PCR for A. flavus and 3 (1.4%) for A. fumigatus.
Conclusion: Positive results of the nested-PCR technique were too high that may reflect contamination. In addition, although the positive results of routine mycological methods and real-time PCR were not significant, but the patients with pulmonary disorders should be followed by further investigations to rapidly confirm the diagnosis.
8th International Congress of Clinical Microbiology, Tabriz; 09/2014
[Show abstract][Hide abstract] ABSTRACT: We investigated the resolving power of the beta tubulin protein-coding gene (BT2) for systematic study of dermatophyte fungi. Initially, 144 standard and clinical strains belonging to 26 species in the genera Trichophyton, Microsporum, and Epidermophyton were identified by internal transcribe spacer (ITS) sequencing. Subsequently, BT2 was partially amplified in all strains, and sequence analysis performed after construction of a BT2 database that showed length ranged from approximately 723 (T. ajelloi) to 808 nucleotides (M. persicolor) in different species. Intraspecific sequence variation was found in some species, but T. tonsurans, T. equinum, T. concentricum, T. verrucosum, T. rubrum, T. violaceum, T. eriotrephon, E. floccosum, M. canis, M. ferrugineum, and M. audouinii were invariant. The sequences were found to be relatively conserved among different strains of the same species. The species with the closest resemblance were Arthroderma benhamiae and T. concentricum and T. tonsurans and T. equinum with 100% and 99.8% identity, respectively; the most distant species were M. persicolor and M. amazonicum. The dendrogram obtained from BT2 topology was almost compatible with the species concept based on ITS sequencing, and similar clades and species were distinguished in the BT2 tree. Here, beta tubulin was characterized in a wide range of dermatophytes in order to assess intra- and interspecies variation and resolution and was found to be a taxonomically valuable gene.
[Show abstract][Hide abstract] ABSTRACT: Most of cutaneous leishmaniasis cases occur in only 7 countries, including Iran. Leishmania tropica is the main cause of anthroponotic cutaneous leishmaniasis in Iran. In order to study the heterogeneity and phylogeny of L. tropica in southern Iran, a total of 61 isolates were obtained from Bam district and the cities Kerman and Shiraz. The internal transcribed spacer (ITS) from the ribosomal DNA locus was amplified and then analysed by sequencing. Analysis of the ITS sequences showed four haplotypes in the isolates, including 3 haplotypes among the 58 isolates from the south eastern region, including Bam district and Kerman city, and 2 haplotypes among the 3 isolates from Shiraz city. The results showed a monophyletic structure for the south eastern population. In comparison to GenBank sequences of L. tropica from different countries, most of the southeast Iranian and Indian isolates are comprised in one cluster, while isolates from other countries and few other Iranian isolates group in a different cluster. Analysis of ITS sequences of south eastern L. tropica showed a homogeneous population which could be the basis for other molecular epidemiology studies using more discriminative markers and tracing possible changes in the population structure of L. tropica.
[Show abstract][Hide abstract] ABSTRACT: Background: Determination of β-D-Glucan (BDG) in the serum aids to diagnose the invasive fungal infections. The current study evaluated the diagnostic potential value of BDG assay in monitoring the disease in experimental systemic candidiasis in a rat model. The results can provide a useful preliminary data to improve this approach in developing countries.
Objectives: The present study aimed to evaluate β-D-Glucan assay in diagnosis and monitoring the systemic candidiasis in a rat model.
Materials and Methods: Twenty one rats were infected with 106 Candida albicans blastospore per rat. Twelve rats were considered as the negative controls (six immunocompromised rats without infection and six intact rats). During a week, every 24 hours the BDG sera level was determined by both Fungitell and Wako kits. To confirm the systemic infection in each rat, the suspensions of their internal organs were cultivated on agar plates and the number of colony forming units (CFU) of C. albicans was counted.
Results: All the infected rats were positive with BDG tests. An increasing level of BDG was observed during early days after injection. The cutoff value for discrimination of BDG positive sera was obtained from the negative sera by the Fungitell kit. The sensitivity, specificity, positive and negative predictive values assessed for the Fungitell kit were 95%, 66.6%, 90.47% and 80%, respectively. These criteria for those of Wako were 90%, 83.3%, 94.7% and 71.4%, respectively.
Conclusions: While BDG assay seems to be a sensitive and specific adjunctive tool to diagnose and monitor the experimental systemic candidiasis, it seems that measuring the positive cutoff value in different laboratory conditions is necessary for favorable establishment of these tests.
