Molecular Identification and Phylogenetic Analysis of Leishmania major Isolated from Zoonotic Cutaneous Leishmaniasis Foci in Northeast Iran.

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. Methods: 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. Results: 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.

Update on dermatophytosis in Mashhad, Northeastern Iran, emergence of infection with Trichophyton persicum.

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.

Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts.

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 < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended.

Frequency of Toxocara Antibodies in Patients Clinically Suspected to Ocular Toxocariasis, Northeast of Iran.

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.

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay.

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.

High incidence of azole resistance among Candida albicans and C. glabrata isolates in Northeastern Iran.

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 discharge specimens were evaluated. Candida isolates detected by polymerase chain reaction-restriction fragment length polymorphism were examined by standard antifungal disk diffusion susceptibility testing method for voriconazole, itraconazole, fluconazole, and ketoconazole. 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 identified as C. albicans, C. glabrata, C. kefyr, C. parapsilosis, and C. tropicalis. The susceptibility rates of Candida isolates to voriconazole, ketoconazole, itraconazole, and fluconazole were 92.6%, 90.7%, 68.5%, and 63.9%, respectively. Moreover, the resistance rates to fluconazole, itraconazole, ketoconazole, and itraconazole were 15.7%, 8.3%, 1.9%, and 1.9%, respectively. The C. glabrata and C. albicans isolates were resistant to antifungal discs among 93% and 20% of the specimens, respectively. Conclusion: The C. glabrata and C. albicans species showed the highest resistance to antifungal drugs. Furthermore, Candida isolates showed the highest sensitivity to voriconazole and ketoconazole and the lowest sensitivity to fluconazole.

Dicrocoelium ova can block the induction phase of experimental autoimmune encephalomyelitis.

AIMS: This study aimed at investigating the impact of Dicrocoelium ova on experimental autoimmune encephalomyelitis (EAE) treatment in C57BL6 mice. METHODS AND RESULTS: Twenty-eight C57BL/6 mice were assigned into four groups as PBS, prophylaxis (P), treatment1 (T1) and treatment2 (T2). Prior to induction of EAE in prophylaxis group and on days 7 and 18 in T1 and T2 groups, respectively, Dicrocoelium eggs were injected intraperitoneally to each mouse. The clinical score, weight changes and incidence time of EAE were recorded. IFN-γ and IL-4 expression is quantified on spleen cells. Also, histopathological study by (H&E) and Toluidine-Blue (TB), and Luxol Fast Blue (LFB) were performed. The data were analysed using SPSS version 21. Mean disease scores were significantly lower in P and T1 groups than the PBS group (P = .01). IFN-γ was lower in P and T1 groups than the PBS group. The highest level of IL-4 was observed in T1 group. The total number of neuroglia cells of corpus callosum was similar in all groups, but the density increased in T1 group compared to the PBS group (P = .03). CONCLUSIONS: Dicrocoelium eggs have a great potential to stimulate immunomodulation towards treatment of EAE during the initial phase.

The rK39 Antigen from an Iranian Strain of Leishmania infantum: Detection of Anti-Leishmania Antibodies in Humans and Dogs.

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<0.05). CONCLUSION: New rk39 recombinant antigen from an Iranian strain of Leishmania infantum seems to be used for diagnosis of VL in humans and dogs. Further extended field studies are recommended.

Changes in the Epidemiology of Cutaneous Leishmaniasis in Northeastern Iran

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.

Clinical and Laboratory Findings of Visceral Leishmaniasis in Children Hospitalized in Mashhad, Northeastern Iran: A Twenty-Year Retrospective Study.

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: A total of 35 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.

Molecular diagnosis and genotyping of Pneumocystis jirovecii in bronchoalveolar lavage samples obtained from patients with pulmonary disorder.

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.

Fish tank granuloma: An emerging skin disease in Iran mimicking Cutaneous Leishmaniasis.

