Genetic approach toward linkage of Iran 2012-2016 cholera outbreaks with 7th pandemic Vibrio cholerae.

Vibrio cholerae, as a natural inhabitant of the marine environment is among the world-leading causes of diarrheal diseases. The present study aimed to investigate the genetic relatedness of Iran 2012-2016 V. cholerae outbreaks with 7th pandemic cholera and to further characterize the non-ST69/non-ST75 sequence types strains by whole-genome sequencing (WGS).Twenty V. cholerae isolates related to 2012, 2013, 2015 and 2016 cholera outbreaks were studied by two genotyping methods - Pulsed-field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST)-and by antimicrobial susceptibility testing. Seven sequence types (STs) and sixteen pulsotypes were detected. Sequence type 69 was the most abundant ST confirming that most (65%, 13/20) of the studied isolates collected in Iran between 2012 and 2016 belonged to the 7th pandemic clone. All these ST69 isolates (except two) exhibited similar pulsotypes. ST75 was the second most abundant ST. It was identified in 2015 and 2016. ST438, ST178, ST579 and STs of 983 and 984 (as newfound STs) each were only detected in one isolate. All strains collected in 2016 appeared as distinct STs and pulsotypes indicative of probable different originations. All ST69 strains were resistant to nalidixic acid. Moreover, resistance to nalidixic acid, trimethoprim-sulfamethoxazole and tetracycline was only observed in strains of ST69. These properties propose the ST69 as a unique genotype derived from a separate lineage with distinct resistance properties. The circulation of V. cholerae ST69 and its traits in recent years in Iran proposes the 7th pandemic strains as the ongoing causes of cholera outbreaks in this country, although the role of ST75 as the probable upcoming dominant ST should not be ignored.Genomic analysis of non-ST69/non-ST75 strains in this study showed ST579 is the most similar ST type to 7th pandemic sequence types, due to the presence of wild type-El Tor sequences of tcpA and VC-1319, VC-1320, VC-1577, VC-1578 genes (responsible for polymyxin resistance in El Tor biotype), the traits of rstC of RS1 phage in one strain of this ST type and the presence of VPI-1 and VSP-I islands in ST579 and ST178 strains. In silico analysis showed no significant presence of resistance genes/cassettes/plasmids within non-ST69/non-ST75 strains genomes. Overall, these data indicate the higher susceptibility of V. cholerae non-ST69/non-ST75 strains in comparison with more ubiquitous and more circulating ST69 and ST75 strains.In conclusion, the occurrence of small outbreaks and sporadic cholera cases due to V. cholerae ST69 in recent years in Iran shows the 7th pandemic strains as the persistent causes of cholera outbreaks in this country, although the role of ST75 as the second most contributed ST should not be ignored. The occurrence of non-ST69/non-ST75 sequence types with some virulence factors characteristics in border provinces in recent years is noteworthy, and further studies together with surveillance efforts are expected to determine their likely route of transport.

Multidrug-resistant Salmonella strains from food animals as a potential source for human infection in Iran.

This study aimed to determine the frequency and characterization of Salmonella isolates from food-producing animals and human diarrheal samples in Shiraz, Iran. Overall, 105 Salmonella isolates were obtained from chicken meat (70/100, 70.0%), beef (19/100, 19.0%), lamb (11/100, 11.0%), and human stool (5/295, 1.7%). S. Infantis (40.9%), S. Enteritidis (29.5%), and S. Paratyphi B (8.6%) were the most prevalent serotypes. Totally 59.1% of the isolates were multidrug-resistant. High resistance to nalidixic acid (67.6%), tetracycline (62.9%), and trimethoprim-sulfamethoxazole (42.3%) were observed. Mutations in the gyrA and parC genes were detected in nalidixic acid-resistant isolates from chicken meat and human stool. No qnrA, qnrB or qnrS genes were detected. The blaTEM and blaCMY-2 genes were detected in ß-lactam-resistant isolates from beef and lamb. Despite the high genetic diversity of PFGE patterns, some isolates from different sources and times showed identical PFGE patterns suggesting specific Salmonella species circulate between various sources over time.

