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Background: Vulvovaginal candidiasis (VVC) is a prevalent infectious disease that affects the majority of women. While Candida albicans is the most common cause of VVC, the prevalence of non-albicans species is increasing, and mixed infections have made treatment more challenging. This study aimed to identify Candida species and detect mixed infections in women with VVC in a tropical region of northeastern Iran, employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Methods: In the prospective study, a total of 270 specimens of vaginal discharge were collected using sterile swabs from patients suspected of having VVC. After extracting DNA from Candida colonies, the genome was amplified using PCR with specific primers. Species identification was conducted through PCR-RFLP analysis of the ribosomal DNA internal transcribed spacer (ITS) regions, using the MspI restriction enzyme. Results: Out of the 270 specimens suspected of VVC, 167 tested positive for Candida cultures. Among these positive Candida cultures, Candida species were identified in 150 specimens, with 44 (29.3%) showing evidence of mixed Candida infections. The species of Candida in 17 specimens were not identified. Out of the 190 identified Candida isolates included, the distribution was as follows: 85 (44.8%) C. albicans, 39 (20.5%) C. glabrata, 28 (14.7%) C. guilliermondii, 11 (5.8%) C. kefyr, 11 (5.8%) C. parapsilosis, 8 (4.2%) C. tropicalis, and 8 (4.2%) C. krusei. The mixed Candida species combinations observed were as follows: C. albicans/C. guilliermondii 26 (65%), C. albicans/C. kefyr 4 (10%), C. parapsilosis/C. glabrata 4 (10%), C. parapsilosis/C. tropicalis 4 (10%), C. krusei/C. tropicalis 2 (5%), C. albicans/C. parapsilosis 1 (2.5%), C. albicans/C. krusei 1 (2.5%), C. glabrata/C. guilliermondii 1 (2.5%), and C. kefyr/C. tropicalis 1 (2.5%). Conclusions: In women with VVC in the tropical region of northeastern Iran, the prevalence of clinical non-albicans species is higher than that of C. albicans. Furthermore, there is a notable high prevalence of clinical specimens containing mixed Candida infections.

Background: Epidemiological studies, classification and genetic studies of Leishmania species are effective in treatment, control and prevention in endemic areas. We aimed to investigate the genetic diversity and phylogeny of Leishmania in Zoonotic foci located in northeastern Iran using nagt gene for the first time. Method: DNA of 100 confirmed positive slides collected from the health centers of Sarkhes, Darghez, Fariman, Esfarayen, and Sabzevar were extracted during 2020-2021. The partial sequence of kDNA was amplified to identify the species. Twenty-five DNA samples were randomly subjected to amplify by nagt gene primes and were sequenced. The sequences were aligned with reference sequences in National Center for Biotechnology Information (NCBI). Then, the genetic similarities of the sequences were checked using Clustalx2.1 software and the phylogenetic tree was drawn by Mega 7 software. Result: All the positive samples were diagnosed as L. major. Approximately, half of the sequences of species were similar to two reference genes JX103550.1:404-712 L. major Esfahan and KX759012.1:568-807 L. Major Ilam (more than 90% similarity). According to the results of the phylogeny tree, the closest genotype to our study samples was JX103550.1:404-712 L. major Esfahan. Conclusion: The most causative agent CL in these areas was L. major. The genetic diversity of L. major was high such as other zoonotic foci in Iran. Due to the high similarity of the strains in the study areas with the strains of Isfahan and Ilam, similar control and prevention methods is suggested in these areas.

Abstract Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p

Background and Purpose: Resistance to antifungal drugs is increasing among Candida isolates from patients with vulvovaginal candidiasis (VVC). Lack of correct diagnosis of Candida causing VVC and the experimental use of antifungal drugs are the main causes of this resistance. This study aimed to determine the susceptibility of antifungal drugs against Candida species isolated from VVC in Northeastern Iran.Materials and Methods: Among women suspected of VVC, 189 vaginal dischargespecimens were evaluated. Candida isolates detected by polymerase chain reactionrestriction fragment length polymorphism were examined by standard antifungal diskdiffusion susceptibility testing method for voriconazole, itraconazole, fluconazole, andketoconazole. The susceptibility pattern of these antifungals was reported as sensitive,susceptible dose-dependent, and resistant. The results were evaluated by SPSS software and analyzed by Pearson chi-squared test.Results: Among the vaginal specimens, 108 out of 189 Candida isolates were identifiedas C. albicans, C. glabrata, C. kefyr, C. parapsilosis, and C. tropicalis. The susceptibilityrates of Candida isolates to voriconazole, ketoconazole, itraconazole, and fluconazolewere 92.6%, 90.7%, 68.5%, and 63.9%, respectively. Moreover, the resistance rates tofluconazole, itraconazole, ketoconazole, and itraconazole were 15.7%, 8.3%, 1.9%, and1.9%, respectively. The C. glabrata and C. albicans isolates were resistant to antifungaldiscs among 93% and 20% of the specimens, respectively.Conclusion: The C. glabrata and C. albicans species showed the highest resistance toantifungal drugs. Furthermore, Candida isolates showed the highest sensitivity tovoriconazole and ketoconazole and the lowest sensitivity to fluconazole.

