Comparison of Real-Time PCR with Disk Diffusion, Agar Screen and E-test Methods for Detection of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the ``gold standard'' comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 mu g/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 mu g/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost

Screening of Myo7A mutations in Iranian patients with autosomal recessive hearing loss from west of Iran

Hearing loss (HL) is the most frequent neurosensory impairment. HL is highly heterogeneous defect. This disorder affects 1 out of 500 newborns. This study aimed to determine the role of DFNB2 locus and frequency of MYO7A gene mutations in a population from west of Iran. Methods: Thirty families investigated in Shahrekord University of Medical Sciences in 2014, genetic linkage analysis via four short tandem repeat markers linked to MYO7A was performed for two consanguineous families originating from Hamedan (family-13) and Chaharmahal-Bakhtiari (family-32) provinces of Iran, co-segregating autosomal recessive HL and showed no mutation in GJB2 gene in our preliminary investigation. All 49 coding exons and exon- intron boundaries of the MYO7A gene were amplified by PCR and analyzed using direct DNA sequencing. Results: Two of families displayed linkage to DFNB2. Family-13 segregated a homozygous missense mutation (c.6487G>A) in exon 48 that results in a p.G2163S amino acid substitution in C-terminal domain of the myosin VIIA protein. While family-32 segregated a homozygous nonsense mutation (c.448 C>T) in exon five, resulting in a premature truncation at amino acid position 150 (p.Arg150X) in the motor domain of this protein. Conclusion: Mutation frequency of MYO7A gene in different populations of Iran as well as cause of HL in most cases are still unknown and more extensive studies have to be done. © 2017, Iranian Journal of Public Health. All rights reserved

Genetic linkage analysis of DFNB24 locus in a group of families with autosomal recessive non-syndromic hearing loss in Khouzestan province of Iran

زمینه و هدف: ناشنوایی رایج ترین اختلال حسی عصبی با بروز یک در هر هزار نوزاد می باشد. حدود 70 از موارد ژنتیکی ناشنوایی را موارد غیر سندرومی تشکیل می دهند. بیش از 100 لوکوس در ناشنوایی غیر سندرومی مغلوب اتوزومی(ARNSHL) درگیر می باشند. هدف از این مطالعه بررسی آنالیز پیوستگی به لوکوس DFNB24 (ژن رادیکسین) در خانواده های مبتلا به ARNSHL می باشد. روش بررسی: در این مطالعه توصیفی- آزمایشگاهی 400 نمونه از 25 خانواده مبتلا به ناشنوایی غیر سندرومی مغلوب اتوزومی با ازدواج خویشاوندی و دارای حداقل سه فرد ناشنوا، از استان خوزستان انتخاب شدند. در نهایت23 خانواده از نظر جهش در ژن GJB2 (لوکوس DFNB1) منفی گزارش و به مطالعه وارد شدند. شش نشانگر STR (Short Tandem Repeat) انتخاب شد و پس از انجام واکنش PCR، تعیین ژنوتیپ با استفاده از بررسی نمونه ها بر روی ژل پلی اکریل آمید، انجام شد. نرم افزارهایی همچون Easy Linkage، SimWalk و HaploPainter برای تجزیه و تحلیل ژنتیکی مورد بررسی قرار گرفت. یافته ها: در راستای بررسی پیوستگی لوکوس DFNB24 در جمعیت ناشنوایان استان خوزستان، نتایج مطالعه ما نشان داد که هیچ مورد پیوستگی بین لوکوس DFNB24 و ناشنوایی در هیچ یک از خانواده ها وجود ندارد. نتیجه گیری: نتایج مطالعه حاضر نشان از آن دارد که احتمالاً جهش های این ژن نقشی ناچیز در بروز ناشنوایی در جمعیت ناشنوای استان خوزستان دارد

Comparison of Agar screen and duplex-PCR methods in determination of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from nasal carriage

Methicillin-resistant Staphylococcus aureus strains (MRSA) have become a serious health issue in engendering nosocomial infections. Due to the heterogeneity of this type of resistance, the conventional antibiotic susceptibility tests may fail to detect MRSA strains. The purpose of this research was to compare the phenotypic agar screen method with polymerase chain reaction (PCR) for detection of MRSA strains isolated from the nasal samples of hospital personnel. Totally, 52 coagulase positive S. aureus strains were isolated from nasal samples of 204 hospital personnel of Hajar Hospital affiliated to Shahrekord University of Medical Sciences. Susceptibility to oxacillin in the strains was evaluated by the phenotypic agar screen method. The presence of the methicillin resistance gene, mec A, was studied through duplex PCR method. The results of both methods were compared and the sensitivity and specificity of the methods were determined. Totally, 23 out of the 52 isolated S. aureus (44%) were phenotypically resistant to oxacillin, but 27 (52%) carried mecA gene. The sensitivity and specificity of the phenotypic agar screen method for determination of MRSA strains were found to be 81.5 and 96%, respectively. As compared to duplex PCR, oxacillin agar screen method is a simple, inexpensive, and practical phenotypic method with relatively low false positive results and thus may be suitable for verification of suspicious MRSA strains. However, for the relatively high false negative results, it may not be recommended for the primary screening of MRSA strains from the nasal samples of healthy carriers working at hospitals

