EMAST status as a beneficial predictor of fluorouracil-based adjuvant chemotherapy for Stage II/III colorectal cancer.

BACKGROUND: Elevated microsatellite alteration at selected tetranucleotide repeats (EMAST) is a type of microsatellite instability that occurs in ∼60% of colorectal cancers (CRCs) and associated with MSH3 dysfunction. A 5-fluorouracil (5-FU)-related cytotoxicity is attenuated in MSH3-deficient colon cancer cells. Reported here is the predictive value of EMAST in CRCs with Stage II or III disease treated with 5-FU-based chemotherapy. METHODS: EMAST status was analyzed in 157 patients with CRC with Stage II or III disease and MSH3 expression was analyzed using immunohistochemistry. The patients treated with 5-FU-based chemotherapy were studied in terms of the links of EMAST status with MSH3 expression, clinicopathological features, and overall survival (OS). RESULTS: A total of 63 patients (40.1%) had EMAST positive (EMAST+ ) CRC and 77 patients (49.0%) had low MSH3 expression. EMAST+ tumors were associated with advanced TNM stage and poor and moderately differentiated tumor. EMAST CRC was more frequently observed in tumors with low expression of MSH3 in the nucleus (n = 53; 84.1%, p < .001). On multivariate analysis, patients with EMAST+ status had a worse OS (hazard ratio: 2.489, 95% confidence interval [1.149-5.394], and p = .021). Worse OS in EMAST+ patients who received 5-FU-based chemotherapy was significantly more common compared with EMAST- CRCs. CONCLUSION: There is a link between EMAST and reduced nuclear expression of MSH3. There is worse survival in patients with EMAST+ CRC after 5-FU-based chemotherapy. According to our findings, adjuvant 5-FU-based chemotherapy might not be advantageous in EMAST+ CRCs with Stage II or III disease.

Plasma Levels and Gene Expression of RANK in Non-Alcoholic Fatty Liver Disease.

BACKGROUND: The pathobiology of initiation and progression of nonalcoholic fatty liver disease (NAFLD) has not been completely elucidated. It seems that the RANK/RANKL/OPG cytokine system play an etiologic role in pathogenesis of this disease. This study aimed to investigate the plasma content and gene expression of RANK in NAFLD patients as compared to healthy individuals. METHODS: This case-control work was performed on 63 patients with NAFLD and 25 healthy subjects. The plasma levels of RANK and biochemical parameters were measured using ELISA and colorimetric methods, respectively. Also, RANK mRNA content was evaluated by quantitative RT-PCR in peripheral blood mononuclear cells. RESULTS: RANK plasma contents were shown to be lower in NAFLD patients than in control subjects (1.02 ± 0.75 and 1.41 ± 1 ng/mL, respectively (p = 0.008)). The differences in gene expression of RANK between NAFLD patients and controls were significant (p = 0.001). In the NAFLD patients, RANK was inversely correlated with HDL. Logistic regression showed the association of RANK plasma content with the risk of NAFLD. Moreover, ROC curve analysis showed that RANK has a great ability to differentiate between NAFLD patients and controls. CONCLUSIONS: This study for the first time showed lower plasma and mRNA levels of RANK in NAFLD patients compared to control individuals. These results recommend a possible association between RANK and pathobiology of NAFLD.

Coexistence of genes encoding aminoglycoside modifying enzymes among clinical Acinetobacter baumannii isolates in Ahvaz, Southwest Iran.