Background: The genus Acanthamoeba is a free-living opportunistic protozoan parasite, which widely distributed in soil and fresh water. Acanthamoeba keratitis, which causes a sight-threating infection of the cornea, is going to rise in Iran and worldwide. The aim of this study was to compare direct microscopy, culture and PCR for detection of Acanthamoeba spp. in clinical samples and to determine the genotypes of Acanthamoeba spp. by sequencing 18SrRNA gene. Methods: Among patients clinically suspected to AK referred to a tertiary ophthalmology center at Mashhad, northeastern Iran. During 2017-18, twenty corneal scrapes specimens obtained. The samples were divided into three parts, subjected to direct microscopic examination, culture onto non-nutrient agar and PCR technique. Sensitivity, specificity, accuracy and likelihood ratio were evaluated. Results: Among 20 persons clinically suspected to amoebic keratitis, 13(69.2%) patients definitely diagnosed as Acanthamoeba keratitis. Wearing contact lens, eye trauma due to foreign particle and swimming in fresh water were the main predisposing factors. Most of patients suffered from pain and photophobia. Corneal ring infiltration and epithelial defect were common clinical sings. Direct examination had the lowest sensitivity and sensitivity of both Nelson-PCR and JDP-PCR methods were equal and highest. In addition, the results of sequencing identified that all strains belonged to T4 genotype. Conclusion: Amoebic keratitis is a sporadic parasitic keratitis, which is mainly seen in contact lens user in Mashhad. PCR based on 18S ribosomal DNA with JDP primers is a reliable and highly sensitive method for diagnosis of Acanthamoeba keratitis in clinically suspected cases.

Introduction: Candida vulvovaginitis is one of the most common vaginal infections in women and in women with diabetes. Although C. albicans has been reported to be the most common cause of this infection, but there have been recent changes in the pattern of Candida causing Candida vulvovaginitis. This study was performed with aim to investigate the frequency of dominant Candida isolates in patients with Candida vulvovaginitis in Torbat-e Jam and its relationship with diabetes. Methods: In this cross-sectional study, 428 women with suspected Candida vulvovaginitis referred to Torbat-e-Jam medical centers were assessed in 2019. Two specimens of vaginal secretions were taken from each patient. The first specimen was cultured on sabouraud dextrose agar (SDA) medium, and the second one was directly examined using wet mount microscopy. The Candida colonies were passaged on CHROMagar Candida medium to identify the species. Demographic and diabetes characteristics of the patients were evaluated. Data were analyzed by SPSS software (version 16) and Chi-square test. p

Background and Purpose: Keratinophilic fungi play an important role in the decomposition of keratinous substances in nature. This capacity induces dermatomycosis in both humans and livestock. The soil of livestock stables can be a reservoir of keratinophilic fungi. Therefore, the present study was conducted to isolate and identify keratinophilic fungi in the soil of the livestock stables located in Qayen, South Khorasan Province, Iran. Materials and Methods: This study was conducted on 62 soil samples collected from livestock stables. The samples were cultured by means of hair bait technique (HBT). The identification of the isolates was performed based on their morphological characteristics and then confirmed by polymerase chain reaction and sequencing of the ITS regions of ribosomal DNA. Results: A total of 118 isolates of 7 species from 5 genera were identified. Aphanoascus verrucosu (n=70, 59.36%) was detected as the dominant keratinophilic fungus, followed by Arthroderma quadrifidum (n=13, 11.01%), Aphanoascus terreus (n=12, 10.16%), Acremonium (n=12, 10.16%), Arthroderma gertleri (n=5, 4.23%), Fusarium equiseti (n=3, 2.54%), and Uncinocarpus reesii (n=3, 2.54%). Conclusion: Different keratinophilic fungi were isolated from the soil of livestock stables; however, Aphanoascus verrucosu was found to be the dominant species.

Background: Nowadays, the prevalence of onychomycosis caused by non-dermatophyte molds is increasing. As Aspergillus is the most common etiologic agents of the disease, this study was performed to evaluate the susceptibility of clinical isolates of Aspergillus as the cause of onychomycosis to itraconazole, voriconazole, and amphotericin B. Methods: This cross-sectional study was performed on 50 Aspergillus strains isolated from the patients with onychomycosis referred to diagnostic laboratories of university hospitals in Mashhad, Iran. According to the phenotypic and molecular analysis of Aspergillus isolates, the most frequent species was Aspergillus flavus (A. flavus) with 34 cases. The drug susceptibility test was performed according to Clinical and Laboratory Standards Institute-M38-A2 (CLSI-M38-A2) protocol, and the minimum inhibitory concentration (MIC) of the drugs was determined. Findings: From 50 patients with dystrophic nails, 13 were men and 37 were women. 15 patients had fungal infection of fingernails, and 33 patients had fungal infection on toenails; 2 patients had both infections of finger and toe nails. 14.7% of A. flavus isolates with MIC > 2 μg/ml to amphotericin B were considered as resistant clinical isolates. The sensitivity to itraconazole and voriconazole was 100%. MIC90 of amphotericin B, itraconazole, and voriconazole were obtained by broth microdilution method for A. flavus species as 4, 0.25, and 1 μg/ml, respectively. Significant difference was observed in MIC between itraconazole and amphotericin B (P < 0.050). Conclusion: All Aspergillus species are susceptible to itraconazole and voriconazole, and these two drugs are more effective than amphotericin B on Aspergillus species. Due to the different susceptibility of these species to antifungal agents, it is necessary to perform drug susceptibility testing in order to select the appropriate drug.

The province of Khorasan-Razavi in the North East of Iran is an endemic area for anthroponotic cutaneous leishmaniasis (ACL caused mainly by Leishmania tropica) and zoonotic cutaneous leishmaniasis (ZCL caused mainly by Leishmania major). Based on clinical signs, some cities were considered as ACL foci while others were considered to be endemic for ZCL. This paper reviews studies performed on patients diagnosed with cutaneous leishmaniasis (CL) via the use of direct slide examination, ELISA, electrophoresis isoenzyme, RAPD PCR and PCR in Mashhad; the study also includes cases of CL in other cities of the Khorasan-Razavi province where only PCR used as a diagnostic tool. The data show that both Leishmania tropica and Leishmania major caused CL in most of the cities investigated. Our review shows that Leishmania major was found in areas where ACL is prevalent and Leishmania tropica was observed in areas with high incidence of ZCL. This distribution represents a major change in the epidemiological pattern of Leishmania in the Khorasan-Razavi province.