Evaluation Frequency of Human Papillomavirus and Its Related Genotypes in Women of the General Population Living in 11 Provinces of Iran.

Introduction: Persistent infection with one of the most high-risk genotypes of human papillomavirus causes all cases of cervical cancer and a significant proportion of other genital cancers. The HPV virus, unlike any other infection that leads to cancer, is transmitted only through sexual intercourse and is less affected by the general changes and development in lifestyle and medical standards, so only vaccination and screening can prevent the HPV virus and cancers caused by it. Therefore, determining the prevalence and distribution of HPV genotypes are of utmost importance in screening strategies regarding cervical cancer and vaccination decisions against HPV that vary based on the geographical and cultural characteristics of the study area. As a result, this study aimed to determine the frequency of human papillomavirus and the distribution of this virus's genotypes in the general population of women living in 11 provinces of Iran. Materials and Methods: This study is a community-based survey study. Sampling was done by the cluster sampling method. Women aged 15-59 years old from the general population living in 11 provinces of Iran were included in the study after considering the inclusion and exclusion criteria. Data were collected using a questionnaire and vaginal examination. The study was performed on 2562 vaginal specimens that were referred to the laboratory of the present study. HPV genome was detected by the nested MY-GP method and papillomavirus genotyping was performed using the PCR multiplex method to identify 19 papillomavirus genotypes. Results: The general prevalence of HPV in the 11 provinces was obtained at 2.4% (108 out of 2562 people). The highest prevalence of the virus was in the age group of 25-34 years. The prevalence of HPV was statistically significant among different provinces. Hormozgan province with 22 cases (5.9%) had the highest and Isfahan province with 6 cases (2.2%) had the lowest incidence of HPV. The prevalence of high-risk HPV and medium-risk HPV is 3%, and the prevalence of low-risk HPV was estimated to be 2.1% of the total female population. Also, the highest prevalence was related to genotype 16. Conclusion: According to the high prevalence of the HPV virus in young age groups in Iran, it is necessary to pay attention to screening programs to reduce the incidence of cervical cancer.

Clonal Relationship and Resistance Profiles Among ESBL-Producing Escherichia coli.

AmpC ß-lactamases hydrolyze all ß-lactams except cefepime and carbapenems. The study of AmpC-producing E. coli has high priority for the infection control committee. This research is aimed to investigate the resistant urinary AmpC-generating E. coli isolates and identify their genetic variety. Some 230 E. coli isolates from patients suffering urinary tract infection symptoms were studied in 2017-2018 to assess their susceptibility toward antimicrobial agents. AmpC gene was evaluated by PCR and molecular typing using the 10-loci MLVA method. MLVA images were examined by BioNumerics 6.6 software through the use of the UPGMA algorithms. Thirty-eight AmpC-generating E. coli isolates were detected. The most abundant determinant was blaCIT and blaEBC , blaFOX , and blaDHA had the next ranks, respectively. Six major clusters and a singleton were identified by MLVA. AmpC beta-lactamases in urinary isolates of E. coli in the hospital under study and high rate of additional resistance to gentamicin, cotrimoxazole and ciprofloxacin. The most frequent gene determinant of AmpC beta-lactamase was blaCIT and vary depending on time and geographical location.

Subtyping ß-lactamase-producing Escherichia coli strains isolated from patients with UTI by MLVA and PFGE methods.