Aminoglycosides are widely recommended for treatment of Acinetobacter baumannii infections in combination with ß-lactams or quinolones. This cross-sectional study was aimed to investigate the coexistence of aminoglycoside modifying enzyme (AME) genes among A. baumannii isolates from clinical samples in Ahvaz, Iran. A total of 85 clinical A. baumannii isolates typed by ERIC-PCR were investigated for the presence of AME genes, including ant(3″)-Ia, aac(6')-Ib, aac(3')-Ia, ant(2″)-Ia, and aph(3')-VIa by PCR. The resistance rates to aminoglycoside agents were evaluated by disk diffusion. In this study, 84 out of 85 A. baumannii isolates were resistant to at least one of the aminoglycosides and harbored at least one AME gene. The most common gene encoding AMEs was aph (3')VIa, followed by aac(3')-Ia, ant(3″)-Ia, ant (2″)-Ia, and aac(6')-Ib. The aminoglycoside-resistant genotypes were completely matched to resistant phenotypes to each one of the aminoglycoside agents. There was a clear association between AME gene types and the phenotype of resistance to aminoglycosides with their ERIC-PCR types. Our findings highlight the coexistence of AME genes and clonal dissemination of multiresistant A. baumannii in hospital setting.

CagA and vacA allelic combination of Helicobacter pylori in gastroduodenal disorders.

BACKGROUND: Allelic variation of the virulence genes, vacA and cagA, as the most important virulence associated genes play an important role in the pathogenesis of severe gastrointestinal disease. OBJECTIVE: The aim of the present study was to identify the diversity of the virulence genes in patients with Gastric Cancer (GC), who were referred to the gastro-endoscopy unit of Imam Khomeini Hospital, Ahvaz Jundishapur University of medical science, Ahvaz, Iran. METHODS: Gastric biopsy specimens from 301 patients suspected to gastrointestinal disorders, were analyzed for H. pylori using molecular and phenotypical methods (culture, and biochemical test (catalase, oxidase and urease tests)). RESULTS: Among 201 PCR positive for H. pylori, using histopathological methods, 22 (10.9%) patients had GC. Presence of vacA gene in our H. pylori strains was 100% (201/201), while the most virulent vacA s1 allele was detected in 82.6% isolates, and the mid region vacA m1 was found in 39.8% isolates. The vacA s1/m1 genotype was the most virulent allelic combination in GC and Peptic Ulcer Disease (PUD) in 68.2% and 50%, respectively. The cagA gene was detected in 66.7% isolates. Among the cagA positive isolates, EPIYA-ABC motif was the most common motif in the GC (66.7%), PUD (55.6%) and Erosive Gastroduodenitis (EG) samples (55.2%), while EPIYA-ABCC was the most common motif (58.7%) in the Non-Ulcer Dyspepsia (NUD) samples. The vacA s1m1/cagA+ combination was detected in GC (73.3%) and PUD (51.9%), while vacA s1m2/cagA+ presented in the NUD and EG samples in 77.8% and 62.1%, respectively. CONCLUSION: In this work, Western type (EPIYA-ABC and ABCC motifs) cagA, vacA s1m1 combinations have been demonstrated as the dominant genotype in the tested Ahvazian H. Pylori strains. Also the participation of cagA gene and vacA s1m1 genotype in development and severity of gastric disorder was well evident. Therefore, infection with H. pylori strain containing the cagA gene or the vacA s1m1 genotypes could be associated with increased risk of GC. This is the first study in our area that reports the high incidence and diversity of allelic combination of cagA and vacA genes in gastroduodenitis patients.

Sex determination using free fetal DNA at early gestational ages: a comparison between a modified mini-STR genotyping method and real-time PCR.

OBJECTIVE: Recently the use of free fetal deoxyribonucleic acid (DNA) in maternal plasma and serum has been applicable for noninvasive prenatal genetic diagnosis. In this study, we applied a new algorithmic base conventional polymerase chain reaction (PCR) genotyping method and also real-time PCR for detecting fetal X and Y-chromosome sequences in maternal plasma to determine fetal sex in pregnant women in their early gestational ages (5-13 weeks). Finally, we compared the efficiency of each method in sex determination. STUDY DESIGN: DNA was extracted from 106 pregnant women and their husbands' blood samples. Fetus mini-short tandem repeat (STR) genotyping was accomplished through amplification of 19 mini-STRs and 3 non-STR markers using conventional PCR followed by polyacrylamide gel electrophoresis analysis. Simultaneously, TaqMan real-time PCR was done with the use of DYS14-specific primers and probe. RESULTS: In conventional PCR method, 47 cases were diagnosed to be male and 49 to be female. In comparison, real-time PCR amplified DYS14 (Y-marker) sequences in 45 pregnant women plasma samples. Sensitivity and specificity were calculated to be 95.9% and 98% for conventional PCR and 91.8% and 100% for real-time PCR method, respectively. CONCLUSION: According to our study, the conventional PCR method was more sensitive than real-time PCR and it could be employed in future clinical diagnostics singly or in combination with real-time PCR.