Background and Purpose: Pneumocystis pneumonia (PCP) is one of the most common and life-threatening fungal diseases in patients with human immunodeficiency, treated with immunosuppressive medications. Immunocompetent people can also be a spreading agent for PCP. Regarding this, the aim of the present study was to diagnose and identify Pneumocystis jirovecii in bronchoalveolar lavage (BAL) samples obtained from patients with pulmonary disorder using a molecular method. Materials and Methods: For the purpose of the study, BAL samples (n=138) were collected from patients, undergoing bronchoscopy at the different departments of university hospitals affiliated to Mashhad University of Medical Sciences, Mashhad, Iran, during a period of one year (i.e., April 2014 until May 2015). Giemsa staining and molecular identification were carried out for each sample. The samples were also subjected to nested polymerase chain reaction (PCR), sequencing, and genotyping based on mitochondrial ribosomal large subunit (mtLSU rRNA) of P. jirovecii. The phylogenic tree was constructed by MEGA6 software. Results: The results of direct microscopic examination revealed the presence of P. jirovecii in 3 (2.2%) out of 138 samples; in addition, nested PCR and sequencing led to the detection of species in 17 (12.3%) samples. Out of patients with positive results, 10 (25%) and 7 (7.1%) cases were immunosuppressed and immunocompetent, respectively. The most common clinical symptoms among patients with pneumocystis were fever, dyspnea, and dry cough. In addition, genotypes III and II were the dominant genotypes in our dataset. Conclusion: Nested PCR and sequencing methods showed higher sensitivity and specificity as compared with a direct staining technique. Genotype III was identified as the most dominant type in patients with pulmonary disorder in Mashhad.

Background and Purpose: Dermatophytes as the causative agents of dermatophytosis (ringworm) are widely spread around the world. Accurate identification of dermatophytes in one area can be particularly important for epidemiological studies. Regarding this, the aim of the present study was to describe the species spectrum of dermatophytes, isolated from patients in Mashhad city, Iran, using the molecular-based method. Materials and Methods: This study was conducted on 79 dermatophyte isolates obtained from the human skin, hair, and nail specimens. Species identification was performed by the polymerase chain reaction-restriction fragment length polymorphism analysis of ribosomal DNA internal transcribed spacer regions using MvaI restriction enzyme. Results: The identified species included Trichophyton mentagrophytes/T. interdigitale species complex (n=37, 46.8%), Epidermophyton floccosum (n=12, 15.2%), T. rubrum (n=8, 10.1%), Microsporum canis (n=8, 10.1%), T. violaceum (n=5, 6.3%), T. tonsurans (n=4, 5.1%), Nannizzia gypsea (n=3, 3.8%), T. benhamiae (n=1, 1.3%), and T. verrucosum (n=1, 1.3%). The clinical forms of infection were tinea corporis (n=26, 32.8%), tinea cruris (n=22, 27.8%), tinea capitis (n=10, 12.6%), tinea unguium (n=7, 9%), tinea manuum (n=6, 8%), tinea pedis (n=5, 6.3%), and tinea faciei (n=3, 3.5%). Conclusion: As the findings indicated, T. mentagrophytes/T. interdigitale species complex had the highest prevalence, and T. benhamiae appeared to be a new emerging agent of dermatophytosis in Mashhad, northeastern Iran.

Background: Dermatophytes are a group of fungi that attack keratinous tissues of the skin, hair, and nail in humans and animals, and cause infections called dermatophytosis (tinea). Since identification of pathogenic fungi at the species level is essential for the detection of the source, control and prevention, and identifying epidemiology of infection, it is necessary to use specific and sensitive diagnostic methods to identify the causes of dermatophytosis. Methods: The clinical samples (skin, nail, and hair) of patients with dermatophytosis in Mashhad City, Iran, were cultured in Mycosyl Agar culture media, and the DNA of obtained dermatophyte colonies were extracted by specific kit. The internal transcribed spacer (ITS) gene was amplified and sequenced by ITS1, ITS4 primers. Finally, the sequencing results were analyzed using SeqMan software, and were compared with the data of the global genebank. Findings: In this study, 80 dermatophyte isolates were sequenced, which included 9 dermatophyte species as 23 (28.8%) Trichophyton (T.) interdigital, 18 (22.5%) T. tunsorans, 10 (12.5%) Epidermophyton fluccosum, 10 (12.5%) of T. mentagrophytes, 8 (10%) Microsporum canis, 4 (5%) T. rubrum, 4 (5%) T. benhamiae, 2 (2.5%) Nannizzia (N.) fulvum, 1 (1.2%) N. persicolor. Conclusion: According to report the rare species of dermatophytes in this study, the use of molecular methods such as sequencing of the ITS gene can determine the diversity of dermatophytes in a region more precisely than morphological methods.

Background: Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. Methods: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. Results: The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. Conclusion: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future.

