Phenotypes characterization and ABC genotypes distribution of clinical Candida albicans isolates.

BACKGROUND: Candidemia and vaginitis are the most common types of candidiasis mostly caused by Candida albicans species. C. albicans has several genotypes and the potential ability to form different phenotype colonies on specific media. This study aimed to evaluate the genotype distribution of blood and vaginal C. albicans isolates and phenotype characteristics on Spider and yeast peptone dextrose agar medium. METHODS: A total of 40 clinical Candida albicans isolates comprising vagina (20) and blood (20) were used. ABC typing using CA-INT-R and CA-INT-L primers was performed to span the transposable group I intron of the 25S rDNA gene. For colony phenotypic characteristics, the Spider and YPDA media were used. RESULTS: Among the blood and vaginal isolates, genotype A (12/60%) and genotype C (10/50%) were the most common types, respectively. The highest phenotype shape frequency of the colonies in blood and vaginal samples was the ring and the lowest was the hat/ring. The dominant color phenotype in blood and vaginal samples was gray. There was a significant relationship between genotype and phenotype forms in the blood sample on YPDA medium (p = 0.02). In the Spider medium, there were no significant differences between genotypes and phenotypes. CONCLUSION: In this study, genotype A and genotype C were predominant in blood and vaginal samples, respectively. In both groups, YPD agar medium demonstrated the most variety of phenotypes that was related to genotypes A and C. The variety of phenotypes in both groups was the same in genotypes A and C on the Spider medium.

Sublingual versus vaginal misoprostol for second trimester termination: a randomized clinical trial.

PURPOSES: To evaluate the efficacy of two routes of administration of misoprostol (sublingual and vaginal) for medical termination of second trimester pregnancies. METHODS: One hundred and thirty-four women referred for second trimester termination were enrolled in this randomized clinical trial. They were divided to receive 400 µg every 6 h misoprostol either sublingually or vaginally. They were followed for 48 h, at which point they underwent D&C if the termination was not complete. Efficacy was defined as successful termination without the need for interventions. RESULTS: There were no differences between the vaginal and sublingual groups in terms of tablets mean dose of misoprostol applied (1360 ± 2.4 vs. 1320 ± 2.3) or endometrial thickness after termination of pregnancy (13.02 ± 5.2 vs. 13.3 ± 6.6 mm). The success rate was 61.2 % (n = 41) in the vaginal group versus 70.1 % (n = 47) in the sublingual group (p = 0.3). Twenty-six patients (38.8 %) in the vaginal group underwent D&C due to retained tissue, compared with 20 patients (29.8 %) in the sublingual group. In primigravids, the success rate was significantly higher in the sublingual group than vaginal group. There was no significant difference with regards to complications between the two groups. CONCLUSION: The sublingual route of misoprostol administration has the same efficacy as the vaginal route and can be applied for second trimester pregnancy termination in primigravid women in outpatient settings due to its simple administrations.

Evaluation of building washing machines as an extreme environment for potentially pathogenic fungi.

Washing machines are commonly used in households and are considered indispensable appliances for maintaining cleanliness and hygiene. Environmental conditions within household washing machines are ideal for fungal colonization, which may pose risks to human health and contribute to sick building syndrome. This study aimed to investigate the fungal species contamination in the building washing machines. A total of 50 building washing machines were swab-sampled at three locations: the detergent drawer, the inner and outer parts of the rubber door seal. The housekeeping conditions of these appliances were assessed through a questionnaire. The isolated fungi were identified using standard mycological diagnostic procedures and molecular analysis based on the ITS1/ITS4 and ß-tubulin gene regions. The possibility of fungal agents transferring from contaminated washing machines to autoclaved clothes during laundry cycles was investigated. Fungi were detected in 82% of the sampled appliances, with the inner rubber door seal being the most frequently colonized area. Using conventional and molecular techniques, we identified 122 fungal isolates, encompassing 17 diverse genera of molds, yeast-like, and yeast fungi. The mold fungi included 14 genera of hyaline and black genus. Among these, the most frequently identified genera of hyaline and black fungi were Aspergillus (27.7%), and Cladosporium (10.7%), respectively. This study demonstrates that building washing machines may serve as suitable ecological niches for fungal growth and transmission. Therefore, regular cleaning and disinfection of these devices are necessary.

Quantitative analysis of in vitro biofilm formation by clinical isolates of dermatophyte and antibiofilm activity of common antifungal drugs.