Mutation in second exon of myo15a gene cause of nonsyndromic hearing loss and its association in the Arab population in Iran

Hearing loss is a genetically and clinically heterogeneous defect and more than 140 loci and 65 genes have been identified to cause autosomal recessive non-syndromic hearing loss (ARNSHL). According to the previous studies, mutations in GJB2 are estimated to be involved in 18.17% of ARNSHL cases in the Iranian population; as a result, the remaining 81.83% of this disorder is yet ambiguous. This study aimed to determine the contribution of DFNB3 in hearing loss as well as the frequency of gene mutations in a population (Arab tribal origin) in the Southwest of Iran. In this descriptive laboratory study, we included 25 families from the Southwest of Iran and negative GJB2 gene. Linkage analysis was performed by DFNB3 (MYO15A) molecular markers (STR). The families with hearing loss linked to this locus were further analyzed for mutation detection. MYO15A gene exons were amplified and analyzed using direct DNA sequencing. In studied families, one family displayed linkage to DFNB3 locus. Identified mutations include substitution and substitute C for A in 1047 location of coding region of MYO15A gene (c.1047 C > A) in exon 2 which cause to change Tyrosin to stop codons (P.Y349X), results in the premature truncation at amino acid position 349

Effect of Oxidized Low Density Lipoprotein on the Expression of Runx2 and SPARC Genes in Vascular Smooth Muscle Cells.

BACKGROUND Vascular calcification is an important stage in atherosclerosis. During this stage, vascular smooth muscle cells (VSMC) synthesize many osteogenic factors such as osteonectin (encoded by SPARC). Oxidative stress plays a critical role in atherosclerosis progression, and its accumulation in the vascular wall stimulates the development of atherosclerosis and vascular calcification. The osteonectin overexpression has been observed in the arterial wall during the course of atherosclerosis. However, the regulatory mechanism of oxidized low density lipoprotein (oxLDL)-mediated vascular calcification remains to be clarified. The aim of this study was to investigate the effect of oxLDL on the osteonectin gene expression through the Runx2 transcription factor. METHODS In this experimental study, VSMC were cultured in F-12K media and then treated with oxLDL. The expression of Runx2 and osteonectin genes was determined by real-time PCR method. Protein levels were investigated by the western blotting technique. The Runx2 gene was knocked down using siRNA in order to determine whether Runx2 regulates the osteonectin expression in VSMC induced by oxLDL. Then transfected cells were treated with oxLDL, and the expression levels of Runx2 and osteonectin were determined again. RESULTS oxLDL was found to increase Runx2 and osteonectin gene expression (4.8±0.47- and 9.2±1.96-fold, respectively) after 48 h. Western blotting analysis confirmed the induced levels of Runx2 and osteonectin proteins. However, oxLDL-induced osteonectin expression was not observed to be blocked by Runx2 knockdown. CONCLUSION The up-regulation of osteonectin by oxLDL is independent of Runx2, and it may be mediated by other transcription factors

Comparison of the performance of Disk diffusion, Agar screening and E-test methods with Real- time PCR for the detection of methicillin resistant coagulase negative Staphylococcus strains isolated from clinical samples of Shahrekord university hospitals, 2008

