Regulation of ERG3, ERG6, and ERG11 genes in antifungal-resistant isolates of candida parapsilosis

Background: Candida parapsilosis is one of the five common strains of yeasts involved in invasive candidiasis. The expression analysis of sterol biosynthesis pathway genes, which are associated with resistance, can assist the better understanding of antifungal resistance mechanisms

Methods: The antifungal susceptibility of 120 clinical C. parapsilosis isolates was examined. The changes in the gene expression related to resistance were analyzed

Results: Eight strains were resistant to fluconazole [FLC], itraconazole [ITC], and amphotericin B [AMB]. The regulation variations included increased mRNA levels of ERG3, ERG6, and ERG11 and decreased mRNA levels of ERG3 and ERG6 in response to FLC. ERG11 mRNA level increases in response to ITC and AMB

Conclusion: The mechanism of resistance to azoles in C. parapsilosis is very similar to C. Albicans. This feature may help to design new treatment strategy for candidiasis

Genotyping of echinococcus gtanulosus isolates from human clinical samples based on sequencing of mitochondrial genes in Iran, Tehran

The present study was aimed to investigate molecular diversity of Echinococcus gmnulosus isolates collected from human clinical samples using two mitochondrial genes cox1 and nod1 in Iran. Forty seven human hydatid cysts were collected through surgery from two hospitals in Tehran during 2010-2012. To determine the fertility of protoscoleces, the cyst fluids were subjected to morphological microscopic examinations. Protoscoleces were removed from each cyst and their total genomic DNAs were extracted. PCR was performed to amplify fragments of 450 and 400 base pair [bp] for cox1 and nod1 genes, respectively. Genotype diversity and sequence variation of the strains were studied by bioinformatics software and in comparison with those mtDNA sequences already dposited in GenBank. Sixteen, [53.3%], 13 [43.3%], and 1 [3.3%] samples were related to lung, liver, and spleen, respectively. The remained 17 unfertile samples were excluded from the study. From the 29 isolates, 86.7% [n=26] and 10% [n=3] were related to G1, and G3 genotypes, respectively. The sole isolate with G6 genotype was obtained from lung sample. Analysis of concatenated sequences of cox1+nad1 indicated the presence of 11 haplotypes among our strains that were related to genotypes G1 [n=9], G3 [n=1] and G6 [n=1]. In consistent to other reports from Iran, genotypes G1, G3, and G6 were observed in our human isolates. The rate of G3 genotype was however higher than other studies implying that human can be considered as a new appropriate host for G3 genotype. Further studies with more sample size from different geographic areas of Iran are needed for E. granulosus mapping

Pneumocystis jirovecii colonization in non-HIV-infected patients based on nested-PCR detection in bronchoalveolar lavage samples

Pneumocystis jirovecii causes Pneumocystis pneumonia [PCP] in immunocompromised patients with a high rate of morbidity and mortality. Colonization with this fungus may stimulate pulmonary inflatnmation or lead to PCP in susceptible patients. The epidemiology of this infection and routs of its transmission has poorly studied in Iran. We examined Pnaumocystis colonization in patients with various lung underlying diseases. 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 UNA gene was used for investigating P. jirovecii in the specimens. 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. 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

In-vitro activity of 10 antifungal agents against 320 dermatophyte strains using microdilution method in Tehran