OBJECTIVES: Strain subtyping is an important epidemiological tool to trace contamination, determine clonal relationships between different strains, and the cause of outbreaks. Current subtyping methods, however, yield less than optimal subtype discrimination. Pulsed-field gel electrophoresis is the gold standard method for Escherichia coli and Multiple-Locus Variable-number tandem repeat Analysis is a rapid PCR-based method. The purpose of this study was to evaluate MLVA and PFGE methods for subtyping ß -lactamase-producing E. coli strains isolated from urinary tract infections. MATERIALS AND METHODS: Overall, 230 E. coli isolates from patients with urinary tract infections were examined for antimicrobial susceptibility testing. 10-loci and 7-loci MLVA and PFGE methods were used for molecular typing of ß -lactamase-producing E. coli isolates. RESULTS: Out of 230 isolates, 130 (56.5%) ß -lactamase-producing E. coli isolates were found in this study. The diversity indices of the VNTR loci showed an average diversity of 0.48 and 0.54 for 7-loci and 10-loci MLVA, respectively. The discriminatory power of PFGE showed a value of 0.87. The discordance between the methods was high. CONCLUSION: Our study showed that PFGE is more discriminatory than MVLA. MLVA is a PCR- based method and can generate unmistakable data, in contrast to PFGE. Optimization of polymorphic VNTR is essential to improve the discriminatory power of MLVA based on geographical region.

Deregulation of miR-21 and miR-29a in Cervical Cancer Related to HPV Infection.

BACKGROUND: Early diagnosis is an important factor to improve the survival of Invasive Cervical Cancer (ICC) patients. Molecular biomarkers such as micro RNA (miRNA) can be used in the early detection of ICC. The expression of miR-21 and miR-29a are deregulated in many types of human cancers. OBJECTIVE: The aim of this study was to investigate the differences in miR-21 and miR-29a expression patterns in the Human Papilloma Virus (HPV) infection and various grades of cervical cancer among Iranian women. METHODS: Small RNAs were extracted from positive for HPV, cervical cancer and healthy samples from 43, 50 and 46 individuals, respectively. Expression levels of miR-21 and miR-29a were analyzed by SYBR Green real-time RT-PCR using specific primers, and 5s rRNA as the internal reference gene. RESULTS: Results have shown a significant increase in miR-21 and decrease in miR-29 in cancerous samples in comparison with the control groups (P < 0.0001). CONCLUSION: This study illustrated that miR-21 and miR-29a could be operated as an oncogene and tumor-suppressor in cervical cancer progression. More studies are needed to demonstrate the role of miR-21 and miR-29a as potential biomarkers for the diagnosis of cervical cancer in future investigations.

Glutathione S-transferase omega gene polymorphism as a biomarker for human papilloma virus and cervical cancer in Iranian women

Objective: Human papillomavirus (HPV) infection is an important sexually-transmitted infection worldwide. Persistent infections with different high-risk HPV genotypes may cause cervical intraepithelial neoplasia and cervical cancer. Single nucleotide polymorphisms of glutathione S-transferase omega (GSTO) 1 and 2 play an important role in cancer progression. To evaluate GSTO gene polymorphism influence on women's susceptibility to low-risk or high-risk HPV infections and also risk of cervical cancer development. Material and Methods: We examined 50 patients with cervical cancer, 43 patients who were positive for HPV, and 43 healthy individuals as negative controls. We used polymerase chain reaction-restriction fragment length polymorphism to determine GSTO1 A140D and GSTO2 N142D variants in study participants. Results: We found a significant association between the GSTO1 A140D gene polymorphism and HPV 6, 16, 18, 16/18 infections and cervical cancer in Iranian women. We noted a significant difference for the 140AD/142NN combination genotype between patients in the cervical cancer group and healthy controls. There were no significant differences for the GSTO2 N142D genotype and allele frequencies between the patient (i.e., cervical cancer and HPV-positive) groups and controls. Conclusion: The 140AD genotype, 140D allele, and 140AD/142NN combination genotype seem to confer a protective property in women's susceptibility to HPV 6, 16, 18, 16/18 infections and cervical cancer. However, the GSTO2 N142D polymorphism is not associated with HPV infections and cervical cancer. It would appear that GSTO1 A140D SNPs likely play a role in the level of susceptibility to HPV-related cervical cancer.