Spastic paraplegia 51: phenotypic spectrum related to novel homozygous AP4E1 mutation.

AP-4-associated hereditary spastic paraplegia (HSP), also known as AP-4 deficiency syndrome, is a genetically diverse group of neurologic disorders defined by complex spastic paraplegia. Different forms of AP-4-associated HSP are classified by chromosomal locus or causative gene. Spastic paraplegia 51 (SPG51) is a neurodevelopmental condition that is caused by autosomal recessive mutations in the adaptor protein complex 4 complex subunit 1 (AP4E1) gene. Further, previous studies described an autosomal dominant mutation in the AP4E1 gene has also been linked to persistent stuttering. Here, we describe a patient from a consanguineous marriage who manifested severe intellectual disability (ID), absent speech, microcephaly, seizure, and movement disorders. Exome sequencing identified a novel homozygous frame-shift variant (NM_007347.5:c.3214_3215del, p.Leu1072AlafsTer10) in the AP4E1 gene, which was confirmed by Sanger sequencing. In this study, we also reviewed the phenotype of the former cases. Our findings added to the knowledge of little-studied homozygous AP4E1 mutation.

Targeting the MALAT1 gene with the CRISPR/Cas9 technique in prostate cancer.

BACKGROUND: The MALAT1 lncRNA acts as an oncogene in Prostate cancer (PC); thus, it can be severe as a cancer biomarker. METHODS: Using bioinformatics datasets including (HTSeq-Counts, GDC, and TCGA) 5501 gene expression profiling specimens were gathered. Then, expression profiles and sample survival of lncRNA were investigated using COX regression analyses, ROC curve analysis. The Database for Annotation, Visualization, and Integrated Discovery was used to conduct GO and KEGG studies on the lncRNA-related PCGs. After MALAT1 Knockout via CRISPR/Cas9 technique, the MALAT1 expression was assessed in DU-145 cells. The deletion of the target fragment was examined by polymerase chain reaction (PCR). Also, the expression of apoptosis genes was investigated by qRT-PCR. The viability and cell proliferation were measured using the MTT assay. Cell migration capability was determined using the cell scratch assay. The results of qRT-PCR were assessed by the ΔΔCt method, and finally, statistical analysis was performed in SPSS software. RESULTS: A maximum of 451 lncRNAs were discovered to reflect different expressions between PC and non-carcinoma tissue samples, with 307 being upregulated and 144 being down-regulated. Thirty-six lncRNAs related to OS were carefully selected, which were then subjected to stepwise multivariate Cox regression analysis, with 2 lncRNAs (MALAT1, HOXB-AS3). MALAT1 is highly expressed in PC cells. MALAT1 Knockout in DU-145 cells increases apoptosis and prevents proliferation and migration, and DU-145 transfected cells were unable to migrate based on the scratch recovery test. Overall, data suggest that MALAT1 overexpression in PC helps metastasis and tumorigenesis. Also, MALAT1 knockout can be considered a therapeutic and diagnostic target in PC. CONCLUSION: Targeting MALAT1 by CRISPR/Cas9 technique inhibit the cell proliferation and migration, and in addition induce apoptosis. Thus, MALAT1 can act as a tumor biomarker and therapeutic target.

A combination of two novels homozygous FCSK variants cause disorder of glycosylation with defective fucosylation: New patient and literature review.