Dermatophyte fungi are the etiologic agents of skin infections commonly referred to as ringworm. These infections are not dangerous but as a chronic cutaneous infections they may be difficult to treat and can also cause physical discomfort for patients. They are considered important as a public health problem as well. No information is available regarding the efficacy of antifungal agents against dermatophytes in Tehran. Therefore, in this study we evaluated the efficacy of 10 systemic and topical antifungal medications using CLSI broth microdilution method [M38-A]. The antifungal agents used included griseofulvin, terbinafine, itraconazole, ketoconazole, fluconazole, voriconazole, clotrimazole, ciclopiroxolamine, amorolfine and naftifine.Fifteen different species of dermatophytes which were mostly clinical isolates were used as follows; T. mentagrophytes, T. rubrum, E. floccosum, M. canis, T. verrucosum, T. tonsurans,M. gypseum, T. violaceum, M. ferruginum,M. fulvum, T. schoenleinii, M. racemosum, T. erinacei,T.eriotriphon and Arthrodermabenhamiae. The mean number of fungi particles [conidia] inoculated was 1.25 x 10[4] CFU/mL. Results were read after 7 days of incubation at 28 °C. According to the obtained results,itraconazole and terbinafine showed the lowest and fluconazole had the greatest MIC values for the most fungi tested. Based on the results, it is necessary to do more research and design a reliable standard method for determination of antifungal susceptibility to choose proper antibiotics with fewer side effects and decrease antifungal resistance and risk of treatment failure

molecular epidemiological survey of clinically important dermatophytes in Iran based on specific RFLP profiles of betatubulin gene

Surveillance of dermatophytosis is essential to determine the likely changes in etiological trends and distribution profile of this infection. In this study beta tubulin gene [BT2], was used as the first time in a PCR-RFLP format to clarify the distribution of dermatophytosis agents in some parts of Iran. A total of 603 clinical isolates was obtained from 500 patients in Tehran, Isfahan, Mazandaran and Guilan provinces. The isolates were identified using macro/micro-morphological criteria and electrophoretic patterns of PCR amplicons of BT2 after digestion with each of the restriction enzymes. FatI, Hpy CH4V, MwoI and Alw21L. Among the patients, 59.2% were male and 40.8% female. The most prevalent clinical form was tinea pedis [42.4%], followed by tinea cruris [24.2%], tinea unguium [12.3%], tinea corporis [10.8%], tinea faciei [4%], tinea manuum [3.14%], tinea capitis [3%] and tinea barbae [0.16%], respectively. Trichophyton interdigitale ranked the first, followed by T.rubrum, Epidermophyton floccosum, Microsporum canis, T. tonsurans, T. erinacei and T. violaceum [each 0.49%] and the less frequent species were T. schoenleinii, M. gypseum and T. anamorph of Arthroderma benhamiae [each 0.16%]. A case of scalp infection by E. floccosum was an exceptional event in the study. No case of T.verrucosum was found. Trichophyton species and E. floccosum are yet the predominant agents of infection in Iran, while Microsporum species are decreasing. T.interdigitale and Tinea pedis remain as the most causal agent and clinical form of dermatophytosis, respectively. it seems that BT2 can be a useful genetic marker for epidemiological survey of common pathogenic dermatophytes

[ identification of nocardia in BAL specimens of bronchoscopic patients by using classical and molecular methods]

Nocardiosis is a rare and potentially life-threatening infection caused by several species of the Nocardia genus. The objective of this study was to develop and evaluate a rapid and new method to clinically identify relevant Nocardia species. Rapid and accurate diagnosis of Nocardia species is essential for the treatment of severe infections and prevention of cerebral abscess. One hundred and eighty patients, 103 [57.22%] male and 77 [42.78%] female, with severe symptomatic pulmonary infection were studied in the course of a 12-month period in Dr. Shariati Teaching Hospital affiliated to Tehran University of Medical Sciences in 2010. The specimens were cultured and identified using microbiological and biochemical tests. Polymerase chain reaction [PCR] was used to directly identify the organism in the broncoalveolar lavage samples collected from the patients. NG1 and NG2 primers were used to amplify a Nocardia genus-specific 598-bp fragment of 16S rRNA. Nineteen samples [10.56%] were positive with PCR and 5 samples [2.78%] with conventional methods. All samples with positive cultures were also positive by PCR. The results of this study showed that PCR has a high sensitivity and accuracy for the detection of Nocardia compared with culture and biochemical tests. Considering the rapidity, precision, high sensitivity and specificity of molecular techniques, use of these techniques is suggested in conjunction with conventional methods for the detection of Nocardia phenotypes in clinical laboratories and research centers