Background: Human toxocariasis is a neglected parasitic disease in most countries including Iran. Among different clinical forms of toxocariasis, ocular toxocariasis (OT) is an important disease resulting in severe vision loss. However, the prevalence and incidence of OT are currently unclear in Iran. This study aimed to determine the prevalence of ocular toxocariasis among patients with uveitis in the Northeast of Iran. Methods: From 2015 to 2017, 510 patients with uveitis referred to Khatam-al-Anbia, a tertiary eye hospital at Mashhad, Iran were examined for OT. Serum samples of the suspected patients were obtained and evaluated for IgG against Toxocara canis using ELISA test. Anti-Toxocara IgG positive serums were further investigated using confirmatory Western blotting (WB) analysis. Results: Twenty patients had pathologic changes and clinical presentations in the anterior and posterior segments of their eyes and they were clinically diagnosed ocular toxocariasis. Among the 20 patients, 2 (10%) patients showed IgG antibody against Toxocara canis on ELISA as well as on WB test. The calculated prevalence of ocular toxocariasis was about 0.4%. Conclusion: Ocular toxocariasis can be diagnosed both clinically and serologically in Mashhad, northeastern Iran. Although OT is a rare pathologic eye disease, it should be considered as one of the important cause of infectious posterior uveitis.

Background: Over the last decade, a few cases of visceral leishmaniasis (VL) have been reported in some provinces of northeastern Iran. We aimed to investigate clinical and laboratory findings of VL among children who admitted to the pediatric ward in a referral hospital in Mashhad, northeastern Iran. Methods: A retrospective study, between 1997 and 2017, was performed on the data sheet registered for children with confirmed VL at the referral Emam Reza Hospital in Mashhad. Hematological and biochemical profiles of the patients were analyzed. Results: Thirty-five children with VL, confirmed by the presence of amastigotes of Leishmania in Giemsa stained smears of the bone marrow, had been recorded through 20 yr. The mean age of patients was 3.7±4 yr. The majority of the patients suffered from hepatosplenomegaly (100%, n=35/35), followed by prolonged fever and pallor (91%, n=32/35), weight loss (85%, n=30/35). The main laboratory findings were anemia (94.1%), leukopenia (52.9%) and thrombocytopenia (70.5%). Almost one-third (37.1%; 13/35) of VL patients inhabited in rural areas of the Bojnoord district as a known VL endemic focus in northeastern Iran. Conclusion: Our preliminary data showed that the origin of VL is still in some districts other than Mashhad, where VL just will be diagnosed.

Background: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis in Iran with high mortality rates in the case of inaccurate diagnosis and treatment. This study aimed to prepare and evaluate a new rk39 recombinant antigen from an Iranian strain of Leishmania infantum for diagnosis of VL in humans and dogs. Methods: rK39-based enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for the detection of anti L. infantum antibodies. We screened 84 human sera and 87 dog sera from clinical cases in the endemic area of Meshkin-Shahr, Iran along with 176 sera from healthy controls (collected from 86 humans and 90 dogs) during 2013 -2016. Results: Using the rK39 ELISA, a sensitivity of 85.7% (95% CI, 95-99%) and a specificity of 86.0% (95% CI, 95%-99%) were detected in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 96.6% (95% CI, 95%-99%) and specificity of 94.4% (95% CI, 95%-99%) were found at a 1:80 (cut-off) titer compared with DAT. Kappa analysis indicated agreement between the rK39 ELISA and DAT (0.718) when using human sera at a 1:800 (cut-off) titer as well as (0.910) at a 1:80 (cut-off) titer when using dog sera (P

Background: Dermatophytosis is a common global superficial mycosis caused by a group of keratinophilic moulds known as dermatophytes that invade the skin and keratinized tissues such as hair and nails of humans and animals. This study takes identification of a collection of clinical dermatophyte isolates by using partial sequencing of translation elongation factor-1α (Tef-1α) gene aiming both to update the epidemiological status of dermatophytosis in Mashhad, Northeastern Iran and to corroborate the efficacy of Tef-1α for species-level identification of dermatophytes. Method: The demographic data related to 87 culture-positive dermatophytes isolated from patients clinically suspected to have dermatophytosis were collected. The dermatophyte isolates were subjected to a partial polymerase chain reaction (PCR)-sequencing of Tef-1α gene by using specific pan-dermatophyte primers. The data were analysed by SeqMan software, the sequences were compared and aligned with the GenBank database and the isolates were identified. Results: Identification based on Tef-1α partial sequence was successful for all isolates. The identified dermatophyte isolates in decreasing order were as Trichophyton interdigitale 19 (22%), T. tonsurans 19 (22%), T. mentagrophytes 13 (15%), T. persicum 10 (11.5%), Epidermophyton floccosum 9 (10.3%), Microsporum canis 7 (8%), T. rubrum 5 (5.7%), T. violaceum 2 (2.2%), Nannizzia fulva 2 (2.2%) and N. persicolor 1 (1.1%). The isolates have been associated with clinical forms of tinea corporis (n = 38; 43.7%), tinea faciei (n = 13; 15%), tinea cruris (n = 12; 13.9%), tinea manuum (n = 7; 8%), tinea unguium (n = 7; 8%), tinea capitis (n = 7; 8%) and tinea pedis (n = 3; 3.4%). Conclusion: Dermatophytosis has yet remained a public health problem in Northeastern Iran, and infection with new and less frequent species, e.g., T. persicum, N. fulva and N. persicolor have emerged. The Tef-1α gene partial sequencing reconfirmed the resolution power of this locus for the determination of species boundaries in dermatophytes.