OBJECTIVE: Mycobacterium 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/METHODS: One 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. RESULTS: Twenty-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. CONCLUSION: Patients 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.

Epidemiology of dermatophytosis in northeastern Iran; A subtropical region.

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.

In Vitro and In Vivo Effectiveness of Carvacrol, Thymol and Linalool against Leishmania infantum.

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.

Generation of a CRISPR/Cas9-Based Vector Specific for Gene Manipulation in Leishmania major.

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.

Pediatric Vaginal Leech Infestation with Severe Bleeding: A Case Report and Review Article.

BACKGROUND: Vaginal leech infestation is a rare event with vaginal bleeding being its prominent sign. Few cases have been reported in young children. CASE: In this article, we present a case of vaginal leech infestation in a 2-year-old girl who presented with significant vaginal bleeding that caused severe acute anemia, prompting transfusion. On examination, she appeared anemic, but healthy, without any signs of pubertal development. The leech was seen during examination with the patient under anesthesia and was removed. With removal of the leech, bleeding decreased significantly and stopped completely after 2 hours. She was discharged the next day in good condition. SUMMARY AND CONCLUSION: The important concern in vaginal leech infestation is early diagnosis to prevent severe acute anemia and shock.

Investigation of Visceral Leishmaniasis among 192 Dog Carcasses Killed by Road Accidents in Khorasan Razavi, Northeastern Iran during 2014-2016.

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.

Kinetics of antifungal activity of home-generated ozonated water on Candida albicans.

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.

The Golden jackal (Canis aureus) as an indicator animal for Trichinella britovi in Iran.

Nematodes of the genus Trichinella are zoonotic parasites causing trichinellosis. In Iran, these parasites occur in several animal species and rare cases have been recorded in humans. To monitor the epidemiological pattern of these parasites in the Khorasan-e-Razavi province, Northeastern Iran, muscle tissues were collected from the tongues of roadkill animals between 2016 and 2017: 295 stray dogs, one red fox (Vulpes vulpes), 12 golden jackals (Canis aureus), and one wild boar (Sus scrofa). Trichinella spp. larvae were retrieved using the artificial digestion method and identified to the species level by multiplex PCR. Larvae identified as Trichinella britovi were detected in five stray dogs (1.7%) and one golden jackal (8.3%). The results confirm the circulation of T. britovi in animals of the Khorasan-e-Razavi province, as previously documented. A review of the literature on Trichinella spp. in animals in Iran showed that these parasites were previously detected in 20.02% and 0.04% of carnivore and omnivore mammals, respectively, and that golden jackals can be screened as indicator animals for these zoonotic nematodes. Convenient sampling of Trichinella susceptible roadkill animals may provide a suitable method of monitoring the circulation of these parasites within any given region.

Predominance of non-fumigatus Aspergillus species among patients suspected to pulmonary aspergillosis in a tropical and subtropical region of the Middle East.

Non-fumigatus Aspergillus species are the leading cause of Aspergillus infections in the tropical and subtropical regions of the world. In a prospective study between 2015 and 2016, a total of 150 bronchoalveolar (BAL) specimens was collected from patients suspected to pulmonary aspergillosis (PA) underlying immunodeficiencies in Mashhad, Northeastern Iran, located in the Middle East. All Aspergillus strains were phylogenetically identified at the species level by PCR-sequencing of partial ß-tubulin gene. Overall, Aspergillus species were isolated from 20 specimens originating from 10 (50%) patients with cancer, 5 (25%) patients receiving corticosteroid therapy, 3 (15%) organ transplant recipients and 2 (10%) patients admitted to intensive care unit (ICU). A. flavus complex was the predominant 15 (75%) cause of probable invasive pulmonary aspergillosis (IPA), followed by A. tubingensis 3 (15%), and 2 (10%) A. fumigates complex. In conclusion, distribution of clinical Aspergillus species in the tropical region of the Middle East shows predominance of the non-fumigatus Aspergillus spp., which warrants further attention by health care professionals.