Jundishapur Journal of Microbiology 06/2014; · 0.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The study was conducted to determine the sequence variation in two mitochondrial genes, namely cytochrome c oxidase 1 (pcox1) and NADH dehydrogenase 1 (pnad1) within and among isolates of Toxocara cati, Toxocara canis and Toxascaris leonina. Genomic DNA was extracted from 32 isolates of T. cati, 9 isolates of T. canis and 19 isolates of T. leonina collected from cats and dogs in different geographical areas of Iran. Mitochondrial genes were amplified by polymerase chain reaction (PCR) and sequenced. Sequence data were aligned using the BioEdit software and compared with published sequences in GenBank. Phylogenetic analysis was performed using Bayesian inference and maximum likelihood methods. Based on pairwise comparison, intra-species genetic diversity within Iranian isolates of T. cati, T. canis and T. leonina amounted to 0-2.3%, 0-1.3% and 0-1.0% for pcox1 and 0-2.0%, 0-1.7% and 0-2.6% for pnad1, respectively. Inter-species sequence variation among the three ascaridoid nematodes was significantly higher, being 9.5-16.6% for pcox1 and 11.9-26.7% for pnad1. Sequence and phylogenetic analysis of the pcox1 and pnad1 genes indicated that there is significant genetic diversity within and among isolates of T. cati, T. canis and T. leonina from different areas of Iran, and these genes can be used for studying genetic variation of ascaridoid nematodes.
Journal of Helminthology 04/2014; · 1.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background and Objectives: Candida species, especially C. albicans, are commensals of human mucosal surfaces. But, increasing of patients receiving antibiotics or immunocompromised individuals cause Candida spp. became invasive pathogens. However, the accurate diagnosis of Candida species in patients with pulmonary symptoms is important.
Materials and methods: A total of 75 clinical isolates of Candida species were included in this study. They obtained from immunocompromised and immunocompetent patients with pulmonary symptoms. Candida cultures identified based on ITS1-ITS2 rDNA sequence analysis by PCR-RFLP.
Results: The molecular identification indicated that most Candida isolates belonged to C. albicans (49.3%), followed by C. tropicalis (26.8%), C. glabrata (16%), C. cruzei (5.3%), C. parapsilosis (1.3%) and C. guilermondi (1.3%), respectively.
Conclusions: Because of the increasing complexity in disease profiles of different patients and their management, rapid and accurate identification of Candida species can guide appropriate antifungal therapy.
The 22st Iranian Congress on Infectious Diseases and Tropical Medicine, Tehran; 01/2014
[Show abstract][Hide abstract] ABSTRACT: Identification of dermatophytes at the species level, relying on macro- and microscopic properties of the colonies is time-consuming, questioned in many circumstances, and requires considerable expertise. In this study, we examined the potency of a new genetic marker, β-tubulin (BT2) gene, for differentiation of dermatophytes in an in silico and experimental restriction fragment length polymorphism (RFLP) profile.
The BT2 sequences of dermatophyte species were retrieved from GenBank and analyzed using bioinformatics softwares to choose suitable restriction enzyme(s). Forty reference culture collections and 100 clinical isolates were PCR-amplified using the primers T1 and Bt2b and consequently subjected to virtual RFLP analysis. The dermatophytes were identified according to specific lengths of bands in agarose gel electrophoresis.
After digestion of partially amplified β-tubulin gene with the restriction enzyme FatI, three dermatophyte species, that is, Microsporum gypseum, M. canis, and Trichophyton verrucosum yielded unique restriction maps while the remaining species including T. interdigitale, T. rubrum, T. tonsurans, T. schoenleinii, and T. violaceum, were identified by further restriction digestion by Alw21I, MwoI, and HpyCH4V endonucleases. The length of RFLP products was same as of those expected by computer analysis.
The two-step BT2 restriction mapping used in this study is an effective tool for reliable differentiation of the clinically relevant species of dermatophytes.
Journal of Clinical Laboratory Analysis 01/2014; · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Clinical manifestations of Strongyloides stercoralis are variable from
asymptomatic to hyperinfection and devastating disseminated infections. Hereby,
clinical characteristics of a large series of Iranian strongyloidiasis indigenous cases
Methods: The records of people referred to the Helminthological Diagnostic Laboratory
of School of Public Health, Tehran University of Medical Sciences and
School of Medicine, Gilan University of Medical Sciences, during 2009-2013 were
reviewed. For those patients that were infected with S. stercoralis and their clinical
manifestations and demographic data were available (70 cases) a checklist was prepared
and data analyzed.
