Up-regulation of CDR1 and MDR1 efflux pump genes and fluconazole resistance are involved in recurrence in Candida albicans-induced vulvovaginal candidiasis.

Recurrent vulvovaginal candidiasis (RVVC) due to fluconazole resistance in Candida albicans isolates causes a wide range of complications. A number of 63 Candida albicans isolates obtained from vulvovaginal candidiasis (VVC) were identified by Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (ITS-RFLP). Antifungal susceptibility testing was performed by broth microdilution method according to the CLSI protocol. The role of CDR1 and MDR1 genes in progress of VVC to RVVC was examined and the activity of virulence-related enzymes was assessed. Candida albicans was diagnosed in 62.4 % cases, of which 22.2 % were confirmed as RVVC. Voriconazole was the most active drug among five tested antifungals. The mean expression level of CDR1 and MDR1 was higher in RVVC isolates compared to multidrug azole-resistant VVC isolates. Our results demonstrated that the expression of CDR1 and MDR1 and the level of phospholipase and proteinase activities could be quite important to induce fluconazole resistance in C. albicans and to progress of VVC to become RVVC in involved patients.

Molecular epidemiology, antifungal susceptibility, and ERG11 gene mutation of Candida species isolated from vulvovaginal candidiasis: Comparison between recurrent and non-recurrent infections.

Vulvovaginal candidiasis (VVC) is a prevalent infection of the genitourinary tract affecting millions of women worldwide. In the present study, the importance of virulence factors, ERG11 gene mutations, ERG11 gene expression, and plasma membrane ergosterol content for fluconazole resistance in Candida species was investigated in 200 women suspected of vulvovaginitis. Isolated Candida species were identified using the ITS-restriction fragment length polymorphism (ITS-RFLP) technique. Antifungal susceptibility testing was performed according to the CLSI document. ERG11 gene expression was analyzed using real-time PCR. ERG11 gene mutation analysis was performed using sequencing methods, and the ergosterol content of the cell membrane was determined in fluconazole-resistant isolates. Furthermore, the production of phospholipase and proteinase enzymes was evaluated in recurrent and non-recurrent infections. VVC was diagnosed in 101 (50.5%) of the 200 clinical cases, of which 21 (20.8%) were confirmed as RVVC. Candida albicans was the most prevalent species, followed by C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, and C. guilliermondii. Ketoconazole and fluconazole were the most effective drugs against C. albicans among five tested antifungals with MIC ranges between 0.06 and 16 µg/mL and 0.25-64 µg/mL. Substitutions of A114S, Y257H, T123I and A114V were detected in fluconazole-resistant C. albicans. The ergosterol content of the fungal cell membrane and the mean levels of ERG11 gene expression transcript were higher in fluconazole-resistant C. albicans isolates obtained from RVVC than in those obtained from VVC cases. Phospholipase and proteinase were produced in different amounts in all Candida species isolated from VVC and RVVC cases. In this review, our results demonstrated that several molecular mechanisms, including ERG11 gene expression, changes in the cell membrane ergosterol content, and mutations in ERG11 gene alone or simultaneously involved in fluconazole resistance of C. albicans species and the recurrence of VVC.

Antibacterial Properties of Ag-Cu Alloy Nanoparticles Against Multidrug-Resistant Pseudomonas aeruginosa Through Inhibition of Quorum Sensing Pathway and Virulence-Related Genes.

Objective: To evaluate the application of Ag-Cu NPs as quorum sensing (QS) inhibitors and attenuate virulence expression to overcome the global crisis of multidrug-resistant (MDR) P. aeruginosa. Methods: Ag-Cu NPs were synthesized by co-reduction of silver-nitrate and copper-nitrate (Ag:Cu = 1:1 0.75 µM). In this cross-sectional study, a total of eighty clinical strains of P. aeruginosa were collected from patients with burns. The antibacterial and resistance pattern of the clinical isolated was determined using the microdilution and Kirby Bauer disk methods. The effect of sub-MIC of Ag-Cu NPs on the expression of lasI, exoS and toxA in five clinical isolates of imipenem-resistant P. aeruginosa was performed using qRT-PCR. Results: The characterization methods confirm the formation of the Ag-Cu alloy NPs with agglomerated spherical morphology and particle sizes of about 30-40 nm. We observed that the MIC and MBC of Ag-Cu alloy NPs against MDR P. aeruginosa was found to be 2.5 and 5 µg ml-1, respectively. The effects of a sub-inhibitory concentration of Ag-Cu NPs on MDR P. aeruginosa QS and virulence-related genes showed that the expression level of QS regulatory and virulence genes significantly decreased in both MDR P. aeruginosa and reference strain under Ag-Cu sub-MIC treatment. Conclusion: Ag-Cu NPs could be potentially used as a promising QS inhibitor and anti-virulence compound against P. aeruginosa.

Investigations concerning the impact of consumption of hot beverages on acute cytotoxic and genotoxic effects in oral mucosa cells.