Background and Purpose: Candida-associated denture stomatitis is one of the most common forms of oral candidiasis among denture wearers. Regarding this, the aim of the present study was to evaluate the antifungal effects of home-generated ozonated water on the adhesion of the C. albicans attached to the surface of the denture base acrylic resins. Materials and Methods: For the purpose of the study, different concentrations of C. albicans were added to the tubes containing acrylic resin blocks, and then incubated for 2 h at 35°C. The samples were assigned into three groups, each of which contained 42 samples, including normal saline (NS) solution as the negative control, nystatin (N) solution as the positive control, and ozonated water as the test group. The samples were washed and placed in an ultrasonic bath. Subsequently, the saline solution was cultured on Sabouraud dextrose agar. The concentrations of Candida were evaluated during the contact times. Results: The test group (i.e., ozonated water) with 114 colony-forming units (CFU) showed a significant reduction of Candida colonies, compared to the NS group with 2,172 CFU. The 120- and 1-minute incubation with ozonated water showed the highest and lowest effects on the viability of Candida adhered to the acrylic resin, respectively. Conclusion: Based on the findings, home-generated ozonated water can be applied to remove the Candida attached to the surface of the denture plates.

Background and purpose: Cutaneous leishmaniasis is endemic parasitic disease in different regions of the world especially in Iran. Khorasan province is endemic for anthroponootic and zoonotic cutaneous leishmaniasis with unequal outbreak. Identification of species could have important implications for the disease and reservoirs control and finding the epidemiologic map. There is no study on this protozoa disease in Fariman, northeast Iran. The aim of this study was to determine the protozoa genotyping in this area. Materials and methods: A cross-sectional study was done in 50 suspected cases in Fariman 2015-2016. After light microscope checking, scrapings from all smears were done for DNA extraction and species identification was done using kDNA- PCR. Results: Among 50 suspected cases, 40 patients (80%) were found positive with direct smears and 42 (84%) were identified by PCR. Leishmania tropica was identified in 13 (31%) and 29 (69%) were infected with Leishmania major. Two negative smears were positive with PCR. The disease mainly involved those aged 11-20 years (28.6%) and the most common site of lesions was hands (38.1%) as ulcerative form (58%). From 13 cases of Leishmania tropica 75% and 25% were in urban and rural districts, respectively. Also, from 29 cases of Leishmania major 17.2% and 82.8% were in urban and rural districts, respectively. This difference was statistically significant (P= 0.004). Conclusion: Fariman is a zoonotic leishmaniasis foci in northeast Iran. Further studies on disease reservoirs and vectors seems necessary to control and reduce the number of people affected.

Background and Purpose Vulvovaginal candidiasis (VVC) is a common problem in women. The purpose of this study was to identify of Candida species isolated from vulvovaginitis woman suffering vulvovaginitis refered to Ghaem Hospital, Mashhad, Iran, by use of MALDI-TOF mass spectrometry. Materials and Methods The 65 clinical samples isolated from Vulvovaginitis women were collected in Ghaem Hospital. All specimens were identified using phenotypic techniques such as microscopy and culture on Sabouraud dextrose agar and corn meal agar medium,Then, All isolates were detected and were processed for MALDI TOF MS identification. Results Of the 65 isolates analyzed, 61 (93.8%) were recognised by MALDI-TOF mass spectrometry and for four isolates (6.1%) only not relabile identifications were achieved. In this study, the most frequently isolated species were Candida albicans (58.5%), followed by Candida tropicalis (16.9%), Candida glabrata (7.7%), Candida parapsilosis (7.7%) and Candida guillermondii (3.1%). Conclusion presented results demonstrate that the MALDI TOF mass spectrometry is a fast and reliable technique, and has the potential to replace conventional phenotypic identification of Candida species and other yeast strains routinely isolated in clinical microbiology laboratories.

Background and Purpose: Rhinosinusitis is a common disorder, influencing approximately 20% of the population at some time of their lives. It was recognized and reported with expanding recurrence over the past two decades worldwide. Undoubtedly, correct diagnosis of fungi in patients with fungal rhinosinusitis affects the treatment planning and prognosis of the patients. Identification of the causative agents using the standard mycological procedures remains difficult and time-consuming. Materials and Methods: Based on clinical and radiological parameters, 106 patients suspected of fungal rhinosinusitis were investigated in this cross-sectional prospective study from April 2012 to March 2016 at an otorhinolaryngology department. In this study, internal transcribed spacer (ITS) and calmodulin (CaM) sequencing were respectively validated as reliable techniques for the identification of Mucorales and Aspergillus to species level (both agents of fungal rhinosinusitis). Results: Of these, 63 (59.4%) patients were suspected of allergic fungal rhinosinusitis (AFRS), 40 (37.7%) patients suspected of acute invasive fungal rhinosinusitis (AIFRS), and 3 (2.8%) patients suspected of fungus ball. In patients suspected of AFRS, AIFRS, and fungus ball only 7, 29, and 1 had positive fungal culture, respectively. After ITS and CaM sequencing, Aspergillus flavus was the most common species isolated from non-invasive forms, and A. flavus and Rhizopus oryzae were more frequently isolated from invasive forms. Conclusion: Aspergillus flavus is the most common agent of fungal rhinosinusitis in Iran, unlike most other reports from throughout the world stating that A. fumigatus is the most frequent causative agent of this disease.

Introduction: Vulvovaginal candidiasis (VVC) is one of the most common vaginal infection in women of childbearing age. In some cases, due to the intervention of resistant strains of Candida albicans and non-albicans species, recurrent vulvovaginal candidiasis (RVVC) occurs. This study was performed with aim to identify the Candida isolated from patients with vulvovaginal candidiasis by PCR-RFLP method. Methods: This descriptive study was performed on 189 vaginal discharge obtained from women with clinical presentation of VVC referred to Ghaem Hospital, Mashhad University of Medical Sciences during 2015-2016. The vaginal discharge was examined by direct examination and culture on Sabouraud's dextrose agar medium for definitive diagnosis. The genomic DNA of Candida colonies have grown in culture were extracted by boiling lysis method. The Candida isolates were identified by PCR-RFLP technique. The results were analyzed by SPSS software (version 16) and using Pearson and Chi-square test. PResults: Among 189 sampled patients, 108 positive Candida cultures were acceptable. The Candida species which were identified included C. albicans 84 (77.8%), C. glabrata 10 (9.3%), C. kefyr 4 (3.7%), C. parapsilosis 1 (0.9%) and C. tropicalis 1 (0.9%). About 8 (7.4%) of the specimens contained two species of Candida. RVVC form was observed in 5 (4.6%) of the patients. Conclusion: The most common cause of VVC was C. albicans; and C. glabrata had the highest frequency among the non-albicans species. Most of patients had non- RVVC.