Pathogenic variants in FCSK cause Congenital Disorder of Glycosylation with Defective Fucosylation-2 (FCSK-CDG; MIM: 618,324). It is a rare autosomal recessive genetic disease caused by defects in the L-fucose kinase, which is necessary for the fucose salvage pathway. Herein, we report two novel variants in an Iranian patient, the fourth individual with FCSK-CDG described in the literature. Two homozygous variants in FCSK (rs376941268; NM_145059.3: c.379C > A, p. Leu127Met and rs543223292; NM_145059.3: c.394G > C, p. Asp132His) were identified in the proband. Sanger sequencing conducted on his unaffected parents revealed that they were heterozygous for the same variants. The proband, a four-and-a-half year old Iranian male born to consanguineous parents, manifested Intellectual disability, growth delay, ophthalmic abnormalities, seizures, speech disorder, and feeding difficulties.

MicroRNAs take part in pathophysiology and pathogenesis of Male Pattern Baldness.

Male Pattern Baldness (MPB) or androgenetic alopecia is a common form of hair loss with androgens and genetics having etiological significance. Androgens are thought to pathophysiologically power on cascades of chronically dramatic alterations in genetically susceptible scalp dermal papillas, specialized cells in hair follicles in which androgens react, and finally resulting in a patterned alopecia. However, the exact mechanisms through which androgens, positive regulators of growth and anabolism in most body sites, paradoxically exert their effects on balding hair follicles, are not yet known. The role of microRNAs, a recently discovered class of non-coding RNAs, with a wide range of regulatory functions, has been documented in hair follicle formation and their deregulation in cancer of prostate, a target organ of androgens has also been delineated. Yet, there is a lack of knowledge in agreement with microRNAs' contribution in pathophysiology of MPB. To investigate the role of microRNAs in pathogenesis of MPB, we selected seven microRNAs, predicted bioinformatically on a reverse engineering basis, from previously published microarray gene expression data and analyzed their expression in balding relative to non-balding dermal papillas. We found for the first time upregulation of four microRNAs (miR-221, miR-125b, miR-106b and miR-410) that could participate in pathogenesis of MPB. Regarding microRNAs' therapeutic potential and accessibility of hair follicles for gene therapy, these microRNAs can be considered as good candidates for a new revolutionized generation of treatments.

Frontier Therapeutics and Vaccine Strategies for SARS-CoV-2 (COVID-19): A Review.

COVID-19 is considered as the third human coronavirus and has a high potential for transmission. Fast public health interventions through antibodies, anti-virals or novel vaccine strategies to control the virus and disease transmission have been extremely followed. SARS-CoV-2 shares about 79% genomic similarity with SARS-CoV and approximately 50% with MERS-CoV. Based on these similarities, prior knowledge in treating SARS-CoV and MERS-CoV can be used as the basis of majority of the alternatives for controlling SARS-CoV-2. Immunotherapy is an effective strategy for clinical treatment of infectious diseases such as SARS-CoV-2. Passive antibody therapy, which decreases the virus replication and disease severity, is assessed as an effective therapeutic approach to control SARS-CoV-2 epidemics. The close similarity between SARS-CoV-2 genome with the SARS-CoV genome caused both coronaviruses to bind to the same angiotensin-converting enzyme 2 (ACE2) receptors that found in the human lung. There are several strategies to develop SARS-CoV-2 vaccines, which the majority of them are based on those developed previously for SARS-CoV. The interaction between the spike (S) protein of SARS-CoV-2 and ACE2 on the host cell surface leads to the initiation of SARS-CoV-2 infection. S protein, which is the main inducer of neutralizing antibodies, has been targeted by most of these strategies. Vaccines that induce an immune response against the S protein to inhibit its binding with the host ACE2 receptor, can be considered as effective vaccines against SARS-CoV-2. Here, we aimed to review frontier therapeutics and vaccination strategies for SARS-CoV-2 (COVID-19).