Results: Forty-three patients (61.4%) were male and 27 (38.6%) female. Gastrointestinal,
cutaneous and pulmonary symptoms were present in 71.4%, 25.7%, and
15.7% of patients, respectively. None of them had larva currens eruption. Eosinophilia
was the most prevalent reason for suspicious on S. stercoralis, but the mean
was lower in elderly patients. Hyperinfection were recorded in 8 patients (11.4%),
and 2 cases had disseminated infection.
Conclusion: Eosinophilia is common both in asymptomatic and symptomatic cases
of strongyloidiasis, but the mean tend to lower with increase in age.
Iranian Journal of Parasitology 01/2014; 9(2):155-162. · 0.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the identification of medically important Aspergillus species. Our subjects included nine standard Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital indoor sources. First of all, Aspergillus isolates were identified in the level of species by using morphologic method. A twenty four hours culture was performed for each isolates to harvest Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method. PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The electrophoresis data were analyzed and compared with those of morphologic identifications. Total of 205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for identification of at least the most medically important Aspergillus species.
Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]. 01/2014; 45(2):503-7.
[Show abstract][Hide abstract] ABSTRACT: To investigate the antifungal drug-susceptibility of fungi responsible for dermatomycoses, we performed minimum inhibition concentration (MIC) tests in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, and Epidermophyton floccosum with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole, and bifonazole) by broth micro dilution assay according to Clinical Laboratory Standard Institute protocol. Six possible dermatomycosis-causing non-dermatophytic fungi were also tested. The two major etiological pathogens of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to six antifungal drugs. However, non-dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. The MIC values of the non-azole drugs in Trichophyton spp. had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, we calculated the fractional inhibitory concentration (FIC) index for the combination of amorolfine and itraconazole as representative external and internal drugs in dermatophytes. The results indicated that the combination of amorolfine and itraconazole had synergistic or additive effects on most dermatophytes, and had no antagonistic effect. The variation of susceptibility seen in clinical fungal isolates indicated that identification of etiological fungi is indispensable for suitable choice of effective antifungal drugs in the early stages of infection. The results of combination assay suggested that the use of multiple drugs with different antifungal mechanism for growth of dermatophytes will be available for the treatment of refractory dermatomycosis, especially onychomycosis.
Microbiology and Immunology 11/2013; · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Due to the epidemiological alteration in distribution of Candida species as well as significant increasing trend of either intrinsic or acquired in resistance of some of these fungi, the precise identification of Candida species is necessary for effective antifungal therapy and also for prevention of nosocomial infections. PCR-RFLP method is indicated to be a reliable, rapid and simple technique which is able to differentiate the Candida species. In the present study, we applied this method to evaluate the distribution of Candida species in patients affected with cutaneous candidiasis in the Guilan province. 896 clinical cutaneous samples were collected from different parts of skin and nail of suspected patients referred to clinical centers all over the Guilan province during 24 months. Samples were examined directly with 15% KOH and cultured on fungal specific media. Genomic DNA was extracted and the restriction enzyme Msp1 was applied for polymorphism analysis. Totally, 47 yeast strains were successfully isolated from different clinical samples and identified by conventional as well as PCR-RFLP methods. The results indicated that Candida albicans (36.17%) was the most frequent species followed by C. parapsilosis (25.53%), C. tropicalis (19.14%), C. guilliermondii (14.89%), C. famata (2.12%) and C. krusei (2.12%). Female finger nails were the most common location to be affected by Candida species. In conclusion, PCR-RFLP method was successfully used for recognition of clinical Candida species within the Guilan province and obtained results revealed C. albicans as the predominant causative agent of cutaneous candidiasis. However, distribution of other Candida species did not completely consist with the reported distribution of Candida species in other parts of Iran with different climate to the Guilan province.
[Show abstract][Hide abstract] ABSTRACT: Toxocara is a common nematode of cats in different parts of Iran. Despite the close association of cats with human, no attempt has been done so far for molecular identification of this nematode in the country. Therefore, current study was performed on identification of some isolates of Toxocara from stray cats in Shiraz, Fars Province, Southern Iran, based on morphological and molecular approaches, and also determination of intensity of infection.
This cross-sectional study was carried out on 30 stray cats trapped from different geographical areas of Shiraz in 2011. Adult male and female worms were recovered from digestive tract after dissection of cats. Morphological features using existing keys and PCR-sequencing of ITS-rDNA region and pcox1 mitochondrial l gene were applied for the delineating the species of the parasites.