Background: Cutaneous leishmaniasis is a parasitic skin disease that is endemic in many parts of the world, particularly in Iran. Although the microscopic technique is a common method of diagnosis of leishmaniasis, species of leishmania cannot be differentiated by this method. So, molecular methods have special importance. Methods: In this study, kinetoplast-DNA polymerase chain reaction (kDNA-PCR) method was used to diagnose the leishmania species in Dargaz city, Iran. Direct slide smears were obtained from skin lesions of 94 patients suspected to the leishmaniasis referred to health centers of this city. Smears were stained using Gimsa method. PCR method was performed using specific kDNA primers. Data were analyzed using SPSS software. Findings: Among 94 subjects with skin ulcers suspected to cutaneous leishmaniasis, 82 (87.2%) were positive by direct microscopic smear and 85 (90.4%) by PCR in which 22 patients (26%) had Leishmania tropica and 63 (74%) had Leishmania major. Sensitivity and specificity of direct microscopic examination were calculated as 96% and 100%, respectively. There were statistically significant differences between disease occurrence and seasons (P = 0.045), habitat (P = 0.004), new or old building (new or old) (P = 0.003), and distance to rodents' living sites (P = 0.025). Conclusion: Dargaz city is a known site of zoonotic leishmaniasis in Iran; but this study shows that there are some new sites of anthroponotic leishmaniasis in this city, too. Leishmania major is the dominant species in Dargaz city but there is Leishmania tropica foci in this area, too.

ObjectiveMycobacterium marinum causes a rare cutaneous disease known as fish tank granuloma (FTG). The disease manifestations resemble those associated with Cutaneous Leishmaniasis (CL). The aim of this study was to determine whether FTG was the cause of cutaneous lesions in patients who were referred to the Parasitology laboratory of Imam Reza Hospital in Mashhad to be investigated for CL.Materials/methodsOne hundered patients, clinically diagnosed with CL between April 2014 and March 2015, were included in this study. Ziehl-Neelsen staining was performed to identify acid-fast Mycobacterium in addition to bacterial cultures using Löwenstein-Jensen medium. Skin lesion samples were also collected and kept on DNA banking cards for PCR testing.ResultsTwenty-nine of the 100 individuals with skin lesions, and therefore suspected of suffering from CL, tested positive for Mycobacterium marinum by PCR. Of these, 21 (72.4%) were male and 8(27.6%) were female. In 97% of these cases the lesions were located on hands and fingers. These patients had a history of manipulating fish and had been in contact with aquarium water. A sporotrichoid appearance was observed in 58.6% of the patients with mycobacterial lesions; 67% of patients had multiple head appearance.ConclusionPatients suspected to have CL and who test negative for CL could be affected by FTG. Therefore, after obtaining an accurate case history, molecular diagnosis is recommended for cases that give a negative result by conventional methods.

Background: Visceral leishmaniasis (VL), so-called Kala-azar is a life threating parasitic infectious disease caused by Leishmania spp. L. infantum is the main causative agent for Mediterranean form of Kala-azar which is endemic in northeastern Iran. This study attempted to investigate existence of canine visceral leishmaniasis (CVL) in Khorasan Razavi. Methods: Between 2014 and 2016, tissue samples collected from spleen and liver of 192 stray dogs were examined to investigate existence of L. infantum. Kinetoplast DNA (k-DNA) PCR was performed to identify the species of parasites. The positive PCR products were sequenced in both directions to confirm the kDNA PCR results Results: Among samples obtained from 192 dogs, kinetoplast DNA of L. infantum was detected in two female dogs. L. infantum was confirmed by sequence analysis of PCR products. Conclusion: Our data confirm stray dogs play as potential reservoirs for VL in this province. Further investigation will be necessary to clear role of stray dogs in the transmission of L. infantum to human and domestic dogs.

Background and Purpose: Pneumocystis pneumonia, caused by Pneumocystis jirovecii, is a fatal disease threatening patients with AIDS or immunosuppression. Assessment of colonization in these patients is of great significance, since it can lead to severe pulmonary disorders. Considering the scarcity of published reports on Pneumocystis jirovecii isolates from patients in Mashhad, Iran, we aimed to evaluate pneumocystis colonization in individuals with different pulmonary disorders. Materials and Methods: We used nested polymerase chain reaction (PCR) method to amplify mitochondrial large subunit-ribosomal ribonucleic acid (mtLSU-rRNA) gene in 60 bronchoalveolar lavage (BAL) samples, obtained from patients, referring to the Department of Internal Medicine (Pulmonary Diseases Section) at Imam Reza Hospital, affiliated to Mashhad University of Medical Sciences, Mashhad, Iran. Results: DNA of Pneumocystis jirovecii was detected in 10 out of 60 BAL samples (16.66%) via nested PCR method. Conclusion: According to the present findings, the colonization rate of Pneumocystis jirovecii was similar to the rates reported in other similar studies in Iran.