Association of the genes encoding Metallo-ß-Lactamase with the presence of integrons among multidrug-resistant clinical isolates of Acinetobacter baumannii.

Background: Metallo-ß-Lactamases (MBL) are usually encoded on the gene cassettes harboring integrons and disseminated easily among Acinetobacter baumannii isolates. This study was aimed to investigate the association of the genes encoding MBL with the presence of class 1 and 2 integrons among multidrug-resistant (MDR) A.baumannii isolates. Methodology: A total of 85 non-duplicated A.baumannii isolates were collected and evaluated for the amplification of blaOXA-51. The presence of genes encoding MBLs, including blaIMP, blaVIM, blaSIM, blaSPM, blaGIM, blaDIM and blaNDM , as well as intI 1 and intI 2 was evaluated by PCR. Also, the production of MBLs was screened phenotypically by the combination of EDTA and meropenem. Results: In this study, 77 out of 85 isolates were MDR. Also, 34 isolates had only intI 1, 10 had only intI 2 and 15 had both intI 1 and intI 2. The phenotypic detection of MBLs was found in 30 isolates, among which blaVIM was as the most common the gene encoding MBL followed by blaIMP, blaSPM and blaSIM . The gene cassettes analysis revealed that class 1 integron is often responsible for transferring the genes harboring MBLs. Conclusion: The production of MBLs among A. baumannii strains is one of the main mechanisms of resistance to carbapenems. Therefore, the development of inexpensive screening methods for the phenotypic detection of MBLs in clinical laboratories settings is essential. Also, our data revealed that the class 1 integron is often responsible for the dissemination of the MBL genes among A. baumannii isolates.

Circulating Levels of Pro-inflammatory Cytokines in Patients with Nonalcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis.

BACKGROUND: Pro-inflammatory cytokines are associated with systemic inflammatory responses. OBJECTIVE: To investigate the levels of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) in patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) compared to healthy individuals. METHODS: This case-control study was conducted on 30 patients with NAFL, 30 patients with NASH, and 30 healthy volunteers. The plasma level of IL-1ß, IL-6, and TNF-α were determined by ELISA, and biochemical parameters were measured using colorimetric methods. RESULTS: IL-1ß and IL-6 levels were significantly higher in patients with NASH compared with NAFL and control group. However, TNF-α levels had no significant variations in NAFL and NASH patients compared to the control group (p=0.903 and p=0.960, respectively). CONCLUSION: Results showed that the levels of ALT activity and pro-inflammatory cytokines were higher in patients with NASH compared to control and NAFL subjects; Therefore, steatosis and inflammation develop as a result of excessive pro-inflammatory factors in NASH.

Genetic basis for metronidazole and clarithromycin resistance in Helicobacter pylori strains isolated from patients with gastroduodenal disorders.

BACKGROUND: The aim of this study was to evaluate the antimicrobial resistance and genetic basis for metronidazole (Mtz) and clarithromycin (Cla) resistance in strains of Helicobacter pylori, isolated from patients with gastroduodenal disorders. PATIENTS AND METHODS: A total of 157 H. pylori isolates (from 22 gastric cancer, 38 peptic ulcer disease, and 97 non-ulcer dyspepsia patients) were analyzed for drug susceptibility to Mtz and Cla, by gradient diffusion test (E-test, MAST). The PCR and sequence analysis of the rdxA and frxA for Mtz-resistant strains and the 23S rRNA for Cla-resistant strains were used to determine the genetic basis of drug resistance in H. pylori strains. Increased expression of TolC homologous genes (hefA) that upregulates efflux pump activity was determined in multidrug-resistant (MDR) strain of H. pylori by real-time PCR technique. RESULTS: Among 157 H. pylori isolates, 32 (20.4%) strains were resistant to at least one of the antimicrobial agents. The highest resistance rate was attributed to Mtz (n=69, 43.94%). Among the resistant strains of H. pylori, 15 cases (9.55%) were detected as MDR. Mutations in the rdxA (85.5%) and A2143G point mutations (63.1%) in the 23S rRNA were the most common cause of resistance to Mtz and Cla in strains of H. pylori, respectively. In MDR strains, the rdxA mutation and A2143G-point mutation in the 23S rRNA were the most abundant mutations responsible for drug resistance. The relative expression of hefA in MDR strains (mean 3.706) was higher than the susceptible strains (mean 1.07). CONCLUSION: Mutational inactivation and efflux pump overexpression are two mechanisms that increase the resistance to H. pylori antimicrobial agents and the rate of MDR strains. In Iran, the mutations of rdxA and frxA in Mtz-resistant strains and A2143G and A2142G of the 23S rRNA in Cla-resistant strains were significant. The screening for these mutations could help to prevent antibiotic resistance, and to determine the most effective anti-H. pylori drugs.