BACKGROUND: The ability of dermatophytes to develop biofilm, as one of the virulence factors in fungal infections which contribute to antifungal resistance, is an outstanding aspect of dermatophytosis that has been noted recently. Because of the paucity of data about the biofilm formation by dermatophytes and their susceptibility to antifungal drugs, this study evaluated the biofilm formation by clinical isolates of dermatophytes and antibiofilm activity of common antifungals widely used to manage dermatophytosis. METHODS: The ribosomal DNA internal transcribed spacer (ITS) regions sequencing for species identification of 50 clinical dermatophyte isolates was performed. The ability of isolates to form biofilm and inhibitory activity of itraconazole, terbinafine, and griseofulvin against biofilm formation was assayed by the crystal violet staining method. Optical microscopy and scanning electron microscopy (SEM) were applied for the visualization of the biofilm structures. RESULTS: Trichophyton (T.) mentagrophytes (n: 14; 28%) and T. rubrum (n: 13;26%) were included in more than half of the dermatophyte isolates. Biofilm formation was observed in 37 out of 50 (74%) isolates that were classified as follows: nonproducers (n: 13; 26%), weak producers (n: 4; 8%), moderate producers (n: 16; 32%), and strong producers (n: 17; 34%) by comparison of the absorbance of biofilms produced by clinical strains with control. The mean IC50 values for terbinafine, griseofulvin, and itraconazole were 2.42, 3.18, and 3.78 µg/ml, respectively. CONCLUSIONS: The results demonstrated that most of the clinical dermatophyte isolates are capable to form biofilm in vitro with variable strength. Moreover, terbinafine can be suggested as the first-line choice for the treatment of biofilm-formed dermatophytosis.

A randomized comparison of the efficacy, side effects and patient convenience between vaginal and rectal administration of Cyclogest(®) when used for luteal phase support in ICSI treatment.

PURPOSE: This study compares the efficacy, side effects and patient convenience of vaginal and rectal routes of administration of progesterone suppositories (Cyclogest) when used for luteal phase support during in vitro fertilization cycles, through the use of antagonist protocols. METHODS: 147 patients who underwent intra-cytoplasmic sperm injection cycle were randomized on the day of the embryo transfer (ET) by a computer-generated randomization program to receive 400 mg of Cyclogest either vaginally or rectally twice daily for up to 8 weeks. A pregnancy test was conducted 2 weeks after embryo transfer. If the pregnancy test was negative, the application was discontinued. On day 14th after embryo transfer, patient's acceptability and side effects were assessed using a questionnaire which was given to the patients on the day of ET prior to performing the pregnancy test. The clinical pregnancy rate at the 8th week of gestation and the level of luteal progesterone were evaluated. RESULTS: There were no substantial differences in the demographics or other characteristics between the two groups. There were no significant differences in serum P concentration 6 days after ET, the clinical pregnancy and abortion rates. The difficulty of administration route, the discomforts experienced following administration, and the proportion leaking out on the 14th day were similar between the two groups. Significantly more patients administering the medication per vagina had perineal irritation (21.3 vs. 2.2 %). The prevalence of tenesmus (35.1 vs. 21.1 %) and rectal itching (26.7 vs. 2.8 %) were significantly more in rectal route. CONCLUSIONS: This study demonstrates that the efficacy of Cyclogest is similar when administered via both the vaginal and rectal routes. Although their side effects differ, the ease of administration for patients and their preference are similar.

Optimizing denaturing HPLC as a robust technique for identification of Short Tandem Repeats (STR) in forensic medicine.

INTRODUCTION: Short Tandem Repeats (STRs) are defined as short lengths of 2-7 base pairs spreading through human genome which due to their highly diverse individually distribution are widely applied for identity detection and other forensic medicine purposes. Burdening considerable costs by the conventional methods such as capillary electrophoresis, we aimed to compare concomitant usage of multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) as cheap, fast, highly accurate, and more accessible methods, with capillary electrophoresis (CE) to evaluate their potential for early screening of STRs. MATERIALS AND METHODS: The present study randomly included 20 blood samples from the subjects referred to forensic medicine of Semnan, Iran. According to the size and allele frequency, we selected 8 major STR loci including CSF1PO, VWA, D18S51, TPOX, Amelogenin, FGA, SE33, and Penta D. A quad-STR multiplex PCR was performed for each locus and the PCR products were then analyzed using DHPLC machine and compared with the basic genetic properties obtained by capillary electrophoresis. RESULTS: By optimizing the PCR and DHPLC conditions, our findings suggest this strategy as an effective method for STR detection. The genotypes were determined using size of loci which led to comparable results with capillary electrophoresis confirming an insignificant variation in the detection of TOPX, Amelogenin, CSF1PO, and D18S5 (p = 0.331), but discrepant results for FGA and VWA loci (p = 0.002). CONCLUSION: Our study proposed DHPLC method as an effective screening method to characterize TOPX, Amelogenin, CSF1PO, and D18S51 as frequently used STR loci during identity detection in forensic medicine.