Molecular Epidemiology of Vancomycin-Resistant Enterococcus faecalis and Enterococcus faecium Isolated from Clinical Specimens in the Northwest of Iran.

This study was conducted to investigate the phenotypic and genotypic characteristics of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium. Antibiotic resistance and virulence genes in the aforementioned resistant isolates were studied using the epsilometer (E)-test and polymerase chain reaction (PCR). These isolates were subjected to typing by pulsed-field gel electrophoresis (PFGE). Thirty vancomycin-resistant enterococci (VRE; 18.75%) were isolated from a total of 160 various clinical specimens cultured for any bacterial growth. Of these, 11 (36.7%) isolates were identified as E. faecalis and 19 (63.3%) as E. faecium. Minimum inhibitory concentrations (MICs) of vancomycin, teicoplanin, and three alternative therapeutic options (linezolid, daptomycin, and quinupristin/dalfopristin) were determined using the E-test. Multiplex PCR was done for confirming species, identification of the resistant genotypes, and the detection of the virulence genes. Finally, the clonal relationship of all VRE strains was studied by PFGE. All VRE strains showed vancomycin MIC ≥256 µg/mL, and 27 (90%) isolates carried the vanA gene, whereas none of the isolates carried vanB. The most common resistance antibiotic pattern observed was toward rifampicin (n = 30 [100%]). Among all virulence genes studied, gelE (n = 28 [93.33%]) was found as the most prevalent virulent gene. VRE isolates exhibited 90%, 46.67%, 100%, and 66.67% resistance to teicoplanin, linezolid, quinupristin/dalfopristin, and daptomycin, respectively. Molecular typing demonstrated 16 PFGE types of VRE isolates (A-P). Although vanA was carried by most of the isolates, PFGE displayed small clonal dissemination among VR E. faecium and VR E. faecalis species.

Comparative Analytical Performance of Various HbA1c Assays in Iran.

INTRODUCTION: Hemoglobin A1c (HbA1c) measurement devices are widely used to evaluate glycemic control in diabetic patients. The aim of this study was to investigate the comparability of various HbA1c instruments used in Iran. METHODS: In the present study, 154 fresh whole blood samples from diabetic patients, with different HbA1c levels (4.0%-10%) and no types of hemoglobinopathy were analyzed by six HbA1c assays including one high performance liquid chromatography (HPLC) method (D10 HbA1c), two immunoassay methods (COBAS INTEGRA 400 and Pars Azmoon kit), one Boronate affinity method (Nycocard Reader II), and two ion exchange methods (Biosystems and DS5). The two National Glycohemoglobin Standardization Programs (NGSP) certified system, D10 and COBAS INTEGRA 400 which are certified as secondary reference measurement procedures, were considered as reference methods. The CLSI document (EP9-A2) - Method comparison and Bias estimation using patient samples, approved guideline - was used to compare the performance of different HbA1c instruments. RESULTS: The mean of HbA1c in all four types of assays was less than the reference methods (P-value < 0.01).The mean of absolute difference between the reference methods was the least (0.11%). Among the other four tests, Biosystems had the smallest mean of difference (-0.21%), while Pars Azmoon had the highest (-1.18%). Pars Azmoon showed the greatest difference (95% confidence interval) when compared to D10 [-15.5%(-5.7%to -25.3%)] and COBAS INTEGRA [-17% (-9.16% to -24.84%)]. The highest regression slope (B) was found in DS5 method (0.96) in regression model with both reference methods. CONCLUSION: It can be concluded that although HbA1c standardization programs have resulted in great improvements in the comparability of HbA1c assays, unacceptable errors still exist and further national and international projects are required for standardization of HbA1c measurement. In this situation, it is recommended to use the same laboratory for HbA1c measurement to monitor diabetic patients.

Comparison of In-House Multiplex Real Time PCR, Diagcor GenoFlow HPV Array Test and INNO-LiPA HPV Genotyping Extra Assays with LCD- Array Kit for Human Papillomavirus Genotyping in Cervical Liquid Based Cytology Specimens and Genital Lesions in Tehran, Iran.