Virulence-associated genes and drug susceptibility patterns of uropathogenic Escherichia coli isolated from patients with urinary tract infection.

Background: Different Escherichia coli phylogenetic groups, such as A, B1, B2, and D, have four functional groups - adhesins, microcins, toxins, and capsules - which can cause urinary tract infections (UTIs). A phylogenetic group with a high virulence content becomes a worldwide health concern. Resistance to antimicrobial agents increasingly complicates the management of E. coli extraintestinal infections, as a major source of illness, death, and increased health care costs. The aim of this study was to determine the virulence content and the antimicrobial susceptibility pattern of different uropathogenic E. coli (UPEC) phylogenetic groups in Ahvaz, Iran. Methods: Phylogenetic groups, virulence-associated genes (VAGs), and antimicrobial susceptibility tests were detected by molecular and phenotypic methods in a total of 232 clinically well-characterized E. coli strains, isolated from two collections of patients with hospital-acquired (HA) and community-acquired (CA) UTIs. Results: Our results revealed that among 232 UPEC strains, the most frequent phylogenetic group was phylogroup D (58%) with the greatest content in virulence factors, including kpsM (23%), neuA (76.3%, capsule), cnf (29.6%, toxin), and Pap (54.8%, adhesin). Phylogroups D and, to a lesser extent, B2 were the most drug-resistant phylogroups. In addition, phylogroup D was responsible for the majority of HA (64.7%) and CA (48.4%) infections. Conclusion: Among UPEC strains causing UTIs, different phylogroups, through different VAGs, could cause severe infection. Knowledge about the distribution of the four functional groups and VAGs belonging to these phylogroups would significantly help to confine and prevent the development of lethal infection caused by these strains.

WITHDRAWN: Phylotyping based virulence associated genes and drug susceptibility of Uropathogenic Escherichia coli in urinary tract infection patients.

Ahead of Print article withdrawn by publisher.

Frequency of rrs and rpsL mutations in streptomycin-resistant Mycobacterium tuberculosis isolates from Iranian patients.

OBJECTIVES: Streptomycin (SM) is one of the most effective drugs for the treatment of multidrug-resistant (MDR) tuberculosis. However, resistance to SM is increasingly reported, mainly due to mutations in the rpsL and rrs genes. This study was designed with the aim of determining the nature of SM resistance and the type and frequency of rpsL and rrs mutations among SM-resistant Mycobacterium tuberculosis (MTB) isolates from Iran. METHODS: A total of 100 clinical monoresistant and MDR MTB isolates were subjected to drug susceptibility testing (DST) for SM. SM-resistant isolates were genotyped by MIRU-VNTR typing. Fragments of the rpsL and rrs genes were amplified to investigate the most common mutations, with subsequent sequence analysis. RESULTS: By DST, 32 isolates (32%) were identified as SM-resistant, of which 50% (16/32) were MDR. By MIRU-VNTR typing, the SM-resistant isolates were classified into 20 different MIRU types and 8 clusters, with Beijing (22%) being the most prevalent genotype. Mutations in the rrs and rpsL genes were identified in 14 (44%) and 10 (31%) of the 32 SM-resistant isolates, respectively. The most common mutations were at rpsL nucleotide 128 (AAG→AGG, Lys43Arg), found in 7 SM-resistant isolates (22%) and nucleotide 263 (A→G, Lys88Arg) in 3 SM-resistant isolates (9%). CONCLUSIONS: The results suggest an association between rpsL mutation and SM-resistant strains of Beijing genotype. The existence of SM resistance in 25% of isolates without mutation in rrs and rpsL suggests the occurrence of further mechanisms associated with SM resistance in these isolates.