Background: Visceral leishmaniasis (VL) caused by Leishmania infantum is the most severe form of leishmaniasis in Iran, which causes a high mortality rate in the case of inaccurate diagnosis and treatment. This study aimed to clone of K26 gene from Iranian strain of L. infantum and register the sequencing results in Genbank to facilitate the preparation a new K26 antigen for the detection of L. infantum infection. Methods: L. infantum was obtained from an infected domestic dog in Meshkin-Shahr area from northwestern Iran in 2015. Canine visceral leishmaniasis was confirmed by direct agglutination test (DAT), rK39 dipstick and parasitological methods. L. infantum was confirmed by N-acetyl glucosamine -1-phosphate transferase (nagt)–PCR and its sequencing. The band of interest for k26 form Iranian strain of L. infantum was purified by gel extraction kit after PCR amplification and then ligated into pBluescript II SK (+) and pET-32a (+), respectively. The sequences of recombinant plasmids were analyzed and submitted to Genbank. Results: The submission of rk26 nucleotide sequence was performed to the GeneBank/NCBI Data Base under accession number KY212883. The related gene was showed a homology about 99% to L. chagasi and L. infantum k26 gene, while the level of homology in comparison with different strains of L. donovani ranged from 84-94%. Conclusion: The successful rk26 cloning into an expression vector performed in this study could help to produce a new recombinant antigen for serodiagnosis of VL especially in areas where L. infantum is the main causative agent.

Background: Chemotherapy still relies on the use of pentavalent antimonials, amphotericin B, paromomycin, miltefosin, and allopurinol. In this study, we explained about the native plant that grows in different regions of Iran and used as anti-leishmanial in Iran and even many other countries. Methods: This narrative review covers all information about local herbal medicine in Iran that used in treatment of cutaneous leishmaniasis in all the worlds, published in local and international journals from 1996 to 2015 using various databases including PubMed, SID, Google Scholar, Scopus, and Science Direct. Results: Overall, 150 articles in databases were identified. Many local plants grown in some places of Iran were used to treat this endemic disease. Conclusion: The cutaneous leishmaniasis is also a major health problem in Iran, especially in Mashhad (Northeast of Iran). Therefore, many patients seek for herbal therapy that is cheaper and readily available. This review provides information regarding plant that exists in Iran and exhibiting effects on anti- Leishmania activity. Among the anti-leishmanial mentioned in this review, most have never been tested for cytotoxicity and very few have been tested for in vivo activity.

Background: Being exposed to house dust mites intensifies atopic dermatitis. This study has investigated the con­tamination rate with Dermatophagoides mites in patient's residential home with atopic dermatitis. Methods: In this cross-sectional study, 40 patients took part with atopic dermatitis (positive or negative for mites by prick Dermal Test). Samples were collected from 3 locations (living room, bedroom and bed) by vacuum cleaner. Dust samples (transferred to freezer -20 ˚C) were examined by direct method and flotation. The data were analyzed using statistical SPSS vr.20 software. Results: Twenty patients of positive prick test included 8 (40%) male and 12 (60%) female. The results of direct observation of mites: 7 cases (35%) in bedding sheets, 6 cases (30%) bedrooms' carpet, 3 cases (15%) living room's carpet. Twenty patients of negative prick test included 8 (40%) male and 12 (60%) female. Only mites were found (5%) in living room's carpets of negative prick test patients. Dermatophagoides pteronyssinus was more frequent than Dermatophagoides farinae. (98% vs 83%). Conclusion: Fifty-five percent of residential homes of prick test positive patients and only 5% of residential homes of prick test negative patients were positive for mite. Sunshine provided home had fewer mites than home where sunshine is not provided. Prick test positive patients used handmade carpets more than machine made ones. In posi­tive prick test patients, mites were found in bed sheet and bedroom’s carpet more than negative prick test patient's sheets and carpets.

Background: Psychiatric patients have an increased risk of some infections like toxoplasmosis. Investigations on Toxoplasma gondii infection among psychiatric patients have been limited in Mashhad, Northeast of Iran. In this case-control study, prevalence of T. gondii was investigated by serological method. Methods: This case-control study was performed among psychiatric patients admitted to Avicenna Hospital in Mashhad, Northeast of Iran. Three hundred and fifty inpatients and 350 controls were examined in 2012-2013 for detection of IgG and IgM antibodies against T. gondii in their blood sera by ELISA. Socio-demographic and clinical manifestations of the patients were obtained. Results: Anti-T. gondii IgG antibodies was found in 164 (46.85%) of 350 psychiatric inpatients and 120 (34.28%) of 350 controls. Seventeen (4.85%) of psychiatric individuals and 3 (0.85%) of control group were IgM+/IgG- indicating acute form of toxoplasmosis. There were no statistically significant differences between the case and control groups. In patient group, schizophrenic patients had the highest positive rate (46.28%) and bipolar mood disorder had the second most prevalent rate (20%). Of 162 schizophrenia patients, 65 (40.1%) had latent infection which was higher than that observed in controls. Conclusion: The prevalence of T. gondii infection among psychiatric patients suffering from schizophrenia was more in Mashhad, compared with control group.