Gdn 3+@CNTs-PEG versus Gadovist®: In Vitro Assay.

OBJECTIVES: Carbon nanotubes (CNTs) are allotropes of carbon with a length-to-diameter ratio greater than 106 with the potential uses as medical diagnostic or therapeutic agents. In vitro studies have revealed that gadolinium (Gd) nanoparticle-catalyzed single-walled carbon nanotubes (SWCNTs) possess superparamagnetic properties, which enable them to be used as contrast agents in magnetic resonance imaging (MRI). Our study synthesized Gd-CNT for use as MRI contrast agents. METHODS: To reduce the toxicity and solubility of CNTs, it was functionalized, and after loading with Gd was coated with polyethylene glycols (PEG). We then synthesized different concentrations of Gdn 3+@CNTs-PEG and Gadovist® to be evaluated as MRI contrast agents. RESULTS: The analysis showed that the Gd concentration in Gadovist® was 12.18% higher than synthesized Gdn 3+@CNTs-PEG, but the mean signal intensity of the Gdn 3+@CNTs-PEG was approximately 3.3% times higher than Gadovist®. CONCLUSIONS: Our findings indicate that synthesized Gdn 3+@CNTs-PEG has the potential to be used as an MRI contrast agent in vitro, but in vivo assessment is necessary to determine the bio-distribution, kinetic, and signal enhancement characteristics.

Evaluation of the Effect of Promoter Type on the Immunogenicity of the Live Recombinant Salmonella Vaccines Expressing Escherichia Coli Heat-labile Enterotoxins (LTB).

Enterotoxigenic Escherichia coli (ETEC)-induced diarrhoea is the second most common cause of death in children in the developing countries. Heat labile toxin (LT) is responsible for ETEC-induced diarrhoea. In the present study, a novel live ETEC vaccine based on subunit B of LT (LTB) expression in attenuated PhoPc Salmonella strain was developed. Herein, we aimed to compare the in-vitro activity of promoters including constitutive tac, IPTG inducible trc, and in-vivo-inducible (nirB and nirB78-23) in PhoPc. Additionally, the ability of these recombinant PhoPc/pLTBs to induce LTB-specific antibody responses in BALB/c mice after nasal immunization was evaluated. In-vitro studies demonstrated that PhoPc has the ability to produce rLTB. Furthermore, nirB promoter directed significantly more LTB expression in PhoPc/pnirBLTB under anaerobic condition without induction compared to the amount of rLTB secreted by PhoPc/ptrcLTB in bacterial soup under uninduced condition (6.06 ± 0.05 vs. 1.4 ± 0.46 µg/109 cfu, p < 0.01). In addition, the constitutive rLTB expression from tac promoter was more than its expression from uninduced trc promoter in bacterial soup (4.2 ± 0.92 vs. 1.4 ± 0.46 (µg/109 cfu)) and pellet (27.4 ± 0.89 vs. 13.4 ± 1.42 (µg/109 cfu), p < 0.0001). However, the mice immunized with PhoPc/ptrcLTB elicited the superior anti-LTB responses among the PhoPc containing the examined prompters, which were significantly higher than those induced by PhoPc/pnirB78-23LTB and PhoPc/pnirB, 6 weeks after the first immunization. Totally, it could be concluded that in-vitro analysis of promoters for LTB expression in PhoPc may not necessarily predict the recombinant PhoPc immunogenicity.

Induction of Apoptosis and Inhibition of Invasion in Gastric Cancer Cells by Titanium Dioxide Nanoparticles.