Genetic diversity of multidrug-resistant Mycobacterium tuberculosis strains isolated from tuberculosis patients in Iran using MIRU-VNTR technique.

Tuberculosis (TB) is considered as one of the most important infectious diseases in the world, and recent rise and spread of multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains, have made the matter worsened. Due to the importance of TB prevalence in Iran, this study was designed to investigate the genetic diversity among MDR strains of MTB by MIRU-VNTR typing scheme. A total of 88 drug resistant M. tuberculosis isolates belong to pulmonary TB cases were collected from several TB reference centers of Iran. Drug susceptibility testing for Isoniazid and Rifampin was performed using the agar proportion method and MDR isolates were underwent genotyping by using 12-locus- based MIRU-VNTR typing. On performing proportion method, 22 isolates were identified as MDR. By typing of MDR isolates using 12-loci MIRU-VNTR technique, high diversity were demonstrated in MDR strains and these were classified into 20 distinct MIRU-VNTR genotypes. MIRU loci 10 and 26 were the most discriminatory loci with 8 and 7 alleles respectively; while MIRU loci 2, 20, 24 and 39 were found to be the least discriminatory with 1-2 alleles each. We noticed a mixed infection in isolate 53, as this isolate comprised simultaneous two alleles in MIRU loci 40, 10, 16 and 39. In conclusion, this result represents MIRU-VNTR typing as a useful tool for studying genetic diversity of MDR-MTB in regional settings, and will help the health sectors to construct a preventive program for MDR-TB. Additionally, it can detect mixed infection which can facilitate management of treatment.

Pathogen Identification in Suspected Cases of Pyogenic Spondylodiscitis.

Pyogenic spinal infection continues to represent a worldwide problem. In approximately one-third of patients with pyogenic spondylodiscitis, the infectious agent is never identified. Of the cases that lead to organismal identification, bacteria are more commonly isolated from the spine rather than fungi and parasites. This study applied universal prokaryotic 16S rRNA PCR as a rapid diagnostic tool for the detection of bacterial agents in specimens from patients suspected of pyogenic spondylodiscitis. Gram and Ziehl-Neelsen staining were used as a preliminary screening measure for microbiologic evaluation of patient samples. PCR amplification targeting 16S rRNA gene was performed on DNA extracted from 57 cases including specimens from epidural abscesses, vertebral, and disc biopsies. Positive samples were directly sequenced. MRI findings demonstrated that disc destruction and inflammation were the major imaging features of suspected pyogenic spondylodiscitis cases, as 44 cases showed such features. The most common site of infection was the lumbar spine (66.7%), followed by thoracic spine (19%), the sacroiliac joint (9.5%), and lumbar-thoracic spine (4.8%) regions. A total of 21 samples amplified the 16S rRNA-PCR product. Sanger sequencing of the PCR products identified the following bacteriological agents: Mycobacterium tuberculosis (n = 9; 42.9%), Staphylococcus aureus (n = 6; 28.5%), Mycobacterium abscessus (n = 5; 23.8%), and Mycobacterium chelonae (n = 1; 4.8%). 36 samples displayed no visible 16S rRNA PCR signal, which suggested that non-bacterial infectious agents (e.g., fungi) or non-infectious processes (e.g., inflammatory, or neoplastic) may be responsible for some of these cases. The L3-L4 site (23.8%) was the most frequent site of infection. Single disc/vertebral infection were observed in 9 patients (42.85%), while 12 patients (57.15%) had 2 infected adjacent vertebrae. Elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) inflammatory markers were noted in majority of the patients. In conclusion, microbiological methods and MRI findings are vital components for the proper diagnosis of pyogenic spondylodiscitis. Our findings suggest that molecular methods such as clinical application of 16S rRNA PCR and sequencing may be useful as adjunctive diagnostic tools for pyogenic spondylodiscitis. The rapid turnaround time of 16S rRNA PCR and sequencing submission and results can potentially decrease the time to diagnosis and improve the therapeutic management and outcome of these infections. Although S. aureus and M. tuberculosis were the most common causes of pyogenic spinal infections in this study, other infectious agents and non-infectious etiologies should be considered. Based on study results, we advise that antibiotic therapy should be initiated after a definitive etiological diagnosis.