Comparison of enzymatic method rapid yeast plus system with rflp-PCR for identification of isolated yeast from vulvovaginal candidiasis

To compare two identification methods, i.e., restriction fragment length polymorphism [RFLP]-PCR analysis and enzymatic method Rapid TM Yeast Plus System to identify different species causing vulvovaginal candidiasis [VVC]. Vaginal discharges of women who had attended the gynecology outpatient clinic of Mobini Hospital in Sabzevar, Iran were collected using cotton swabs and were cultured on Sabouraud dextrose agar. Isolated yeasts were identified by germ-tube testing and Rapid TM Yeast Plus System [Remel USA]. For molecular identification, the isolated DNA was amplified with ITS1 and ITS4 universal primers and PCR products digested with the enzyme HpaII followed by agarose gel electrophoresis. Epidemiological and clinical features of women with respect to identified species were also evaluated. Out of 231 subjects enrolled, 62 VVC cases were detected. The isolated species were identified as follows: Candida albicans, 24 [38.7%], C. glabrata, 15 [24.2%], C kefyr 13 [21.0%] C. krusei, 9 [14.5%], and Saccharomyces cerevisiae, 1 [1.6%] by RFLP-PCR method; whereas findings by Rapid TM Yeast Plus System were C albicans, 24 [38.7%], C glabrata, 5 [8%], C. kefyr, 11 [17.7%] C. krusei, 2 [3.2%], S. cerevisiae, 9 [14.5%], and C. tropicalis, 6 [9.6%] as well as other nonpathogenic yeasts, 4 [6.9%]. Statistical comparison showed that there is no significant difference in identification of C. albicans by the two methods; although, in this study, it was not true about other species of yeasts. A correlation between clinical and laboratory findings is important as it enables us to administer an appropriate treatment on time

Genetic structure of Plasmodium vivax population assessed by sequence analysis of the merozoite surface protein 3 beta gene

The endemicity and transmission intensity levels of malaria are related to genetic diversity of the parasites. Merozoite surface protein 3beta [MSP3beta] is an important marker for assessing the polymorphic nature of Plasmodium vivax while it is also a vaccine candidate against the parasite. In this study we investigated the genetic structure of P. vivax population by sequence analysis of a polymorphic region of the P. vivax MSP3beta gene in isolates from Iran. Blood samples were collected from 100 patients with clinical symptoms. DNA was extracted and the target gene was amplified by polymerase chain reaction [PCR]. The sequences of 17 samples were used for sequence analysis using nucleotide Blast search and ClustalW multiple alignment. Phylogenetic tree was derived to describe the geographical branching and relationships. A large number of nucleotide insertions and deletions were observed in the sequences of polymorphic region of PvMSP3beta gene that were not specific in each biotype. Single nucleotide polymorphism [SNP] was found extensively in the sequences. The phylogenetic analysis did not show any significant geographical branching. The lack of any geographical branching and extensive polymorphism in MSP3beta gene of P. vivax isolates suggests that more investigations are needed to find a more suitable gene in order to develop a vaccine

[Identification of pathogenic yeasts isolated from onychomycosis in Tehran, using polymerase chain reaction and enzymatic digestion]