Objective: Leishmaniasis is one of the most important parasitic infectious diseases in the world. Since last century, many efforts have been made to control and treat the disease, but appropriate vaccines, pesticides and medicines are not available or even eligible. The purpose of this study was to evaluate the effect of hydro-alcoholic extract of Seidlitzia rosmarinus on the lesions of experimental Cutaneous Leishmaniasis (CL) in Balb/c mice. Materials and Methods: The population study was 60 Ballb/c mice which divided to 6 groups, all infected with Leishmania major [MRHO/75/IR]. Soon after the ulcer started to appear in the early stage, a dose of provided herbal extract with 5, 10 and 15% concentration applied on each lesion. The surface area of the lesions measured during an interval of 10 days. Direct Giemsa stained smears prepared two and four weeks after treatment. Results: Increasing the mean size of the lesions was statistically significant compared to those in control group (p>0.001). Visceral Leishmaniasis (VL) developed in all of the mice including the control group that received Eucerine alone. Survival rate in group receiving 15% S. rosmarinus extracts showed significantly higher compared to mice in control group (p

Background: Toxocariasis is the clinical terms applied to infection of human with Ascarid nematodes in the order Ascaridida, named Toxocara canis and T. cati. Because in recent years in Iran many people desire to keep pets (cats and dogs), and lacking of seroepidemiological study of toxocariasis in Mashhad, we decided to determine the seroprevalence of toxocariasis among people who own cats and dogs in comparison with control group. Methods: A serological study for detection antibodies to Toxocara in two groups (93 cat and dog owners and 93 healthy people as control group) was conducted from Feb 2013 to Dec 2013. An ELISA method was employed using determination of IgG antibodies against Toxocara. The serum samples were evaluated for anti-Toxocara antibody, using ELISA technique at Parasitology and Immunology Lab of Imam Reza Hospital of Mashhad. Using a questionnaire, epidemiological factors associated with infection were examined. Data were analyzed using Chi-square test. Results: The seroprevalence of Toxocara antibodies in the pet owners and control group was respectively 20.43% and 1.07%. 47.3% of pet owners were female. Conclusion: Presented data showed the significant difference between seroprevalence of toxocariasis among pet owners and control group. Education of society and in particular pet owners consisting of preventing contamination of the environment with Toxocara eggs is advised.

Background: One of the most important causative agents of visceral leishmaniasis (VL) is Leishmania infantum, which is mainly spread by Phlebotomus and Lutzomyia sandflies in the Old and New World, respectively. Novel and effective drugs to manage this neglected vector-borne disease are urgently required. In this study, we evaluated the toxicity of carvacrol, thymol and linalool, three common essential oil constituents, on amastigotes and promastigotes of L. infantum. Methods: in vitro experiments were performed by 24 h MTT assay. Carvacrol, thymol and linalool at concentrations ranging from 1.3 to 10 μg/mL were tested on promastigotes of L. infantum. For in vivo test, two groups of hamsters (Mesocricetus auratus) received 100 mg/kg of body weight/day of carvacrol and thymol as intraperitoneal injection on day 7 post-infection, followed by a 48 h later injection. The third group was treated with the glucantime as standard drug (500 mg/kg) and the last group (control) just received normal saline. On the 16th day, the number of parasites and histopathological changes in liver and spleen were investigated. Results: 24 h MTT assay showed promising antileishmanial activity of thymol and carvacrol, with IC50 values of 7.2 (48 μM) and 9.8 μg/mL (65 μM), respectively. Linalool at all concentrations did not affect L. infantum promastigote viability. In vivo toxicity data of carvacrol and thymol showed that the former at 100 mg/kg was the safest and most effective treatment with little side effects on the liver. Conclusions: Overall, thymol and carvacrol are highly promising candidates for the development of effective and safe drugs in the fight against VL.

Background: Human cystic echinococcosis (hydatidosis) continues to be an essen­tial cause of morbidity and mortality in many parts of the world. Methods: We studied hydatid cyst pattern in hospitalized adult patients from 2003 to 2012 in Mashhad and Neyshabour, northeast of Iran. Results: Overall, 1342 patients, 711 females (53%) and 631 males (47%) diag­nosed as infected with hydatid cyst were evaluated. Their age was between 1 and 91 yr (mean age 37.75). The most affected age group was 20-30 yr old. Totally, 953 cases (71%) were urban and 375 cases (27.8%) were rural residents. The most com­mon localization of cysts was the liver and lung. The housewives were the most frequently infected occupations. Conclusion: The rate of infection with hydatid cyst is high in Mashhad, northeast of Iran, and the incidence of human hydatidosis tends to increase in recent years so control and prevention programs are recommended.

Introduction: Androgenetic alopecia (AGA) is one of the most common dermatologic disorders with a multifactorial etiology. Inflammatory activators such as Demodex infestation may play a role in the pathogenesis of some cases of androgenetic alopecia that do not respond to common treatments such as minoxidil and finasteride. The goal of this study is to evaluate the relationship between Demodex infestation and AGA. Materials and Methods: In this case-control study, 41 patients with AGA referred to the Dermatology Clinic of Imam Reza Hospital and 33 healthy individuals were evaluated as control. All of them were between 20 and 40 years old men. In order to identify Demodex infestation they were referred to the Parasitology laboratory. Results: Demodex was detected in 19.5% of patients and 15.2% of controls; therefore, there was no significant relationship between them statistically ( P = 0.0787). Most of patients (85.4%) had greasy hair. The most common pattern of baldness was II degree in Hamilton scale. Conclusion: There is no relation between AGA and Demodex.

Background: Toxocariasis is an important disease caused by the larvae of parasitic worms such as Toxocara canis and T. cati. Public parks can be the source of toxocariasis for small children. This survey was conducted to de-termine the prevalence of Toxocara spp. ova in parks of Mashhad and Khaf northeastern Iran. Methods:In this descriptive cross-sectional study, performed in November 2011 to June 2012, overall, 340 soil samples were collected from 39 parks of Mashhad and 29 parks in Khaf city. Flotation method and direct smear were used, and the samples were evaluated using a light microscope. The results were analyzed using SPSS version 19 and Chi-square test. Results:In the evaluation of 195 and 145 soil samples, 18 (9.2%) and 16 cases (11.3%) of contamination with Toxocara spp. eggs were detected, re-spectively. Conclusion:Although the prevalence of Toxocara eggs in soil samples was low, parks can be a source of Toxocara infection of children in these areas.