Comparison of the Antibacterial Properties of Three Mouthwashes Containing Chlorhexidine Against Oral Microbial Plaques: An in vitro Study.

BACKGROUND: The mouth provides an environment that allows the colonization and growth of a wide variety of microorganisms, especially bacteria. One of the most effective ways to reduce oral microorganisms is using mouthwashes. OBJECTIVES: The aim of this study was to investigate the antibacterial effects of chlorhexidine mouthwashes (manufacture by Livar, Behsa, Boht) on common oral microorganisms. MATERIALS AND METHODS: In this in vitro study, isolated colonies of four bacteria, including Streptococcus mutans, S. sanguinis, S. salivarius and Lactobacillus casei, were prepared for an antimicrobial mouth rinse test. The tube dilution method was used for determining the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). RESULTS: The MICs for Kin gingival, Behsa and Boht mouthwashes were 0.14, 0.48 and 1000 micrograms/mL using the tube method for S. mutans, respectively. The MBCs for the mentioned mouthwashes were 0.23, 1.9 and 2000 micrograms/mL for S. mutans, respectively. The MICs for Kin gingival, Behsa and Boht mouthwashes were 0.073, 0.48 and 250 micrograms/mL using the tube method for S. sanguinis, respectively. The MBCs for the mentioned mouthwashes were 0.14, 1.9 and 1000 micrograms/mL for S. sanguinis, respectively. CONCLUSIONS: The Kin Gingival chlorhexidine mouthwash has a greater effect than Behsa and Boht mouthwashes on oral microorganisms and is recommended to be used for plaque chemical inhibition.

Identification of Alloiococcus otitidis, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae in Children With Otitis Media With Effusion.

BACKGROUND: Based on many studies, otitis media with effusion (OME) is one of the major causes of childhood hearing loss, social malformation and medical costs. The pathogenesis still remains unclear, though it is known that this complication is closely related to bacterial infections. Alloiococcus otitidis, Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are the most common bacterial pathogens isolated from middle ear effusions (MEEs). OBJECTIVES: Due to the prevalence of OME in children, we decided to investigate bacterial agents that cause diseases such as A. otitidis, H. influenzae, S. pneumonia and M. catarrhalis in these subjects. PATIENTS AND METHODS: Forty-five children between one and 15 years of age were selected for this study. Seventy specimens were collected from MEE by myringotomy and inoculated in PBS buffer. Conventional culture and PCR methods were used for identification of bacterial agents. RESULTS: The bacterial cultures in 8.6% of samples were positive by conventional culture, with A. otitidis, M. catarrhalis and S. pneumoniae present in 1.4%, 2.9% and 4.3% of samples, respectively. No H. influenzae was isolated. By the PCR method, A. otitidis was the most frequently isolated bacterium, found in 25.7% of samples, followed by S. pneumoniae, M. catarrhalis and H. influenzae, which were identified in 20%, 12% and 20% of samples, respectively. Overall, 55 out of 70 samples were positive by both the PCR and culture method. CONCLUSIONS: It can be concluded that A. otitidis was the major causative agent of MEE in children with OME. Therefore clinicians should be aware that bacterial infection plays an important role in the progression of acute otitis media to OME in children of our region.