Consumption of very hot beverages and foods increases the incidence of oral and esophageal cancer but the mechanisms are not known and the critical temperature is not well defined. We realized a study with exfoliated cells from the oral cavity of individuals (n = 73) that live in an area in Iran which has the highest incidence of EC worldwide. Consumption of beverages at very high temperatures is a characteristic feature of this population. We analyzed biomarkers which are (i) indicative for genetic instability (micronuclei that are formed as a consequence of chromosomal damage, nuclear buds which are a consequence of gene amplifications and binucleated cells which reflect mitotic disturbances), (ii) markers that reflect cytotoxic effects (condensed chromatin, karyorrhectic, karyolitic and pyknotic cells), (iii) furthermore, we determined the number of basal cells which is indicative for the regenerative capacity of the buccal mucosa. The impact of the drinking temperature on the frequencies of these parameters was monitored with thermometers. We found no evidence for induction of genetic damage but an increase of the cytotoxic effects with the temperature was evident. This effect was paralleled by an increase of the cell division rate of the mucosa which was observed when the temperature exceeded 60 °C. Our findings indicate that cancer in the upper digestive tract in drinkers of very hot beverages is not caused by damage of the genetic material but by an increase of the cell division rate as a consequence of cytotoxic effects which take place at temperatures over 60 °C. It is known from earlier experiments with rodents that increased cell divisions lead to tumor promotion in the esophagus. Our findings provide a mechanistic explanation and indicate that increased cancer risks can be expected when the drinking temperature of beverages exceeds 60 °C.

The Effect of Environmental Stresses on lipL32 Gene Expression in Pathogenic Leptospira spp. through Real-Time PCR.

Leptospirosis is a worldwide infectious and zoonotic disease. The incidence of this disease is high in temperate regions, especially in northern Iran. The aim of this study was to investigate the effects of temperature, pH, and Phyllanthus amarus plant extract on the lipL32 gene expression in pathogenic Leptospira spp. Fifty water samples were collected. Culture and PCR technique were used to isolate and identify the bacterium and the presence of the lipL32 gene. The samples were exposed to different temperatures and pH levels for one day and the Ph. amarus plant extract at different concentrations for one and seven days. RNA was extracted, and cDNA synthesis was performed for all the samples. All cDNAs were evaluated by the real-time PCR (SYBR green) technique. Out of the 50 samples, ten samples (20%), using PCR were determined to contain the pathogenic Leptospira. Fold change of the expression of the lipL32 gene associated with stresses was as follows: temperature stress of 40°C, 35°C, and 25°C reduced the lipL32 gene expression in all three isolates, especially in the isolates type 1. The pH stress, i.e., pH values equal to 8 or 9 reduced the gene expression in three types of isolates, and pH = 6 stress increases the lipL32 gene expression in the isolates of type 1. Ph. amarus plant extract stress reduced the mentioned gene expression only in isolates of type 2. Temperature and pH stresses could lead to differences in the expression level and cause the lipL32 gene expression decrease in three pathogenic isolates. The MIC results showed anti-leptospiral effect of Ph. amarus plant extract.

A comparison of culture and PCR methods for identifying Propionibacterium acnes in lesions isolated from patients with acne.

BACKGROUND/AIM: One of the factors that affect the occurrence of acne is the presence of Propionibacterium acnes. The present study was conducted to compare the culture and polymerase chain reaction (PCR) methods for identifying P. acnes in lesions isolated from patients with acne. MATERIALS AND METHODS: To examine the presence of P. acnes, 70 samples of acne lesions were collected. Microbial culture and the PCR molecular technique were used to identify P. acnes. RESULTS: Of the total of 70 samples, 14 cases (20%) were identified as P. acnes positive using microbial culture and 58 cases (82.85%) using PCR. The results obtained showed the lack of a relationship between the frequency of P. acnes and factors such as sex, family history of acne, and history of treatment with either of the techniques examined (i.e. the microbial culture and PCR). In contrast, a significant relationship was observed between the frequency of P. acnes and age with the culture method. CONCLUSION: Given the limitations in the identification of P. acnes using microbial culture, PCR is proposed as a better method with a higher efficiency.

Prevalence of Cytomegalovirus in Patients With Multiple Sclerosis: A Case-Control Study in Northern Iran.

BACKGROUND: Multiple sclerosis (MS) is a chronic debilitating disease known as one of the most common neurological dysfunctions in young adults. Recent studies suggest that infections with herpesviruses play a critical role in the pathogenesis of MS. OBJECTIVES: The present investigation aimed to detect the presence of cytomegalovirus (CMV) in patients with MS using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) methods. PATIENTS AND METHODS: Plasma and peripheral blood mononuclear cells (PBMCs) were collected from MS patients (n = 82) and from blood donors as control group (n = 89). They were tested for the presence of CMV antibodies and DNA by ELISA and PCR, respectively. RESULTS: Anti-CMV was positive in 65 (79.3%) and 69 (77.5%) of the MS patients and healthy subjects, respectively (P= 0.853). Similarly, 23 (28%) and 2 (2.2%) patients were positive for CMV DNA among the MS and control groups, respectively. Statistical analysis showed that the frequency of CMV DNA in the MS patients was significantly higher than in the healthy controls (P < 0.001). CONCLUSIONS: The results of this study showed a possible association between CMV infection and MS. Further experimental and epidemiological studies using case-control approaches are needed to confirm this association.