BACKGROUND: Human papillomavirus is a major etiologic agent for some human common cancers. Cervical precancer and cancer is the most prevalent dysplasia by HPV genotypes. Various rapid and sensitive methods have been developed into readily HPV genotyping. METHODS: In the present study, we compared the performance of Real Time PCR, GenoFlow HPV Array, and INNO-LiPA HPV Genotyping Extra Assays with LCD- Array. RESULTS: From 108 cervical samples, HPV was detected in 33 women (30.55%). Among detected HPV genotypes, HPV 6 and 11 were dominant genotypes. Comparing these methods revealed that for Real Time PCR, Genoflow, and INNO-LiPA in comparison with LCD Array, sensitivity and specificity were 94.2%, 93%; 76.7%, 93%; 64%, 96.5%, respectively. Overall, accuracy and precision of these methods were more than 80% and 90%, respectively. CONCLUSIONS: It seems that these methods are reliable and suitable for detection and genotyping of HPVs in cervical disorders and other dysplasia associated with human papillomaviruses.

Effect of the different assays of HbA1c on diabetic patients monitoring.

BACKGROUND: HbA1c test is widely used for glycemic monitoring of diabetic patients. This study aimed to evaluate clinical performance of different assays for classification of patients into controlled and uncontrolled group base on ADA recommendations. METHOD: A total of 154 samples from patients with diabetes type 2 with HbA1c concentration covering the whole clinical range were analyzed by four commercially methods; D-10 Hb A1c (Bio-Rad Laboratories), Cobas Integra 400 (Roche Diagnostics), NycoCard Reader II (Axis-Shield) and DS5 (Drew Scientific). For each individual assay, patient's results were classified into controlled and uncontrolled groups (less or more than three decision levels; 6.5 %, 7 % and 8 %) compared to D10 results as reference method. The frequency of each group and also sensitivity, specificity, negative and positive predictive value were estimated. RESULTS: We found a significant correlation between assays (r: 0.937-0.945). Sensitivity, specificity, and positive and negative predictive values of the evaluated method to identify uncontrolled patients were as follows: 49.2-95.7 %, 86.5-100 %, 89.1-100 %, and 52.9-93.3 %; respectively. CONCLUSIONS: Results show that some HbA1c assays capability to classify diabetic patients according to HbA1c level is still unacceptable.

Priority Setting for Improvement of Cervical Cancer Prevention in Iran.

BACKGROUND: Cervical cancer is the fourth most common cancer among women worldwide. Organized cervical screening and vaccination against human papilloma virus (HPV) have been successful interventions for prevention of invasive cervical cancer (ICC). Because of cultural and religious considerations, ICC has low incidence in Iran and many other Muslim countries. There is no organized cervical screening in these countries. Therefore, ICC is usually diagnosed in advanced stages with poor prognosis in these countries. We performed a priority setting exercise and suggested priorities for prevention of ICC in this setting. METHODS: We invited experts and researchers to a workshop and asked them to list important suggestions for ICC prevention in Iran. After merging similar items and removing the duplicates, we asked the experts to rank the list of suggested items. We used a strategy grid and Go-zone analysis to determine final list of priorities for ICC prevention in Iran. RESULTS: From 26 final items suggested as priorities for prevention of ICC, the most important priorities were developing national guidelines for cervical screening and quality control protocol for patient follow-up and management of precancerous lesions. In addition, we emphasized considering insurance coverage for cervical screening, public awareness, and research priorities, and establishment of a cervical screening registry. CONCLUSION: A comprehensive approach and implementation of organized cervical screening program is necessary for prevention of ICC in Iran and other low incidence Muslim countries. Because of high cost for vaccination and low incidence of cervical cancer, we do not recommend HPV vaccination for the time being in Iran.

Development of in-house multiplex real time PCR for human papillomavirus genotyping in Iranian women with cervical cancer and cervical intraepithelial neoplasia.