Eight out of 30 cats (26.7%) were found infected with Toxocara nematodes. All the isolates were confirmed as Toxocara cati based on morphological features and the sequence of ribosomal and mitochondrial targets. Intensity of infection ranged from one to a maximum of 39 worms per cat, with a mean of 10.25±12.36, and higher abundance of female nematodes.
The most prevalent ascaridoid nematode of stray cats in the study area was T. cati and female nematodes were more abundant than that of males. This issue has important role in spreading of eggs in the environment and impact on human toxocariasis.
Iranian Journal of Parasitology 10/2013; 8(4):593-600. · 0.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Invasive aspergillosis continues to be a significant cause of morbidity and mortality in solid organ transplant (SOT) recipients. A reliable and early diagnostic method is needed to improve survival. In this study, four methods direct microscopy, culture, nested PCR on internal transcribed spacer region, and TaqMan real-time PCR targeted β-tubulin gene were examined for the detection of Aspergillus fumigatus and A. flavus in sixty-four bronchoalveolar lavage (BAL) fluids that were obtained from SOT recipients. Direct examination with 20 % KOH (potassium hydroxide) and culture on mycological media were also performed. Of the 64 samples, seven (10.9 %) were positive in direct examination (five with septate hyphae and two with aseptate hyphae), and 15 (23 %) had positive culture including five A. flavus, four A. niger, two Penicillium spp., two Rhizopus spp., one Fusarium spp. and one mixed A. flavus/A. niger. Twenty five (39 %) samples had positive nested PCR with A. flavus and 6 (9.4 %) with A. fumigatus-specific primers. Only eight (12.5 %) had positive real-time PCR for A. flavus and nine (14 %) for A. fumigatus. The incidence of aspergillosis in these patients included proven invasive pulmonary aspergillosis (IPA) in two (3 %), probable IPA in 14 (22 %), possible IPA in 38 (59 %), and not IPA in 10 (16 %). A. flavus was the most common cause of pulmonary aspergillosis (PA) in the study. The results suggest that because nested PCR is too sensitive it may increase the number of false-positive results and is not recommended for BAL samples for diagnosis of PA. Although further studies with significant number of proved positive/negative standard BAL samples are necessary for better evaluation, the novel multiplex real-time PCR developed in the study could be promising as a valid diagnostic method for IPA.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients. METHODS: Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting β-tubulin gene were used for the detection of A. fumigatus and A. flavus. RESULTS: Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5. CONCLUSIONS: Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group.
[Show abstract][Hide abstract] ABSTRACT: Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis (ACL) in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs.
Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products.
Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis.
Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles.
Iranian Journal of Parasitology 04/2013; 8(2):264-72. · 0.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Pneumocystis jirovecii causes Pneumocystis pneumonia (PCP) in immunocompromised patients with a high rate of morbidity and mortality. Colonization with this fungus may stimulate pulmonary inflammation or lead to PCP in susceptible patients. The epidemiology of this infection and routs of its transmission has poorly studied in Iran. We examined Pneumosystis colonization in patients with various lung underlying diseases.
Methods: Bronchoalveolar lavage (BAL) fluids of 458 patients with different underlying diseases or pulmonary signs were collected between August 2010 and January 2012. Patients were divided into four groups: transplant recipients, malignant patients, immunosuppressive drug recipients and patients with other different lung diseases. A sensitive nested-PCR method targeted 18S ribosomal RNA gene was used for investigating P. jirovecii in the specimens.
Results: P. jirovecii DNA was detected in 57 out of 458 (12.5%) BAL samples by nested-PCR. Colonization rate in malignant patients, transplant recipients, immunosuppressive therapy recipients and patients with other various lung diseases was 21.7%, 20.3%, 12.7% and 7.3%, respectively. The enzyme BanI cuts all PCR products producing fragments with the size of 228 and 104 base pair. This finding as well as sequencing of four random positive samples validated and reconfirmed the PCR results. P. jirovecii cysts were found in 5 out of 57 PCR positive samples.
Conclusion: A significant number of patients with pulmonary diseases were colonized by P. jirovecii that can develop to PCP in these patients or they may transmit the fungus to other susceptible patients.
Keywords: Pneumocystis jirovecii, Pneumocystis pneumonia, Colonization, Nested-PCR
Iranian Journal of Public Health 03/2013; 42(3):298-305. · 0.41 Impact Factor