The High Prevalence of the Varicella Zoster Virus in Patients With Relapsing-Remitting Multiple Sclerosis: A Case-Control Study in the North of Iran.

BACKGROUND: Multiple sclerosis (MS) is the most common neurological autoimmune disease, characterized by multifocal areas of inflammatory demyelination within the central nervous system. It has been hypothesized that the stimulation of the immune system by viral infections is the leading cause of MS among susceptible individuals. OBJECTIVES: The aim of this study was to investigate the prevalence of the varicella zoster virus (VZV) in patients with relapsing-remitting multiple sclerosis. PATIENTS AND METHODS: Plasma and peripheral blood mononuclear cells (PBMCs) collected from MS patients (n = 82) and controls (n = 89) were screened for the presence of anti-VZV antibodies and VZV DNA by the ELISA and PCR methods. DNA was extracted from all samples, and VZV infection was examined by the PCR technique. Statistical analysis was used to investigate the frequency of the virus in MS patients and a healthy control group. RESULTS: Of all the MS patients, 78 (95.1%) and 21 (25.6%) were positive for anti-VZV and VZV DNA, respectively. Statistical analysis of the PCR results showed a significant correlation between the abundance of VZV and MS disease (P < 0.001). However, there was no significant correlation between the abundance of anti-VZV antibodies and MS disease by the ELISA method. CONCLUSIONS: These results support the hypothesis that VZV may contribute to MS in establishing a systemic infection process and inducing an immune response.

Prevalence of occult hepatitis C virus infection in Iranian patients with lymphoproliferative disorders.

Occult HCV infection is a form of chronic HCV infection characterized by absence of detectable anti-HCV antibodies or plasma HCV-RNA but presence of HCV-RNA in liver biopsy and/or peripheral blood mononuclear cells (PBMCs). The aim of this study was to determine the presence of HCV-RNA in PBMCs of patients with lymphoproliferative disorders. One hundred and four consecutive patients with lymphoproliferative disorders admitted to Firouzgar Hospital from January 2010 to March 2011 were recruited in this cross-sectional study. A 6-ml sample of whole blood was taken from the patients, the total RNA was extracted from the samples after the separation of plasma and PBMCs. The HCV-RNA of the samples was amplified by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR). The HCV genotypes of the positive samples were tested using the INNO-LiPA™ HCV II kit, and the HCV genotypes were then confirmed by sequencing of the 5'-UTR fragments after the PCR products were cloned into a pJET1.2/blunt cloning vector. The mean age of the patients was 48.3 ± 1.76 years (range: 16-83). HCV-RNA was found in PBMCs from 2 (1.9%) of the 104 patients. Genotyping showed that the patients were infected with HCV subtype 1a. One patient suffered non-Hodgkin's lymphoma and the other suffered chronic lymphocytic leukemia. Patients with lymphoproliferative disorders with negative anti-HCV antibodies and negative plasma HCV-RNA may have occult HCV infection. Therefore, in the absence of a liver biopsy, the testing of PBMCs for the detection of genomic HCV-RNA may be beneficial.

Distribution of hepatitis C virus genotypes amongst the beta-thalassemia patients in North of Iran.

Beta-thalassemia patients have high prevalence for HCV infection. In developing countries, HCV antibody is reported to be high in this group of patients. This study carried out to determine the distribution of HCV genotypes amongst the beta-thalassemia patients in North of Iran. The present study has been carried out between February and March 2010 amongst a group of 245 beta-thalassemia patients (125 male and 120 female) referred to the hospitals Mazandaran and Guilan provinces for a blood transfusion. Qualitative analysis of these samples using ELISA and PCR. The PCR positive samples were subjected to genotyping by RFLP method. Of total 245 beta-thalassemia patients who were the subjects of this study, 28 of these patients were diagnosed through PCR test to have RNA virus. For this reason, the prevalence of this illness in this study group was estimated as 11.42%. By using the RFLP technique, the above genotyping were identified and the prevalence of three genotypes, including 3a, 1a and 1b were proved. The genotype 3a was most prevalent. Out of 28 positive samples, 18 (64.3%) samples had this genotype. After that, genotype 1a with 9 positive occurrences (32.1%) and genotype 1b with only 1 positive occurrence (3.6%) were most prevalent. This study demonstrated that the main reason the beta-thalassemia patients became infected with the genotype of the virus was due to receiving infected blood that entered into Iran during the past two decades.