BACKGROUND: HPV related cervical cancer as one of the most common women cancers in developing countries. Regarding accessibility of commercial vaccines, any long or short term modality for integrating preventive immunization against HPV in a national program needs comprehensive information about HPV prevalence and its genotypes. The important role of selecting most accurate diagnostic technologies for obtaining relevant data is underlined by different assays proposed in the literature. The main objective of the present study was to introduce an in-house HPV typing assay using multiplex real time PCR with reliable results and affordable cost for molecular epidemiology surveys and diagnosis. MATERIALS AND METHODS: 112 samples of formalin fixed paraffin embedded tissues and liquid based cytology specimens from patients with known different grades of cervical dysplasia and invasive cancer, were examined by this method and the result were verified by WHO HPV LabNet proficiency program in 2013. RESULTS: HPV was detected in 105 (93.7%) out of 112 samples. The dominant types were HPV 18 (61.6%) and HPV 16 (42.9%). Among the mixed genotypes, HPV 16 and 18 in combination were seen in 12.4% of specimens. CONCLUSIONS: According to acceptable performance, easy access to primers, probes and other consumables, affordable cost per test, this method can be used as a diagnostic assay in molecular laboratories and for further planning of cervical carcinoma prevention programs.

C13orf18 and C1orf166 (MULAN) DNA genes methylation are not associated with cervical cancer and precancerous lesions of human papillomavirus genotypes in Iranian women.

BACKGROUND: Nowadays, molecular biomarkers have critical roles for cancer diagnosis and prognosis in clinical laboratories. Human papillomaviruses are the main agents for etiology of cervical carcinoma. The present survey was conducted to evaluate the genes methylation in cervical cancer and precancerous lesions involvement with HPV genotypes. MATERIALS AND METHODS: C13orf18 and C1orf166 (MUL1 or Mulan) DNA methylation as potential biomarkers and risk factors was investigated in 112 liquid based cytology and Formalin-Fixed Paraffin- Embedded tissue specimens in Iranian females with cervical intraepithelial neoplasia and dysplasia. RESULTS: In this survey, HPV18 (61.6%) and HPV16 (42.9%) proved to be the most common HPV genotypes identified by In-House Multiplex Real Time PCR. There were no significant relationship between HPV positivity and the methylated DNA genes mentioned above (p>0.05). CONCLUSIONS: Our MethyLight data demonstrated that these genes could not be considered as specific, sensitive and suitable prognostic biomarkers in cervical dysplasia related HPV. It is suggested that further studies with more patients should be done on candidate methylated markers in different countries in order to plan for cervical cancer prevention.

Real-Time Assay as A Tool for Detecting lytA Gene in Streptococcus pneumoniae Isolates.

OBJECTIVE: In-time diagnosis of Streptococcus pneumoniae (S. pneumonia) can play a significant role in decreasing morbidity and mortality rate. Applying molecular methods has gained popularity due to the existing limits of routine diagnostic methods. Examining the expression of different genes of this bacterium through different molecular methods suggests that lytA gene has a higher sensitivity and specificity in diagnosis of Streptococcus pneumoniae. The aim of this study was to evalutate lytA gene expression in diagnosis of invasive S. pneumonia in culture positive specimens by real-time polymerase chain reaction (PCR). MATERIALS AND METHODS: IIn this a descriptive study, All received specimens were isolated to identify S. pneumoniae. DNA was then extracted and after optimizing the test and determining the detection limit, samples were tested by real-time PCR using lytA gene primers. RESULTS: Twenty seven isolates were diagnosed as S. pneumoniae. In all, the extracted DNA was positive in real-time method. The electrophoresis of the products also confirmed the presence of single product b along with the 53 base pair fragment. The detection limit of the test was less 6 colony forming unit (CFU). CONCLUSION: Real-Time PCR seems to provide reliable and rapid results. We suggest that this test should be conducted on the preliminary isolated specimens, since applying various biochemical tests need one extra working day.