Background : Coagulase Negative staphylococci (CoNS) are nosocomial pathogens. The main objective of the study was to compare the performance of disk diffusion, E-test and agar screening methods with Real-time PCR technique for detection of methicillin resistance in coagulase negative staphylococci (MRCoNS), using Taqman® Real-time PCR for mecA DV WKH ³ JROG VWDQ GDUG´ FRP SDULVRQ DVVD\ � Methods : 88 coagulase negative Staphylococcus isolates were identified out of 284 Staphylococcus isolates collected from Hajar and Kashani hospitals-shahrekod, Iran. Methicillin resistance strains were identified by several methods: Disk diffusion, Agar screening, E-test and Real-time PCR. The results of the tested methods ZHUH FRP SDUHG ZLWK WKRVH RI WKH 5 HDO� WLP H 3& 5 E\ & KL VTX DUH RU ) LVKHU¶ V H[ DFW WHVWV� Results : Of the 88 coagulase negative Staphylococcus isolates tested, 46 isolates (52.3%) were mecA - positive and 42 isolates (47.7%) were mecA-negative. The results of all the tested methods had a statistically significant agreement with those of Real-time PCR. The E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen (6.0 µg/ml) method were 96% and 98%, respectively and the sensitivity and specificity of oxacillin Disk diffusion method were 91% and 90%, respectively. Conclusion: In the present study, E-test is proposed as the best phenotypic method. In case economic issue matters, the oxacillin agar screening method (6.0 µg/ml), which is suitable for the detection of MRCoNS due to its accuracy and low cost, is recommended. Keywords : methicill

Lack of Association between ESR1 and CYP1A1 Gene Polymorphisms and Susceptibility to Uterine Leiomyoma in Female Patients of Iranian Descent

Uterine leiomyonna (UL) is the most common benign smooth muscle cell tumor with as yet unknown etiology and pathogenesis. This study was carried out to investigate the association of ESR1-351 A>G, ESR1 -397 T>C and CYP1A1 (IIe462Val) polymorphisms with UL in female patients of Iranian origin. In this case-control study, 276 patients with UL and 156 healthy women were recruited. The genetic polymorphisms ESR1-351 A>G, ESR1-397 T>C and CYP1A1 (IIe462Val) were genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). No significant difference were found in frequencies of both genotypes and alleles of ESR1-351 A>G, ESR1-397 T>C and CYP1A1 (IIe462Val) polymorphisms between the two groups (p>0.05). Our findings indicated that these ESR1 and CYP1A1 polymorphisms were not associated with the development of UL in the cases reported here

Genetic linkage analysis of DFNB22 in families with autosomal recessive non-syndromic hearing loss in Khuzestan province

Background and aims: Hearing loss (HL) is the most common sensorineural disorder affecting 1 in 1000 newborns. Autosomal recessive non-syndromic hearing loss (ARNSHL), which is the most common cause of severe HL, is caused by mutations in more than 80 loci. The OTOA gene located on DFNB22 is a rare cause of the disease and the gene studied less in Iranian ARNSHL families. Hence, limited information is available on the frequency and type of OTOA mutations in different populations. In this study, we investigated the role of DFNB22 locus in ARNSHL patients in Khuzestan province, Iran. Materials and Methods: In this descriptive-experimental study, 23 large families with pre-lingual ARNSHL from Khuzestan province were enrolled. Mutations in GJB2 were excluded by DNA sequencing followed by linkage analysis. Homozygosity mapping of DFNB22 was conducted using 6 short tandem repeat polymorphic markers via touch-down PCR and polyacrylamide gel electrophoresis. Homozygosityby-descent was identified by calculating two-point and multi-point LOD score and haplotype reconstruction. Results: Families were negative for GJB2 mutations. Genotyping the STRP markers, haplotype reconstruction, and two-point and multiplepoint LOD scores did not show homozygosity-by-descent in any of the pedigrees. Conclusion: Our findings suggest that OTOA mutations might not contribute significantly to the molecular pathophysiology of ARNSHL in Khuzestan province. However, extending the sample size can illuminate the role of this gene in Khuzestan province. Keywords: Genetic linkage analysis, ARNSHL, DFNB22, OTO

Analysis of CABP2 c.637+1G>T mutation in iranian patients with non-syndromic sporadic hearing loss

The most common sensorineural defect in human is congenital hearing loss and genes have an incontestable role in the development of this defect. Many genetic mutations are known to be responsible in this heterogeneous disease. The most frequent mutations are GJB2 mutations followed by the SLC26A4 mutations. Recently, we published a report regarding the role of c.637+1G>T mutation in CABP2 gene, causing hearing loss in three Iranian families. The present study was launched to analyze the role of this recently reported mutation in patients with sporadic hearing loss. One hundred and eighty three patients with moderate to profound sporadic hearing loss were included in this study. The mutation c.637+1 G>T was investigated in patients using the PCR-RFLP method. PCR-RFLP findings revealed that the considered mutation was absent in subjects with sporadic hereditary hearing loss. The mutation c.637+1 G>T in CABP2 gene did not play any roles in the investigated Iranian patients with sporadic hearing loss. Larger samples of different populations, and assessment of all exons and the promoter region of mentioned gene will help to determine the real role of this gene in producing hearing loss. © 2014, Iranian Neurogenetics Society. All rights reserved