OBJECTIVES: Nanoparticles induce oxidative stress in cells and damage them through the cell membrane and DNA damage, eventually resulting in cell death. This study aimed to evaluate the effect of titanium dioxide (TiO2) nanoparticles on apoptosis induction and invasion of gastric cancer cell line, MKN-45. METHODS: We used the MTT assay to assess proliferation of MKN-45 gastric cancer cells after exposure to different forms of TiO2 nanoparticles including amorph, brookite, anatase, and rutile coated with polyethylene glycol (PEG) and bovine serum albumin (BSA). Ethidium bromide and acridine orange staining were used to visualize cancer cell apoptosis, and the wound healing assay technique (migration test) was used to assay cancer cell invasion. RESULTS: Viability and proliferation of cancer cells in the presence of various forms of TiO2 nanoparticles were reduced (p ≤ 0.050). This reduction in cell proliferation and viability was directly related to concentration and duration of exposure to nanoparticles. Induction of cell death was seen in all groups (p ≤ 0.050). Increased cell invasion was seen in PEG-amorph TiO2 group compared to the control group. Cell invasion was decreased only in the brookite BSA group (p ≤ 0.050). CONCLUSIONS: Various forms of TiO2 nanoparticles reduced cell proliferation and induced apoptosis in cancer cells. Some forms of TiO2 nanoparticles such as brookite BSA also inhibited cell invasion. PEG-amorph TiO2 nanoparticles increased cell invasion. These differences seem to be due to the effects of different configurations of TiO2 nanoparticles. TiO2 may provide a new strategy for cancer treatment and more studies are needed.

Frequency of VanA, VanB and VanH variants amongst vancomycin-resistant enterococci isolated from patients in central region of Iran.

AIM: The aim of this study was to investigate the VRE frequency and the rate of each gene in isolated enterococci from patients with intestinal infection in the central region of Iran. BACKGROUND: Enterococci infections are a public health growing concern due to the glycopeptide antibiotics resistance especially vancomycin. Genes, vanA, B, and H contribute to the influence of vancomycin-resistant enterococci (VRE). PATIENTS AND METHODS: This study was conducted from January to July 2014 in Shahrood university hospital. Enterococci isolation and its antibacterial susceptibility were performed by culturing in Aesculin Azide agar and Kirby-Bauer method, respectively. Vancomycin-resistant genes were screened through conventional PCR, and subsequently sequenced. RESULTS: Among 265 specimens, 100 isolates revealed enterococci, in which E. faecalis (91%) and E. faecium (9%). The isolated enterococci were resistant to vancomycin (6%) and chloramphenicol (21%), whereas their large proportions (94% to 100%) were multi-drug resistant. All VRE isolates belonged to E. faecalis, conversely, the E. faecium were susceptible to the same antibiotic. Both vanA and vanH genes were identified in all VRE isolates, although, no vanB gene was indicated. Homology analysis of sequenced amplicons verified the full length compatibility to the worldwide reported genes. CONCLUSION: The present study revealed VR E.faecalis in gastroenteritis patients and resistance factor for vanA and vanH genes are coordinated. Since enterococci isolates were all multidrug resistance, increase in VR E.faecalis vanA / vanH in this area could be expected.

Evaluating the efficiency of phiC31 integrase-mediated monoclonal antibody expression in CHO cells.

Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site-specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO-S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570-1576, 2016.

Identification of Candida Species Using MP65 Gene and Evaluation of the Candida albicans MP65 Gene Expression in BALB/C Mice.

BACKGROUND: Systemic candidiasis is a major public health concern. In particular, in immunocompromised people, such as patients with neutropenia, patients with Acquired Immune Deficiency Syndrome (AIDS) and cancer who are undergoing antiballistic chemotherapy or bone marrow transplants, and people with diabetes. Since the clinical signs and symptoms are nonspecific, early diagnosis is often difficult. The 65-kDa mannoprotein (MP65) gene of Candida albicans is appropriate for detection and identification of systemic candidiasis. This gene encodes a putative b-glucanase mannoprotein of 65 kDa, which plays a major role in the host-fungus relationship, morphogenesis and pathogenicity. OBJECTIVES: The current study aimed to identify different species of Candida (C. albicans, C. glabrata and C. parapsilosis) using the Polymerase Chain Reaction (PCR) technique and also to evaluate C. albicans MP65 gene expression in BALB/C mice. MATERIALS AND METHODS: All yeast isolates were identified on cornmeal agar supplemented with tween-80, germ tube formation in serum, and assimilation of carbon sources in the API 20 C AUX yeast identification system. Polymerase Chain Reaction was performed on all samples using species-specific primers for the MP65 65 kDa gene. After RNA extraction, cDNA synthesis was performed by the Maxime RT Pre Mix kit. Candida albicans MP65 gene expression was evaluated by quantitative Real-Time (q Real-Time) and Real-Time (RT) PCR techniques. The 2-ΔΔCT method was used to analyze relative changes in gene expression of MP65. For statistical analysis, nonparametric Wilcoxon test was applied using the SPSS version 16 software. RESULTS: Using biochemical methods, one hundred, six and one isolates of clinical samples were determined as C. albicans, C. glabrata and C. parapsilosis, respectively. Species-specific primers for PCR experiments were applied to clinical specimens, and in all cases a single expected band for C. albicans, C. glabrata and C. parapsilosis was obtained (475, 361 and 124 base pairs, respectively). All species isolated by culture methods (100% positivity) were evaluated with PCR using species-specific primers to identify Candida species. Relative expression of Mp65 genes increased significantly after C. albicans injection into the mice (P < 0.05). CONCLUSIONS: The results of the current study showed that the PCR method is reproducible for rapid identification of Candida species with specific primers. Mp65 gene expression of C. albicans after injection into the mice was 2.3 folds higher than before injection, with this difference being significant. These results indicated that increase of Mp65 gene expression might be an early stage of infection; however definitive conclusions require further studies.