Fungal nail infection [onychomycosis] is a common disease in all communities consisting about 50 percent of nail disorders. Yeasts are one of the important causative agents of onychomycosis. Identification of the yeast species is important in the epidemiological and therapeutical point of views. The aim of the present study is the precise species identification of the pathogenic yeast isolated from fungal nail infections, using the DNA-based methods. The isolates were preliminary studied according to study of morphological characteristics. For species identification, the genomic DNA of each sample was extracted by boiling method and the ITS region of ribosomal DNA was amplified by polymerase chain reaction [PCR]. The amplified DNA was digested by the restriction enzyme MspI and each isolate was identified according to the electrophoretic patterns. A new enzymatic profile was used for final differentiation of Candida albicans and C. dubliniensis. A few of yeast isolates were identified by using ITS-sequensin. C. albicans with the prevalence of 45.6% was the most common isolate, followed by C. parapsilosis with 22.5% and C. tropicalis with 21.8%. The less common species were C. glabrata, C. krusei, C. kefyr, C. lusitaniae, C. guilliermondii and C. pulcherrima that consisted 2.72%, 2%, 1.36%, 0.68% and 0.68% of the isolates, respectively. No C. dubliniensis was found among C. albicans isolates. Two isolates [1.36%] were identified as Trichosporon spp. The most common group of the patients was in the age range of 40-70 years old and the majority [83.2%] of the patients were women with finger nail infections. Although C. albicans is still the most prevalent isolates of nail candidiasis, the increasing number of non-albicans species is notable. The study showed that for identification of some rare species, the routine phenotypical approaches are not efficient and application of the ITS-PCR-RFLP can improve the level of differentiation up to 98%. The remaining isolates can be identified by more expensive methods such as sequencing

A Case of Fatal Strongyloidiasis in a Patient with Chronic Lymphocytic Leukemia and Molecular Characterization of the Isolate

Strongyloides stercoralis is a human intestinal parasite which may lead to complicated strongyloidiasis in immunocompromised. Here, a case of complicated strongyloidiasis in a patient with chronic lymphocytic leukemia is reported. Presence of numerous S. stercoralis larvae in feces and sputum confirmed the diagnosis of hyperinfection syndrome in this patient. Following recovery of filariform larvae from agar plate culture of the stool, the isolate was characterized for the ITS1 region of ribosomal DNA gene by nested-PCR and sequencing. Albendazole therapy did not have cure effects; and just at the beginning of taking ivermectin, the patient died. The most important clue to prevent such fatal consequences is early diagnosis and proper treatment.

[Malassezia flora of neonates hospitalized in hospitals affiliated to tehran university of medical Sciences]

Malassezia yeasts are normal flora of humans and warm-blooded animals. These lipophilic yeasts are associated with skin diseases in neonates such as pityriasis versicolor, neonatal postulitis and seborrheic or atopic dermatitis. Moreover in the recent years, these yeasts are increasingly isolated form fatal catheter-related fungemia in premature neonates. Concerning the role of Malassezia species in neonatal diseases and variation in their pathogenesis and sensitivity to antifungal drugs, we investigated the distribution of Malassezia species and related predisposing factors in neonates. 261 skin samples from scalp, chest and ear were collected from neonates in both Children Medical Center and Vali-Asr Hospitals using cellotape method and sterile wet swab. All samples were also inoculated in plates containing Leeming-Notman medium and Malassezia colonies were then sub-cultured on modified-Dixon and SCC media. Malassezia species were identified according to their macroscopic and microscopic morphological features and their physiological properties including tween assimilation test, catalase reaction and splitting of sculine. In this study 36% of samples were collected from Vali-Asr Hospital and the rest from Children Medical Center. The average age of the examined individuals was 11.7 days. 58.7% of neonates were boys and 41.3% were girls. Based on culture results, 68.9% of examined neonates had Malassezia flora. Besides, significant differences in frequency of isolated Malassezia were not seen between either two examined hospitals nor NICU and neonatal wards. M. furfur was the most common isolated species followed in frequency by M. globosa. In addition, M. obtusa and M. slooffia were recovered only once from trunk and head samples, respectively. In contrast to Malassezia flora in adults which is M.globosa, we isolated M. furfur as the dominant flora in neonates. This high prevalence of colonization may put hospitalized neonates in great danger of nosocomial Malassezia infections. Considering high mortality of Malassezia fungemia in neonates, skin should be cleaned effectively from Malassezia flora prior to administration of intra venous lipid or catheters