Construction of a Synthetically Engineered nirB Promoter for Expression of Recombinant Protein in Escherichia coli.

BACKGROUND: Anaerobic-inducible promoters are alternatives of chemical-inducible promoters for expression of recombinant proteins especially in conditions where chemical induction is not possible or anaerobic conditions are preferable. The nirB promoter is the promoter of the first gene of nir operon in Escherichia coli, which encodes NADH-dependent nitrite reductase. This promoter is naturally induced under anaerobic conditions and upregulated by nitrite and nitrate. OBJECTIVES: The current study was carried out to construct a synthetic nirB promoter that does not respond to chemical inducers (nitrite or nitrate), but instead responds to anaerobic induction. For this purpose, a new plasmid was constructed (pFSnirB78-23LTB), which contains a synthetic nirB promoter. The activity of this plasmid was evaluated in E. coli under both aerobic and anaerobic conditions and in response to chemical inducers, nitrite and nitrate. MATERIALS AND METHODS: A synthetic nirB promoter was firstly cloned into a pKK223 derivative plasmid and then the heat labile toxin B subunit gene (LTB) of entrotoxigenic E. coli was cloned under the control of this promoter. The inducibility of this plasmid in E. coli was measured under anaerobic conditions in the presence or absence of nitrite or nitrate by ganglioside GM1 ELISA. RESULTS: Our data showed that this promoter is strongly induced under anaerobic conditions while it showed much lower activity (11%) under aerobic conditions. In contrast to the native promoter, this promoter was not induced by chemical inducers, nitrite or nitrate. CONCLUSIONS: This study showed that the recombinant protein produced under the control of synthetic nirB promoter has critical characteristics such as pentamer formation, receptor recognition ability and conservation of antigenic epitopes. In addition, the data showed anaerobiosis and chemical inducers had no adverse effects on recombinant proteins. Based on the results, this synthetic promoter is suitable for use in live delivery vaccines or drug systems and for production of recombinant proteins especially oxygen sensitive proteins.

Persistent non-visualisation of the fetal stomach: diagnostic and prognostic implications.

DESIGN: All fetuses diagnosed with 'absent stomach' at anomaly screening over an 8-year period were identified using the University College Hospital fetal medicine database. These were cross-referenced with records from the paediatric surgical unit at Great Ormond Street Hospital and pathology department at University College Hospital to ascertain postnatal or postmortem diagnosis and outcome in each case. RESULTS: Of the 84 cases identified, eight were found to have normal stomachs on subsequent antenatal scans, while 76 had persistent non-visualisation of the stomach. Underlying diagnoses included 24 gastro-intestinal tract and/or respiratory anomalies, 22 aneuploidies, six neuromuscular syndromes, three central nervous system anomalies, seven renal anomalies and five genetic syndromes. Seven cases had no identifiable postnatal abnormalities, 26 pregnancies were terminated and nine fetuses died in utero. Of the 33 live births, eight died in the neonatal period and three died in infancy. Only 28 survived into childhood. Two patients were lost to follow up. CONCLUSIONS: Persistent non-visualisation of the fetal stomach in the antenatal period was associated with a wide range of underlying diagnoses. In many cases, prognosis was poor. Only 37% of pregnancies resulted in liveborn infants surviving more than 6 months. The incidence of an abnormal karyotype was 29%. Diagnosis and outcome was normal in only 9.2% of cases. We propose an algorithm for the management of persistent non-visualisation of the fetal stomach on antenatal ultrasound.